2022 Book ProtocolsForTheDiagnosisOfPigV

Rajib Deb · Ajay Kumar Yadav
Swaraj Rajkhowa · Yashpal Singh Malik

Editors
Protocols for
the Diagnosis of
Pig Viral Diseases
SPRINGER PROTOCOLS HANDBOOKS
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Springer Protocols Handbooks collects a diverse range of step-by-step laboratory methods
and protocols from across the life and biomedical sciences. Each protocol is provided in the
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opens with an introductory overview, a list of the materials and reagents needed to complete
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Handbooks are a valuable addition to the laboratory.
Protocols for the Diagnosis of Pig
Viral Diseases
Edited by
Rajib Deb, Ajay Kumar Yadav and Swaraj Rajkhowa

National Research Centre on Pig, Indian Council of Agricultural Research, Guwahati, Assam, India
Yashpal Singh Malik

College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana,
Punjab, India
Editors
Rajib Deb Ajay Kumar Yadav
National Research Centre on Pig National Research Centre on Pig
Indian Council of Agricultural Research Indian Council of Agricultural Research
Guwahati, Assam, India Guwahati, Assam, India
Swaraj Rajkhowa Yashpal Singh Malik

National Research Centre on Pig College of Animal Biotechnology
Indian Council of Agricultural Research Guru Angad Dev Veterinary and Animal Sciences University
Guwahati, Assam, India Ludhiana, Punjab, India
ISSN 1949-2448 ISSN 1949-2456 (electronic)

Springer Protocols Handbooks
ISBN 978-1-0716-2042-7 ISBN 978-1-0716-2043-4 (eBook)
https://doi.org/10.1007/978-1-0716-2043-4
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Preface
Pigs are an adaptable and rapidly growing species that may be attractive for small farms and
beginning farmers seeking to incorporate livestock into their farm. The pig is very much a
vulnerable species from the disease point of view. The species is not so hardy in different
environmental conditions also. There are several diseases of pigs, which affect them in a very
acute manner like African Swine Fever (ASF) or Porcine Reproductive and Respiratory
Syndrome (PRRS). Pigs act as an amplifier host for several zoonotic diseases like Swine
Influenza, Japanese Encephalitis, West Nile Virus, Nipah Virus, and Foot and Mouth
Disease Virus. After the introduction of African Swine Fever in Asian countries, the sector
became very vulnerable and at very high risk to survive in future. One of the contributing
factors in favor of the sustainability of the piggery sector was prompt diagnosis of diseases
and application of preventive measures. There are many advanced diagnostic assays that have
been evolved in recent years. These assays include improved methods of nucleic acid
extraction, polymerase chain reaction (PCR), droplet digital PCR (ddPCR), polymerase
spiral reaction (PSR), cross-priming amplification (CPA), enzyme-linked immunosorbent
assay (ELISA), as well as peptide nucleic acid (PNA) based tools, aptamer-based tools, and
lateral flow assays and different immune assay-based diagnostics for porcine diseases.
The book is a compilation of 26 chapters written by renowned national and interna-
tional veterinary academicians (researchers/young investigators). This book covers new
molecular biological techniques for the detection of both antigens and antibodies of porcine
diseases. Additionally, throughout the book, tables and figures portray the important
diagnostic tools and recommendations, with specific references at the end for readers who
want to obtain further details on each topic. This book aims to build capacity for researchers
who can take advantages from the protocols mentioned in the book. The knowledge
provided in this book will help in developing new technologies for the diagnosis of pig
pathogens.
We believe that owing to the in-depth knowledge on important diagnostics tools, the
present book will be an excellent source of information for scientists, researchers, and
students from different fields.
We, the editors, would like to express our gratitude to all the authors who drafted the
chapters in a very critical way and presented them in a simple form. The editors are
appreciative of Springer Nature for accepting this book proposal, and we extend our special
thanks to David C. Casey, Senior Editor, Springer Protocols, for providing all the editorial
help and high cooperation while processing the manuscripts for its successful publishing.
Guwahati, Assam, India Rajib Deb

Guwahati, Assam, India Ajay Kumar Yadav
Guwahati, Assam, India Swaraj Rajkhowa
Ludhiana, Punjab, India Yashpal Singh Malik
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
1 Requirements and Preparedness for Attending a Viral Disease

Outbreak in Pig Farms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Dipak Deka, Pankaj Kumar Dhaka, Ujjwal Kumar De,
Obli Rajendran Vinodh Kumar, and Yashpal Singh Malik
2 Collection of Samples, Their Preservation and Transportation . . . . . . . . . . . . . . . . 21
Ashok Kumar, Kaushal Kishor Rajak, Ajay Kumar Yadav,
Vishal Rai, Mukesh Bhatt, R. P. Singh, and R. K. Singh
3 Methods for Quantification of Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Mukesh Bhatt, Chris Einstein, Kiran, Arfa Fayaz, Vishal Rai,
Monu Karki, Ashok Kumar, Ajay Kumar Yadav,
and Kaushal Kishor Rajak
4 Protocols for Isolation of Genetic Materials from RNA Viruses . . . . . . . . . . . . . . . 49
Nihar Nalini Mohanty, Vikas Gupta, Laxmi Narayan Sarangi,
Rohini Bhat, and Sathish B. Shivachandra
5 Multiplex PCR for Diagnosis of Porcine Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Manjisha Choudhury, Ajay Kumar Yadav,
Seema Rani Pegu, Rajib Deb, and Swaraj Rajkhowa
6 Protocols for Isolation of Plasmid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Vinod Kumar Singh, Vikas Gupta, and Chayanika Das
7 Recombinant Antigen-Based Diagnostic Assays of Pig Viral Diseases . . . . . . . . . . 109
Rajib Deb, Ajay Kumar Yadav, Gyanendra Singh Sengar,
Seema Rani Pegu, Souvik Paul, Swaraj Rajkhowa,
and Vivek Kumar Gupta
8 RNA-PAGE-Based Diagnosis of Viral Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Naveen Kumar, Geetika Kaur, Shubhankar Sircar,
Zunjar Dubbal, R. S. Sethi, and Yashpal Singh Malik
9 Peptide Nucleic Acid (PNA): A Diagnostic Molecule
for Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Vinay G. Joshi, Anu Kumari, Sushila Maan,
Tarun Kumar, and Satish Kumar
10 Nucleic Acid Sequence-Based Amplification (NASBA) Methods
and CRISPR/Cas13 System to Detect Pig Viral Diseases . . . . . . . . . . . . . . . . . . . . 151
Ajay Kumar Singh, Soumen Naskar,
Pramod W. Ramteke, and Rohit Kumar
vii
viii Contents
11 Aptamers as Diagnostic Markers for Viral Infections

of Veterinary Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Victoria C. Khangembam and Dimpal Thakuria
12 Antibody-Based Sensors for Pathogen Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Nirmita Dutta, Akhil Kumar, Anu Kumari,
Sushila Maan, Gorachand Dutta, and Vinay G. Joshi
13 Lateral Flow Assay for Diagnosis of Pig Viral Diseases . . . . . . . . . . . . . . . . . . . . . . . 195
Aditya Prasad Sahoo and Rajib Deb
14 Droplet Digital PCR-Based Diagnosis for Porcine Viral Diseases . . . . . . . . . . . . . 205
Yoya Vashi and Sachin Kumar
15 Protocols for Immunofluorescence Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Deepika Bisht, Shikha Saxena, Nitish Singh Kharayat,
and Siddharth Gautam
16 Polymerase Spiral Reaction (PSR) for the Diagnosis
of Porcine Viral Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Vikas Gupta, Nihar Nalini Mohanty, and Vinod Kumar Singh
17 Recombinase Polymerase Amplification-Based Diagnostics
of Porcine Viral Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
18 Cell Culture System for Porcine Virus Isolation and Propagation . . . . . . . . . . . . . 251
Vishal Rai, Kaushal Kishor Rajak, Kiran, Arfa Fayaz, Monu Karki,
Chris Einstein, Mukesh Bhatt, Ashok Kumar, and Ajay Kumar Yadav
19 An Overview of Mouse Monoclonal Antibody Production . . . . . . . . . . . . . . . . . . . 265
Kaushal Kishor Rajak, Kiran, Arfa Fayaz, Vishal Rai,
Monu Karki, Chris Einstein, Mukesh Bhatt, Ashok Kumar,
Ajay Kumar Yadav, and R. P. Singh
20 Nucleic Acid Hybridization Techniques for Viral Disease Diagnosis:
A Detailed Perspective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
B. V. Sunil Kumar, Himalaya Bhardwaj, Ankita Gurao,
Naveen Kumar, and Yashpal Singh Malik
21 Ligase Detection Reaction-Fluorescent Microsphere Assay . . . . . . . . . . . . . . . . . . . 291
A. Raja
22 ELISA as a Diagnostic Weapon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Ramya Kalaivanan and Sankar Palanisamy
23 SDS-PAGE and Western Blotting: Basic Principles and Protocol. . . . . . . . . . . . . . 313
Mukesh Bhatt, Vishal Rai, Ashok Kumar, Kiran,
Ajay Kumar Yadav, Kaushal Kishor Rajak, Vikas Gupta,
Vishal Chander, and R. K. Avasthe
24 Immune Assays as Diagnostic for Pig Viral Diseases . . . . . . . . . . . . . . . . . . . . . . . . . 329
Prabhakar Maurya, Jupi Talukdar, Sarmistha Debbarma,
Monuj Kumar Doley, and Luit Barkalita
Contents ix
25 Production of Virus-like Particles Using the Baculovirus Expression

System and Their Application in Vaccines and Viral Disease Diagnosis. . . . . . . . . 351
Hemanta Kumar Maity, Rajib Deb, Sinéad Lyons,
and Ian M. Jones
26 Good Laboratory Practices and Biosafety Containments
in a Virology Laboratory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Yashpal Singh Malik, Anuradha Sharma, Niraj Kumar Singh,
B. T. Naveen Kumar, and Naveen Kumar
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
About the Editors
RAJIB DEB, PHD is currently employed as Scientist at the Indian Council of Agricultural
Research-National Research Centre on Pig, Rani, Guwahati, Assam, India. He has served
more than 9 years at Agricultural Research Service, India. He completed his
postgraduation and PhD at Indian Veterinary Research Institute, Uttar Pradesh, India, in
the subject of Animal Biotechnology. He is currently pursuing the National Post-
Doctoral Fellowship program at National Dairy Research Institute, Karnal, Haryana,
India. He has published more than 35 research papers in peer-reviewed journals. He is
the recipient of several national and international awards including TWAS Fellowship,
Italy; Associate of National Academy of Agricultural Sciences, India; a member of the
National Academy of Sciences, India; and a member of Indian National Young Academy
of Sciences, India. He has filed nine Indian patents and developed several technologies.
He is currently working on the development of diagnostics and vaccines against porcine
disease.
AJAY KUMAR YADAV, PHD is a scientist in veterinary virology at the Indian Council of
Agricultural Research-National Research Centre on Pig, Rani, Guwahati, Assam, India.
He has completed graduation from Govind Ballabh Pant University of Agriculture and
Technology, Uttarakhand, India. Later on, he completed his MVSc and PhD in
veterinary virology from the premier veterinary institute named Indian Veterinary
Research Institute (ICAR-IVRI), Izatnagar, Uttar Pradesh, India. Dr. Yadav joined the
Agricultural Research Services (ARS) in the discipline of veterinary microbiology in the
year 2018. His research interests include disease diagnosis, cell culture, and virus
isolation. He is a member of several societies and has also published more than
20 research articles in peer-reviewed international and national journals. He has more
than 60 GenBank submissions.
SWARAJ RAJKHOWA, PHD joined Agricultural Research Service (ARS) as Scientist in the year
1999. He completed his PhD from Assam Agricultural University in the year 2005. He
did his postdoctoral studies at internationally reputed institutes like Cornell University,
New York, NY, USA, and the University of California, Irvine, CA, USA, where he
learned advanced molecular tools like MLST, SLST, SNP, and DNA Microarray
technology. His area of interest is molecular epidemiology and molecular diagnosis of
infectious diseases of animal origin, including diseases of public health significance. He
has been actively involved in the diagnosis and management of livestock diseases over the
last 20 years and particularly on porcine diseases for the last 12 years. He has published
more than 100 research papers in peer-reviewed national and international journals of
repute, acted as a reviewer of several national and international journals, handled more
than ten externally funded projects as Principal Investigator and several institute-funded
projects, guided master’s and doctoral degree students (including postdoctoral scholar),
and acted as an external examiner of both MVSc and PhD students of SAUs.
YASHPAL SINGH MALIK, PHD serves as Dean, College of Animal Biotechnology at Guru
Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, Punjab,
India, and previously served as ICAR-National Fellow at Indian Veterinary Research
Institute, Izatnagar, India. He works in viral disease epidemiology, virus-host
interactions, microbial biodiversity, characterization, and diagnosis of pathogens. Prof.
xi
xii About the Editors
Malik has acquired advanced training in Molecular Virology from the University of
Minnesota, Minneapolis, MN, USA; Division of Virology, University of Ottawa, Ottawa,
ON, Canada; and Wuhan Institute of Virology, Wuhan, China. He is a recipient of several
prestigious national, state, and academy awards and honors, including the ICAR-
Jawaharlal Nehru Award. He has supervised 5 PhD and 17 MVSc students. He has
authored 5 books, 25 book chapters, and published 217 scientific research and review
articles. Dr. Malik has been the Editor-in-Chief of the Journal of Immunology and
Immunopathology. He has also edited special issues of the Springer Nature journal
VirusDisease and Bentham’s journal Current Drug Metabolism on emerging thematic
areas.
Contributors
R. K. AVASTHE • ICAR Research Complex for NEH Region, Sikkim Centre, Gangtok,
Sikkim, India
LUIT BARKALITA • Department of Animal Biotechnology, Faculty of Veterinary Science,
Assam Agricultural University, Guwahati, Assam, India
HIMALAYA BHARDWAJ • College of Animal Biotechnology, Guru Angad Dev Veterinary and
Animal Sciences University (GADVASU), Ludhiana, Punjab, India
ROHINI BHAT • Department of Biotechnology, University of Agricultural Science, GKVK,
Bengaluru, Karnataka, India
MUKESH BHATT • ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, Uttar
Pradesh, India; Division of Biological Products, ICAR-IVRI, Bareilly, Uttar Pradesh,
India; ICAR-National Organic Farming Research Institute, Tadong, Sikkim, India;
ICAR Research Complex for NEH Region, Sikkim Centre, Gangtok, Sikkim, India
DEEPIKA BISHT • Division of Virology, ICAR-Indian Veterinary Research Institute,
Mukteshwar, Uttarakhand, India
VISHAL CHANDER • ICAR-IVRI, Bareilly, Uttar Pradesh, India
MANJISHA CHOUDHURY • ICAR-National Research Centre on Pig, Guwahti, Assam, India
CHAYANIKA DAS • Centre for Animal Disease Research and Diagnosis (CADRAD), ICAR-
Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India
UJJWAL KUMAR DE • Referral Veterinary Polyclinic & Teaching Veterinary Clinical Complex
(RVP-TVCC), ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh,
India
RAJIB DEB • ICAR-National Research Centre on Pig, Guwahati, Assam, India; Animal
Biotechnology, ICAR-National Research Centre on Pig, Guwahati, Assam, India
SARMISTHA DEBBARMA • ARDD, Govt. of Tripura, Agartala, Tripura, India
DIPAK DEKA • College of Veterinary Science, Assam Agricultural University (AAU),
Guwahati, Assam, India
PANKAJ KUMAR DHAKA • Centre for One Health, Guru Angad Dev Veterinary and Animal
Sciences University (GADVASU), Ludhiana, Punjab, India
MONUJ KUMAR DOLEY • Krishi Vigyan Kendra, Karbi Anglong, Assam Agricultural
University, Diphu, Assam, India
ZUNJAR DUBBAL • Division of Veterinary Public Health & Epidemiology, ICAR-Indian
Veterinary Research Institute, Bareilly, Uttar Pradesh, India
GORACHAND DUTTA • School of Medical Sciences and Technology, Indian Institute of
Technology, Kharagpur, West Bengal, India
NIRMITA DUTTA • School of Medical Sciences and Technology, Indian Institute of Technology,
Kharagpur, West Bengal, India
CHRIS EINSTEIN • Division of Biological Products, ICAR-IVRI, Izatnagar, India
ARFA FAYAZ • Division of Biological Products, ICAR-IVRI, Bareilly, Uttar Pradesh, India
SIDDHARTH GAUTAM • Division of Temperate Animal Husbandry, ICAR-Indian Veterinary
Research Institute, Mukteshwar, Uttarakhand, India
VIKAS GUPTA • CCS-National Institute of Animal Health (NIAH), Baghpat, Uttar
Pradesh, India; National Institute of Animal Health, Baghpat, Uttar Pradesh, India
VIVEK KUMAR GUPTA • ICAR-National Research Centre on Pig, Guwahati, Assam, India
xiii
xiv Contributors
ANKITA GURAO • College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal
IAN M. JONES • School of Biological Sciences, University of Reading, Reading, UK
VINAY G. JOSHI • Department of Animal Biotechnology, College of Veterinary Sciences, Lala
Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, Haryana,
India
RAMYA KALAIVANAN • Faculty of Veterinary Microbiology, Veterinary College and Research
Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal, Tamil
Nadu, India; Faculty of Veterinary Pharmacology and Toxicology, Veterinary College and
Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal,
Tamil Nadu, India
MONU KARKI • Division of Biological Products, ICAR-IVRI, Bareilly, Uttar Pradesh, India
GEETIKA KAUR • College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal
VICTORIA C. KHANGEMBAM • ICAR-Directorate of Coldwater Fisheries Research, Bhimtal,
Uttarakhand, India
NITISH SINGH KHARAYAT • Division of Temperate Animal Husbandry, ICAR-Indian
Veterinary Research Institute, Mukteshwar, Uttarakhand, India
KIRAN • Division of Biological Products, ICAR-IVRI, Bareilly, Uttar Pradesh, India;
ICAR-IVRI, Bareilly, Uttar Pradesh, India
AKHIL KUMAR • Department of Veterinary Microbiology and Immunology, College of
Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar,
Haryana, India
ASHOK KUMAR • ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, Uttar
Pradesh, India; Division of Biological Products, ICAR-IVRI, Bareilly, Uttar Pradesh,
India
B. T. NAVEEN KUMAR • College of Fisheries, Guru Angad Dev Veterinary and Animal
B. V. SUNIL KUMAR • College of Animal Biotechnology, Guru Angad Dev Veterinary and
NAVEEN KUMAR • ICAR-National Institute of High Security Animal Diseases, OIE
Reference Laboratory for Avian Influenza, Bhopal, Madhya Pradesh, India
OBLI RAJENDRAN VINODH KUMAR • Division of Epidemiology, ICAR-Indian Veterinary
Research Institute, Bareilly, Uttar Pradesh, India
ROHIT KUMAR • Department of Biological Sciences, Sam Higginbottom Institute of
Agriculture Technology & Sciences, Allahabad, Uttar Pradesh, India
SACHIN KUMAR • Department of Biosciences and Bioengineering, Indian Institute of
Technology Guwahati, Guwahati, Assam, India
SATISH KUMAR • Retired Principal Scientist, Central Instrumentation Facility, Department
of Veterinary Biotechnology, Indian Veterinary Research Institute, IVRI, Izatnagar,
Bareilly, Uttar Pradesh, India
TARUN KUMAR • Veterinary Clinical Complex, College of Veterinary Sciences, Lala Lajpat
Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, Haryana, India
ANU KUMARI • Department of Animal Biotechnology, College of Veterinary Sciences, Lala
India
SINÉAD LYONS • School of Biological Sciences, University of Reading, Reading, UK
Contributors xv
SUSHILA MAAN • Department of Animal Biotechnology, College of Veterinary Sciences, Lala

India
HEMANTA KUMAR MAITY • Department of Avian Science, West Bengal University of Animal
and Fishery Sciences, Kolkata, India
YASHPAL SINGH MALIK • College of Veterinary Science, Assam Agricultural University
(AAU), Guwahati, Assam, India; College of Animal Biotechnology, Guru Angad Dev
Veterinary and Animal Sciences University (GADVASU), Ludhiana, Punjab, India
PRABHAKAR MAURYA • Consultancy for Environmental, Human Toxicology and Risk
Assessment (India Office), New Delhi, India
NIHAR NALINI MOHANTY • CCS-National Institute of Animal Health (NIAH), Baghpat,
Uttar Pradesh, India
SOUMEN NASKAR • ICAR-Indian Institute of Agricultural Biotechnology, Ranchi,
Jharkhand, India
SANKAR PALANISAMY • Faculty of Veterinary Microbiology, Veterinary College and Research
Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal, Tamil
Nadu, India; Faculty of Veterinary Pharmacology and Toxicology, Veterinary College and
Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal,
Tamil Nadu, India
SOUVIK PAUL • ICAR-National Research Centre on Pig, Guwahati, Assam, India
SEEMA RANI PEGU • ICAR-National Research Centre on Pig, Guwahati, Assam, India
VISHAL RAI • ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, Uttar Pradesh,
India; Division of Biological Products, ICAR-IVRI, Bareilly, Uttar Pradesh, India
A. RAJA • Education Cell, Veterinary College and Research Institute, Tamil Nadu
Veterinary and Animal Sciences University, Namakkal, Tamil Nadu, India
KAUSHAL KISHOR RAJAK • ICAR-Indian Veterinary Research Institute (IVRI), Bareilly,
Uttar Pradesh, India; Division of Biological Products, ICAR-IVRI, Bareilly, Uttar
Pradesh, India
SWARAJ RAJKHOWA • ICAR-National Research Centre on Pig, Guwahti, Assam, India
PRAMOD W. RAMTEKE • Department of Biological Sciences, Sam Higginbottom Institute of
Agriculture Technology & Sciences, Allahabad, Uttar Pradesh, India
ADITYA PRASAD SAHOO • ICAR-Directorate of Foot and Mouth Disease, Nainital,
Uttrakhand, India
LAXMI NARAYAN SARANGI • National Dairy Development Board, Research and Development
Laboratory, Hyderabad, Telangana, India
SHIKHA SAXENA • Division of Animal Genetics, ICAR-Indian Veterinary Research Institute,
Izatnagar, Uttar Pradesh, India
GYANENDRA SINGH SENGAR • ICAR-National Research Centre on Pig, Guwahati, Assam,
India
R. S. SETHI • College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal
ANURADHA SHARMA • College of Animal Biotechnology, Guru Angad Dev Veterinary and
SATHISH B. SHIVACHANDRA • ICAR-National Institute of Veterinary Epidemiology and
Disease Informatics (ICAR-NIVEDI), Bengaluru, Karnataka, India
AJAY KUMAR SINGH • National Institute of Animal Biotechnology (NIAB), Hyderabad,
Telangana, India
xvi Contributors
NIRAJ KUMAR SINGH • College of Animal Biotechnology, Guru Angad Dev Veterinary and
R. K. SINGH • ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, Uttar Pradesh,
India
R. P. SINGH • ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, Uttar Pradesh,
India; Division of Biological Products, ICAR-IVRI, Izatnagar, India
VINOD KUMAR SINGH • Department of Veterinary Microbiology, College of Veterinary Science
and Animal Husbandry, DUVASU, Mathura, Uttar Pradesh, India
SHUBHANKAR SIRCAR • Amity Institute of Virology and Immunology, Amity University,
Noida, Uttar Pradesh, India
JUPI TALUKDAR • Mahisah Perspicientia LLP, Guwahati, Assam, India
DIMPAL THAKURIA • ICAR-Directorate of Coldwater Fisheries Research, Bhimtal,
Uttarakhand, India
YOYA VASHI • Department of Biosciences and Bioengineering, Indian Institute of Technology
Guwahati, Guwahati, Assam, India
AJAY KUMAR YADAV • ICAR-National Research Centre on Pig, Guwahati, Assam, India;
Division of Biological Products, ICAR-IVRI, Bareilly, Uttar Pradesh, India; ICAR-
IVRI, Bareilly, Uttar Pradesh, India
Chapter 1
Requirements and Preparedness for Attending a Viral

Disease Outbreak in Pig Farms
Dipak Deka, Pankaj Kumar Dhaka, Ujjwal Kumar De,
Obli Rajendran Vinodh Kumar, and Yashpal Singh Malik
Abstract
Animal husbandry and livestock rearing are important for rural livelihood and economic development of a
country. The exponential growth of human population demands quality proteins for better health status.
Meat is one of the important sources of high quality proteins, and faster growth and higher feed conversion
efficiency make pig a better animal to bridge the gap of protein demand and population growth. Keeping in
view swine farming is rapidly expanding, both in socio-economically weaker sections of the society for
income source and also emerged as industry for its huge commercial potential. Good husbandry practices
and hygiene are the main factors of animal health and to provide economic benefits through maximized
production. Disease in animals causes negative economic impact in animal production system. Intensive
system of animal rearing poses comparatively greater risk of disease problem than extensive system of
rearing. Pig is a highly prolific animal with greater economic return; however, disease outbreaks are major
point of concern on socio-economical perspective. In recent past swine industry has witnessed significant
numbers of viral disease outbreaks. Proper biosecurity at swine farm level may help in the prevention of
introduction of infectious agents into farm. In addition to proper biosecurity, vaccination of the pigs with
suitable vaccine will keep the infectious diseases at bay. An outbreak investigation requires knowledge on
methods of descriptive and analytical epidemiology. Proper investigation of disease outbreaks is paramount
importance to identify the cause, mode of spread of the disease, and other factors involved in spreading the
outbreak and to control and prevent the spread of devastating diseases. Efficient outbreak investigation
involves proper planning and strategy to answer these important questions. In this chapter we narrated
basics of requirement and preparedness for attending a viral disease outbreak in pig farms.
Key words Epidemic, Biosecurity, Epidemiology, Economics, Health
1 Introduction
Animal husbandry and livestock sectors are very critical for rural
livelihood and economic development of a country. Keeping in
view the rapid expansion of swine farming, presently pig farming
finds not only an important place in socio-economically weaker
sections of the society for income source but it has been also
Rajib Deb et al. (eds.), Protocols for the Diagnosis of Pig Viral Diseases,
Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2043-4_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
1
2 Dipak Deka et al.
emerged as industry for its huge commercial potential. Globally,

the pigs contribute around 2.01% of the total livestock population.
A recent survey reports of January 2020 revealed that there were
about 677.6 million pigs worldwide, from which 310.41 million in
China, 148.2 million in European union, 78.66 million in the USA,
37.85 million in Brazil, 13.88 million in Canada, 11.28 million in
South Korea, 11.05 million in Mexico, and 9.06 million in Japan
has been reported [1]. Among the livestock species, pig has
immense potential to contribute for faster economic return because
of certain inherent traits like better-feed conversion efficiency, high
fecundity, early maturity, and short generation interval. It has enor-
mous role to ensure nutritional and economic security of the soci-
ety as it requires small investment on buildings and farm
equipment. Furthermore, pork consumption being popular
among select populations, the improved pig husbandry programs
have found to be significantly contributed in the poverty alleviation
strategies in many parts of the world. Keeping in view the huge
commercial potential of swine farming, now a days, commercial pig
farming has come up as great business idea, startups and income
source for the people [2].
A sow can be bred as early as 8–9 months of age and can farrow
twice in a year under optimal management conditions. They can
produce 6–12 piglets in each farrowing [3]. It has immense poten-
tial to ensure nutritional and economic security for the weaker
sections of the society. In spite of enormous economic potential
and improved management practices, pig farmers regularly face
huge economic losses due to various infectious diseases which
lead to huge mortality of piglets and reproductive failure among
sows [4]. The mortality of an adult pig or its litter influences the
economic viability and overall profitability of the farm. Piglet mor-
tality in neonatal age has emerged as major hurdle to make pig
production as profitable enterprise. In India, piglet mortality was
observed up to 25% in preweaning stage because of infectious
diseases [5]. The important diseases related with pig husbandry
have been listed in Table 1.
Among the various infectious causes, major viral disease out-
breaks occur continuously and pose a major point of concern for its
socio-economically devastating effect which causes huge economic
loss to the pig industry. With the globalization of economies and
abolition of trade barriers, the increase in national and international
trade of animal and animal products pose multifaceted challenges to
tackle the viral disease outbreaks in pig industry. In last decades,
various countries across the globe experienced several outbreaks of
porcine viral diseases (Table 2). Most recently in 2020, the out-
break of African swine fever has been recorded in India. In devel-
oping countries including India, unavailability of major vaccines
except a few is also another reason of frequent outbreak of viral
diseases, resulting in mortality of pigs.
Requirements and Preparedness for Attending a Viral Disease Outbreak. . . 3
Table 1
Important viral diseases of swine [6, 7]
S. No. Name of the disease Causative agent

1. Classical swine fever Classical swine fever virus, Pestivirus (family:
Togaviridae)
2. Foot-and-mouth disease Foot-and-mouth disease virus (FMDV)-
Aphthovirus; family: Picornaviridae
3. PCV2-systemic disease (PCV2-SD) and Porcine circovirus-2 ( family: Circoviridae)
PCV2-reproductive disease (PCV2-RD)
4. Porcine reproductive and respiratory Porcine reproductive and respiratory syndrome
syndrome (PRRS) virus (PRRSV)-genus arterivirus
5. Porcine epidemic diarrhea Porcine epidemic diarrhea virus (PEDV)-
coronavirus (family: Coronaviridae)
6. African swine fever (ASF) African swine fever virus (ASFV)
Family: Asfarviridae
7. Pseudorabies (Aujeszky’s disease) Suid herpesvirus 1; family: Herpesviridae
8. Stillbirth, mummification, embryonic Porcine enteroviruses ( family: Picornaviridae) and
death, and infertility (SMEDI) Porcine parvoviruses (family Parvoviridae)
9. Blue eye disease Porcine rubulavirus (family: Paramyxoviridae)
10. Japanese B encephalitis Japanese encephalitis virus-Flavivirus ( family:
Flaviviridae)
11. Vesicular exanthema of swine Vesicular exanthema of swine virus-Vesivirus
(family: Caliciviridae)
12. Nipah Nipah virus- Henipavirus (family:
Paramyxoviridae)
13. Encephalomyocarditis Encephalomyocarditis virus (EMCV)
(Cardiovirus, family: Picornaviridae)
14. Bovine viral diarrhea infection Bovine viral diarrhea virus (Pestivirus) [8]
Some of these viral diseases can be effectively controlled while

others become endemic and cause sporadic outbreaks in affected
countries. The highly infectious viral diseases such as African swine
fever, classical swine fever, foot-and-mouth disease, and reproduc-
tive failure due to Porcine Parvovirus, PRRS, Porcine circovirus,
etc. can cause severe economic losses in pig production leading to
shortage of supply and eventually affect global pork prices with
increasing demand. To prevent and control the further spread of
viral diseases during outbreak in effective manner, veterinary pro-
fessionals often are engaged and play a key role in disease investiga-
tion. The important factors related with the disease outbreak (s) in
pig farming are discussed in the following [31–33].
4 Dipak Deka et al.
Table 2
Notable outbreaks of swine viral disease in Asian and other countries [7]
S. No. Name of the disease Country

1. Classical swine fever China [9]
2. Foot-and-mouth disease Taiwan (1997) ([10]; [11])
3. PCV2-systemic disease (PCV2-SD) and Kansas, North Carolina, and Iowa (2005); Canada
PCV2-reproductive disease (PCV2- (2004) ([12]; [13])
RD)
4. Porcine reproductive and respiratory China (2006, 2009–2010); Vietnam (2007);
syndrome (PRRS) Philippines (2008); Laos (2010); Thailand (2010);
Cambodia (2010); Myanmar (2011) ([14]; [15];
[16]; [7])
5. Porcine epidemic diarrhea Thailand (2007), Vietnam (2009), China (2010) and
South Korea (2013) ([17]; [18]; [19])
6. African swine fever (ASF) China (2018), Mangolia (2019), Vietnam (2019),
Cambodia (2019), Hong Kong (2019), North
Korea (2019), Laos (2019), Myanmar (2019),
Philippines (2019), South Korea (2019), Timor-
Leste (2019), Indonesia (2019) and India (2020)
([20]; [7])
7. Pseudorabies (Aujeszky’s disease) Italy (2019) [21]
8. Blue eye disease USA (2001) [22]
9. Japanese B encephalitis Australia (1998); China (2018) ([23]; [24])
10. Vesicular exanthema of swine China (2015) [25]
11. Nipah Malaysia (1998 to 1999) [26]
12. Encephalomyocarditis New South Wales (1970); [27]/Belgium (2007,
2016) ([28]; [29])
13. Bovine viral diarrhea infection England (1992), [30]
1. Level of biosecurity practices: Biosecurity is the implementation

of measures that reduce the risk of the introduction and spread
of disease agents; it requires the adoption of a set of attitudes
and behaviors by people to reduce risk in all activities involving
domestic, captive/exotic, and wild animals and their products
([34]). The biosecurity is implemented in two tier systems:
national level and farm level. Development of strategies to
prevent the spread of disease outbreaks, control or eradication
of endemic diseases, and transboundary disease transmission
are the key factors to contain the disease by biosecurity at
national level. The valuable factors to strengthen the biosecur-
ity at swine farm level are location of pig farm, size and design
of farm, introduction of new pigs, replacement stock, pig
buyers, entry of visitors, fencing, working personnel, quaran-
tine method, vaccination, and management of farm. Small
(back-yard) to medium farms with poor biosecurity practices

remain among the key factors involved in outbreaks. Most of
the smallholders tend to feed the pigs with improperly treated/
processed leftover food or food waste (swill feed) from various
sources such as households, markets, factories, restaurants,
which could contain infectious agents and contaminate the
farm. Moreover, in many places the back-yard and big farms
are frequently found in close proximity to each other which
could be the major factor for facilitating the faster spread of
infectious diseases under such scenarios. However, segregation,
cleaning, and disinfection will be the key measures for effective
biosecurity at farm level.
2. Availability of vaccines: Vaccinations are important tools for
the control of viral disease in pigs like other animals. Few
vaccines are available for major viral diseases of pigs such as
FMD and CSF, but not ASF. Vaccination reduces the pressure
of pathogens, shedding, and disease pressure in the region.
However, the recommended vaccines must have been tested
for efficiency with the existing standards for prophylactic mea-
sures against viral diseases (OIE) [34].
3. Globalization and trade: The rapid and huge trading of pigs
and pork within and between neighboring countries can lead to
the spread of infectious agents. The pork supply chains contain
multiple stakeholders such as pig farmers, brokers, traders,
slaughterhouses, retailers, and consumers. This could facilitate
rapid and long-range disease transmission within countries via
the movement of infected animals and contaminated
vehicles [35].
4. Epidemiological status of disease: The risk of introduction of
new viruses or viral strains in a region due to the increasing
importation of the breeding pigs should also be considered. If
these imported pigs are not carefully monitored, viruses of
exotic origin and other pathogens can be introduced into the
countries which may further lead to disease outbreaks. These
viruses might get established and even become the dominant
circulating viruses causing complicated problems [36]. More-
over, considering that there are already local viruses circulating
in the given region, it could then act as a focus for viral
recombination, especially for RNA viruses. Recombination
between novel and local strains is not unexpected when
co-circulated within the same farm. In fact, these phenomena
have already been shown for some viruses in Asian countries,
for example, NADC30-like PRRSVs in China [37]. However,
the epidemiological points of view, continuous surveillance of
disease, some determinants such as contagiousness, tenacity,
and case fatality rate, and their impact on persistence and
transmission of viruses are also important for future outbreak
of diseases [31, 32].
6 Dipak Deka et al.
5. Consumer’s behaviors: Many consumers still prefer buying pork

from wet animal markets. Poor biosecurity management in
small-scale slaughterhouses and wet markets is responsible for
contamination of pork and its products in the area with various
swine pathogens. Moreover, consumption of wild boar as tra-
ditional delicacy also contributes to spill over of pathogens to
humans and domestic pigs in a direct manner or through
environment contamination.
6. Wildlife as reservoir: In many places, especially where extensive
farming is practiced, the wild boars can play a major role in
disease transmission. Cross-species disease transmission
between wildlife and pigs is an increasing threat to outbreaks.
The threats posed by diseases in wild pigs have been recognized
in all pig raising countries. Wild pigs (Sus scrofa), that include
feral domestic pigs (Sus scrofa domestica), Eurasian wild boar
(Sus scrofa linnaeus), and hybrids between the two, are the
most abundant free-ranging, exotic ungulates of increasing
concern as a potential source for cross-species transmission.
In some parts of the world, wild pigs have been identified as
an important reservoir for disease epidemic such as classical
swine fever virus, African swine fever virus, and foot-and-
mouth disease. These diseases, often termed transboundary
animal diseases, can cause high morbidity and mortality in pig
populations through outbreaks [38].
7. Lack of awareness: During an infectious disease outbreak, it is
vital to learn as much as possible about the concerns, knowl-
edge, attitudes, and behavior of the public. Such information
can be crucial to the improvement of communication efforts by
veterinary disease investigation officers. High concern may not
always translate into a higher compliance with precautionary
recommendations, possibly due to the low or lack level of
knowledge about the disease among the public. Frequent com-
munication between veterinarians and the stakeholders is
recommended to help dispel myths about the disease and to
spread better information about the role that the public can
play in limiting the spread of the disease. Lack of awareness
regarding the diseases in farmers and livestock traders leads to
the spread of diseases; as it is known that the movement of
animals through livestock traders is often the key epidemiolog-
ical factor in the spread of diseases. Therefore, collaborative
efforts orchestrated by the animal health department are
needed and should focus on public education and training
through media resources [39].
2 Infectious Disease Outbreak and Its Spread
As discussed, the globalization, intensive swine production,

increased trade and travel, and changing climate have increased
the risk of catastrophic animal losses due to infectious diseases.
Any outbreak originating from small farms due to the use of con-
taminated swill feeding, poor biosecurity, and lack of awareness
could be a very important starting point of infectious viral out-
breaks in the naı̈ve areas. The routes of virus transmission into these
farms could be varied and include swill feeding or contaminated
fomites and vehicles. For larger farms with good biosecurity man-
agement, the risk of pathogen introduction is generally low. How-
ever, the risk will be increased when the viral load contaminating
environment in the region increases due to outbreaks in neighbor-
ing farms, particularly back-yard farms. Therefore, farm standardi-
zation policy to eliminate the farms with poor biosecurity as well as
compartmentalization or zoning is highly suggested in the affected
regions. Thereby, effective surveillance is considered as prerequisite
for the certification of the disease-free status of an area or country
[7, 40].
3 Epidemiological “Know-How”: An Important Pillar for Development

of Surveillance System and Outbreak Preparedness
To substantiate the risk of any exotic disease(s) or status of endemic

disease in a region for implementation of surveillance system, the
epidemiological “know-how” about the infectious diseases in con-
text of possible risk factors including its geographical distribution,
viruses and its serotypes, vector distribution, host range, reservoirs,
ongoing surveillance activities, climatic change scenario, disease
and/or vector control programs, vaccination status of animal, and
status of targeted disease/pathogens among trading partners, is
essential. The surveillance objectives should depend on the outputs
needed to support decision-making and thus be policy-driven [41].
The impact and epidemiology of various viral pig diseases are widely
differing in different parts of the world; therefore, regions or
countries should adapt the surveillance strategies as per local
requirements.
Key considerations for use of surveys and diagnostic tools in
surveillance system: The sampling strategies, sample size, design
prevalence, and appropriate confidence interval for surveys should
be justified based on prevailing epidemiological situations. On
diagnostic front, proper validation of sensitivity and specificity of
the diagnostic tests for targeted species should be carried out.
Moreover, for the diseases where vaccination is available, it is
important to differentiate “vaccinated versus infected” animals by
8 Dipak Deka et al.
Fig. 1 An overview of the steps involved in designing of a surveillance system
diagnostic tests for unbiased interpretation of the surveillance data

[42]. An overview of important steps for designing of the surveil-
lance system has been provided in Fig. 1.
The important approaches to be planned for surveillance in
order to estimate the prevalence or emergence of any infectious
disease have been described in Table 3.
4 Investigation of Viral Disease Outbreak in Swine
It depends upon the clear-cut objective, availability of facilities,

resources, and tolls of investigations such as specific diagnostic
tests. The main objectives of viral disease outbreak
investigations are: (a) to know the origin and identify the causal
factors of disease and (b) mode of transmission.
4.1 Steps of Disease 1. Reporting of disease outbreak and collection of initial basic
Outbreak Investigation information: The report of any suspected swine disease out-
and Management [43] break can be gathered from different sources or different
Table 3
Approaches for surveillance system with the objective of estimation of disease prevalence [43]
Approach Description
Measure of disease/ Prevalence of the targeted disease/pathogen in the study population and
outbreak associated reservoir and/or vector capacity
Coverage Structured active surveillance to obtain the representative estimates about the
disease/pathogen
Means of data Mainly through active surveillance. The passive surveillance can also be used as
acquisition complementary source of information, but it is often difficult to account for
the possible bias
Frequency of It can be one-off (estimate prevalence) or repeated surveys (estimate changes in
sampling prevalence, e.g., to monitor the effect of control/intervention measures)
Testing method Serological testing is preferred as it is more practical and often less costly as
compared to pathogen detection
Design prevalence According to the expected prevalence; a conservative estimate means choosing a
design prevalence which tends towards 50% to maximize sample size and thus
increase precision
Frequency of analysis After each survey. The risk-based surveillance strategy may also be opted during
high seasonal vector activity or in other risk associated scenario (import of
infected animals, live vaccine related outbreaks, etc.)
For vaccination Serological and virological testing is required to characterize the circulating virus
program serotypes in given regions in order to ensure that all serotypes are included in
vaccination program
peoples such as veterinary workers at village, pig farmers, local/

public authorities, private veterinary practitioners, pork traders
or others. Although reporting of disease outbreak is a very
sensitive issue which can results in huge national and private-
sector costs it should be encouraged by the animal health
authorities otherwise it may discourage future reporting or
the outbreak will be unnoticed resulting in uncontrollable
disease situation. After receiving the report of disease outbreak,
the basic information about the nature of the disease, location,
and the extent and time frame of the outbreak should be
recorded properly.
2. Preparing to attend the field outbreak investigation: It is impor-
tant to obtain certain information such as the age group/breed
of pigs involved, the time of the outbreak, the clinical signs, and
the location of the outbreak prior to field visit which may help
during the investigation process.. After that, a team of experts
for disease investigation is to be prepared and the list of items/
equipment’s required for the investigation is to be decided by
expert team. However, certain items such as disinfection equip-
ment, recording accessories, post-mortem/collection of
10 Dipak Deka et al.
biological and allied sample collection accessories, pig restraint

equipment, paper and pens, and other necessary items which
should always be included. However, this activity can effec-
tively be executed using a standard checklist.
3. Verification of actual problem: To verify the outbreak, firstly, the
probable cause of outbreak is to be identified. After that the
extent of the outbreak is assessed and information is gathered
for further investigation and adopting appropriate control
measures. Accumulation of information like details of animals
involved in the outbreak (species, breed, and age), place (geo-
graphical location, households, farms or villages), and time
(the time of onset of disease) are very important. Apart from
this, the morbidity, mortality, and case fatality rate as well as
number of pig population at risk of disease are also to be
recorded.
4. Amalgamation of all the above information: After gathering all
the possible information, the disease is defined by considering
the following steps.
(a) Classical signs and symptoms of pig before death.
(b) Recording the history and clinical examination of individ-
ual case of both affected and healthy pigs.
(c) Collection of all types of biological/clinical samples from
both healthy and affected pigs.
(d) On-site post-mortem examination of carcasses by expert
veterinary pathologists.
(e) Collection of morbid samples during post-mortem
examination.
(f) Examination and collection of associated factors such as
water, vectors, feeds, etc. to confirm their possible role in
disease outbreak.
5. Analysis: After analysis of all the above-mentioned data, labo-
ratory investigation and post-mortem examination of carcasses,
a statistical association between the cause and possible diseases
need to be established to determine the acceptance or rejection
of hypothesis. In case of rejection, another possible hypothesis
needs to be explored by re-examining the collected data.
6. Preparation of report: This section may comprise of two parts:
(1) details of observations and analysis report and (2) appropri-
ate recommendations to prevent mortality/new cases and
future outbreak.
(a) Details of observations and analysis report: It includes lab-
oratory test, post-mortem examination finding, data anal-
ysis, photographs, and experimental findings, if any.
(b) Appropriate recommendations: The suitable recommenda-

tion after disease outbreak investigations is the utmost
important to control the spread of disease quickly as pos-
sible. The key recommendation should be directed
towards the following aspects:
l Immediate disinfection measures of the farm as it may
vary from virus to virus (e.g., FMD virus is sensitive
to pH).
l Strengthening the biosecurity measures of farm.
l Breaking/hindering of virus transmission pathway.
l Communication of the control measures to different
stakeholder (local authorities, police, municipal offi-
cers, pork traders, pork-market owners, other farmers
and the general public) groups who are responsible for
the implementation.
l The line of treatment required for affected animals in
an outbreak.
l Communication to concerned authorities (State and
Central Government Animal Health Department) in
case of notifiable disease outbreak.
7. To-do-list for an effective outbreak investigation: An outbreak of
exotic animal disease would have widespread impact on animal
farming, export potential, tourism, wildlife, and other sectors
resulting in significant losses if such an outbreak was not dealt
with in an effective and timely manner. In brief, the important
facts to be observed before-, during-, and at the end of out-
break investigation are [44]:
8. Before and early phase of an outbreak:
(a) How vulnerable is the swine population to the disease,
where will the pathogen arrive and where should surveil-
lance be focused?
(b) Which surveillance method should be used?
(c) What is the ongoing outbreak size, and what is the present
pathogen status?
(d) Where and how can interventions be introduced to eradi-
cate the pathogen quickly?
(e) How the healthy animals can be saved from getting
infection?
(f) Where and how the carcasses of the infected animals will
be disposed of?
(g) Which data and resources are required to allow forecasting
and control to be performed effectively?
9. Once a major epidemic is ongoing, the following observations

should be taken into account:
(a) How pathogen can transmit from one animal to another
and by which route?
(b) How many cases will there be reported in 24 h?
(c) How effective are current prevention and control efforts?
(d) Which interventions should be introduced, and how
should they be adapted as the epidemic continues?
10. Observations to be considered at the end of a major epidemic:
(a) Can the epidemic be declared over, or do hidden cases
remain in the animal population (reservoir/carrier status)?
5 Outbreak Investigation Steps for Infectious Diseases
An outbreak investigation is a systematic procedure which is used to

identify the source of cases of infection with a view to control and
prevent possible future occurrence [45]. The outbreak investiga-
tion is important to ensure that appropriate preventive and control
measures have been applied or not. The outbreak investigations
help in the assessment of failures and successes of the intervention
measures, and also in the identification of the changes in the disease
agent, supporting environment or events that might be beyond the
scope of a disease control program. Therefore, it is imperative to
maintain the proper recording of outbreak investigations protocols.
In zoonotic outbreaks, the outbreak investigation should be done
in coordination with public health authorities [45].
5.1 Requirements 1. Laboratory Diagnostic capabilities: The rapid and accurate

for Outbreak diagnosis can only be possible in well-equipped laboratories
Investigation that have range of chemicals/reagents, experienced staff, and
standardized instruments.
2. Collaboration with International laboratories and centers:
There are a network of FAO and OIE reference laboratories
and collaborating centers around the world which are available
to provide advice and assistance to countries during disease
outbreak. In India, Regional Disease Diagnostic Laboratories
(RDDL) often collaborates with Central Disease Diagnostic
Laboratory (CDDL) during the investigations of animal’s dis-
ease outbreaks.
3. Preparation of rapid diagnostic tests (ELISA based, nucleic acid
based, lateral flow assay or other) to obtain results in short time
period (FAO animal production and health manual).
5.2 Descriptive Who?

Epidemiological Steps l How many pigs affected from the outbreak?
of Outbreak
Investigation
l Which breed (including other parameters—age and sex, etc.)
and human population [at-risk occupational and health groups
(pregnancy, immunosuppression, children, elders, etc.)] are
more sensitive?
When?
l Temporal relationship between the disease frequency and related
events. Events that may be associated with infectious disease
outbreaks are as follows.
– Importation of animals from endemic areas.
– Efficacy of used diagnostic and quarantine methods (if any).
– Change in farm management and nutritional practices.
– Seasonal changes and associated vector activity (especially
ticks).
Where?
l The outbreak can be described in terms of spatial distribution to
observe associated risk factor(s). The geographic information
system (GIS) may be used to layout the spatial distribution
parameters such as the following.
– Climate zones and seasonal trends.
– Geographical characteristics.
– Vegetation index.
Why?
l Agent responsible for outbreak?
l Origin of infection?
l Wildlife reservoir?
l Mechanism of transmission?
l Risk factors that can predispose susceptible animals for infection.
The causal association between a factor and outcome can be
established by using analytical and experimental studies. However,
past epidemiological studies can be used to describe the outbreak
characteristics.
5.3 Important Farm In animal population, associated risk factors, such as farm size,
Related Risk Factors stocking density, contact rate, production type, import of infected
for Disease animals, quarantine procedures, nutritional status, infected feed,
Outbreaks Are infected water, immunosuppressive disorders, and other farm man-
agement procedures, may play an important role at farm level
outbreak.
Zoonoses in human population, associated risk factors, such as

contact with animals, use of PPE, at-risk groups, culinary habits
(e.g., uncooked meat), unhygienic habits and immunosuppressive
disorders, may play an important role in outbreak.
5.4 Steps The important steps of outbreak investigation along with the
of Outbreak concerned details have been discussed in the following:
Investigation
1. Establish the existence of outbreak:
Are numbers of observed cases exceeding the expected levels?
Example: In a farm free from any infectious disease, a single

case may also be considered as an outbreak, whereas in endemic
areas the number of cases above threshold level constitutes the
outbreak.
2. Verify the diagnosis using clinical and laboratory diagnosis:
During the suspected outbreak (e.g., high rate of abortions
in pigs), the clinical samples (e.g., aborted material, vaginal
mucus, serum samples, etc.) of the farm animals; associated
human contacts and environmental samples (e.g., farm soil
and sewage) can be collected for laboratory examination.
Various laboratory examinations including conventional
methods, serological diagnosis and molecular techniques can
be employed for detection of the pathogens.
3. Define a case based on its standard elements (e.g., clinical
information, time, place, and affected individuals) and varying
degrees of certainty (with associated risk factors).
4. Identify additional cases linked by similarities to the case
definition.
5. Perform descriptive epidemiology of the outbreak description
of epidemic curve and spatial distribution of the disease.
6. Develop a hypothesis using descriptive epidemiology (e.g.,
animal herds/farms, place, and time with the clinical and labo-
ratory findings) and test the new hypothesis using analytic
epidemiology (e.g., cohort or case-control study).
7. Reconsider the hypothesis by “squaring” the hypothesis to the
clinical, laboratory, and epidemiologic facts. In addition, devel-
opment of a new hypothesis for re-testing may also occur.
8. Perform additional studies (if needed) by better defining the
extent of the epidemic, evaluating new laboratory methods and
case-finding techniques, or conducting an environmental
investigation.
9. Implementation of control measures.
10. Communicate findings for community awareness.
5.5 Emergency As per OIE guidelines, all the countries should develop emergency
Preparedness preparedness and contingency plans for immediate action for dis-
and Contingency eases fulfilling the provisions of Article 1.1.3.1. of the Terrestrial
Planning [46] Code. The emergency response plans should be up-to-date tested
(e.g., simulation exercise) and should be embedded in the legal
framework. There must be proper chain of command and coordi-
nation with relevant support services in order to ensure the execu-
tion of rapid control efforts. A contingency plan is a set of activities,
including immediate actions and longer-term measures, for
responding to an animal health emergency such as disease out-
breaks [46]. The contingency plan should be simple to understand
and implement that involve organizing a team of relevant autho-
rities and stakeholders with proper identification of critical
resources and functions, along with the established plan for recov-
ery. The contingency plan should be properly documented, tested,
and regularly updated. The key components in a contingency plan
include established chain of command, systems for rapid detection
and confirmation, outbreak investigation procedures, rapid con-
tainment measures (e.g., movement control, disinfection, vaccina-
tion, culling), and communication strategy. Following the
confirmation of an outbreak, the control areas/containment
zones may be established. The extent of these zones depends on a
number of factors, in particular, the epidemiological characteristic
of the agent and disease. The control measures imposed in these
areas will often include movement restrictions, intensified surveil-
lance, emergency vaccination, targeted culling (in cases of highly
infectious animal diseases), and other relevant specific measures
applied to affected premises.
6 Implementation of Viral Disease Control Program at Various Levels
Level Control measures

On individual level l Periodic screening of animals
l Segregation and/or culling (in case of highly
infectious diseases) infected animals
l Separation of offspring from the infected dam
On herd level l Implementation of strict biosecurity measures

l Regular screening of the herd
In case of high seroprevalence of infectious diseases,
slaughter of entire herd is feasible option
On regional and l Establishment of regional/national reference
national levels laboratories for diagnosis of important exotic as
well as diseases of economic importance
(continued)
Level Control measures

l Proper implementation of surveillance program and
epidemiological investigation of outbreaks (tracing
of contact flocks)
l Proper reporting of cases at regional and national
level due to its notifiable status
7 Prevention and Control Measures for the Viral Disease Outbreaks in Swine
Population
Occurrence of diseases leads to a huge economic loss in terms of

livestock health and production. As the time passes the advance-
ment that occur in the animal health are expected to play a major
role in the progress of livestock industry. A lot of control on animal
diseases assumes a prime importance in the crucial time when the
animal agriculture is shifting from extensive to intensive and com-
mercial system of management. Presence and accumulation of
infectious agents/pathogens in the environment leads to reduction
in quality and quantity of products produced by animals. Strategic
control and eradication of diseases will result enhancing pig pro-
duction worldwide. The various prevention and control measures
for viral disease outbreak are mentioned in the following.
1. Biosecurity management: The biosecurity management must
not be compromised at any cost in the farm. Good herd bio-
security is essential to maintain herd health status and also for
undertaking control or eradication program of diseases. Biose-
curity is a cornerstone of herd health maintenance.
2. Prevention measures against the virus introduction into farms
through commercial pig feeds or feed ingredients. It has been
shown previously that multiple swine viruses could survive in
certain feed ingredients for a very long time [47, 48]. For
example, one study has shown that the half-life of ASFV in
various feed or feed ingredients ranged from 9.6 days (conven-
tional soybean meal) to 14.2 days (complete feed) [48]. There
are some strategies that might be used for reducing the infec-
tivity of viruses contaminating the feed, such as heat treatment,
e.g., pelleting the feed using higher temperature; chemical
mitigation, e.g., treating the feed with a formaldehyde and
propionic acid solution; and storage period management,
e.g., using the knowledge on the half-life of the virus to adjust
the feed storage time and conditions [7].
3. Decontamination of infected farms with effective disinfectant,
i.e., 2% caustic soda should be made available (FAO animal
production and health manual).
4. Vehicles used during farm visit should be properly disinfected

after use (FAO animal production and health manual).
5. Exit of live pigs should be banned (FAO animal production and
health manual).
6. Perception of risk management of farmers and other stake-
holders could be fundamental to the disease controls.
Continuing education of farmers and other stakeholders in
the supply chains is required to prevent the emergence and
reemergence of infectious disease outbreaks in swine. Collabo-
ration: It takes shared knowledge and communication to pre-
pare for and manage a disease outbreak. The disease eradication
and prevention on the farm might not be successful if the
diseases are still active on neighboring farms. This might also
be applied to the international level. Collaboration among
neighboring countries should be strengthened. Transboundary
disease transmission through smuggled pigs and pork products
has been suspected in many diseases across the globe [31, 32].
7. Livestock traders: They are the important group for public
awareness, as the movement of animals through livestock tra-
ders is often the key epidemiological factor in the spread of
diseases. Therefore, for the prevention of disease there is the
need to build up a climate of trust and confidence between
animal health officials and livestock traders (FAO animal pro-
duction and health manual).
8. Improved control and tracing of consignments of animals and
animal products by development of an integrated database,
introduction of a central alert system, and risk assessment
systems.
9. Isolate the infected animals and immediately slaughter those
who are showing the severe symptoms of disease (FAO animal
production and health manual).
10. Safe disposal of the carcasses of animals that have either slaugh-
tered or naturally died should be done as soon as possible, so
that no longer constitute a risk of further spread of pathogen to
other animals (FAO animal production and health manual).
11. Public awareness campaigns through newspapers, radio, and
television were also useful for prevention and control of disease
(FAO animal production and health manual).
8 Conclusion
The unprecedented increase in the frequency of infectious disease

outbreaks in swine industry requires meticulous planning and
decision-making to curtail the outbreak progression. Biosecurity
at farm level aims to prevent the introduction and spread of
pathogenic organisms, thus ensures better farm health manage-

ment. Vaccination against important infectious diseases also aims
to keep the disease spread at check. An outbreak is designated when
disease rates are higher than normal. The outbreak of viral diseases
in swine industry cause significant economic loss and the outbreak
investigation is one of the critical components of infectious disease
epidemiology and efficiently performed outbreak investigation
helps the identification and removal of a potential cause of the
outbreak and provides post-exposure prophylaxis to affected indi-
viduals. An outbreak investigation requires knowledge of descrip-
tive and analytical epidemiological methods and it includes several
steps. The most critical part in the investigation is to clearly define
the problem, i.e. case definition. Outbreak investigations tries to
find the answer for five Ws such as what (disease event), who
(animal affected), where (place), when (time), and why/how
(causes, risk factors, and modes of transmission). Furthermore,
outbreak investigations frequently result in identification of new
infections and diseases. Effective outbreak investigations play a vital
role in widening the knowledge about infectious diseases, and also
assist in the development of regulations and prevention guidelines.
Since outbreak is different, there is no readymade recipe for success.
By adopting the basic outbreak techniques, the investigator may
efficiently contain the disease progression. In few situations, the
need may arise for intricate analysis demanding the specialists such
as epidemiologists, medicos, laboratory diagnosticians, and others.
The successful outbreak investigation should end in communica-
tion of the outbreak results to public platform or needy people.
References
1. Statista (2020) Number of pigs worldwide in 7. Kedkovid R, Sirisereewan C, Thanawongnu-
2020, by leading country (in million head). wech R (2020) Major swine viral diseases: an
https://www.statista.com/statistics/263964/ Asian perspective after the African swine fever
number-of-pigs-in-selected-countries introduction. Porcine Health Manag 6(1):
2. Bhadauria P, Sharma A, Verma HK, Singh I, 1–11
Singh R (2019) Pig farming: promising agri- 8. King AM, Lefkowitz EJ, Mushegian AR,
business in Punjab. ICAR-Agricultural Tech- Adams MJ, Dutilh BE, Gorbalenya AE,
nology Application Research Institute, Harrach B, Harrison RL, Junglen S, Knowles
Ludhiana NJ, Kropinski AM (2018) Changes to taxon-
3. Mpofu I, Makuza SMM (2003) In: Shonhiwa omy and the international code of virus classifi-
A (ed) Pig production science and technology, cation and nomenclature ratified by the
1st edn. Upfront Publishing, Leicestershire international committee on taxonomy of
4. VanderWaal K, Deen J (2018) Global trends in viruses (2018). Arch Virol 163(9):2601–2631
infectious diseases of swine. Proc Natl Acad Sci 9. Zhou B (2019) Classical swine fever in China-
115(45):11495–11500 an update minireview. Front Vet Sci 6:187.
5. Razia SJ, Vidya B, Sushma K, Raghunandan T https://doi.org/10.3389/fvets.2019.00187
(2017) Pig mortality and its management. 10. Donaldson AI (1997) Foot-and-mouth disease
Theriogenol Insight 7(3):139–144 in Taiwan. Vet Rec 140(15):407
6. Iowa State University. Exotic viral diseases. 11. Dunn CS, Donaldson AI (1997) Natural adap-
https://vetmed.iastate.edu/vdpam/FSVD/ tion to pigs of a Taiwanese isolate of foot-and-
swine/index-diseases/exotic-viral-diseases mouth disease virus. Vet Rec 141(7):174–175
12. DeLay J, McEwen B, Carman S et al (2005) encephalitis in the United States. J Vet Diagn
Porcine circovirus type 2-associated disease is Investig 13(5):428–433. https://doi.org/10.
increasing. AHL Newslett 9(3):22 1177/104063870101300513
13. Cheung AK, Lager KM, Kohutyuk OI, Vincent 23. Johansen CA, Van den Hurk AF, Pyke AT,
AL, Henry SC, Baker RB, Rowland RR, Dun- Zborowski P, Phillips DA, Mackenzie JS,
ham AG (2007) Detection of two porcine cir- Ritchie SA (2001) Entomological investiga-
covirus type 2 genotypic groups in United tions of an outbreak of Japanese encephalitis
States swine herds. Arch Virol 152(5): virus in the Torres Strait, Australia, in 1998. J
1035–1044 Med Entomol 38(4):581–588
14. Ni J, Yang S, Bounlom D, Yu X, Zhou Z, 24. Liu W, Fu S, Ma X, Chen X, Wu D, Zhou L,
Song J, Khamphouth V, Vatthana T, Tian K Yin Q, Li F, He Y, Lei W, Li Y (2020) An
(2012) Emergence and pathogenicity of highly outbreak of Japanese encephalitis caused by
pathogenic Porcine reproductive and respira- genotype Ib Japanese encephalitis virus in
tory syndrome virus in Vientiane, Lao People’s China, 2018: a laboratory and field investiga-
Democratic Republic. J Vet Diagn Investig tion. PLoS Negl Trop Dis 14(5):e0008312.
24(2):349–354 https://doi.org/10.1371/journal.pntd.
15. Nilubol D, Tripipat T, Hoonsuwan T, 0008312
Kortheerakul K (2012) Porcine reproductive 25. Wu Q, Zhao X, Bai Y, Sun B, Xie Q, Ma J
and respiratory syndrome virus, Thailand, (2017) The first identification and complete
2010–2011. Emerg Infect Dis 18(12): genome of Senecavirus A affecting pig with
2039–2043 idiopathic vesicular disease in China. Trans-
16. Tornimbene B, Frossard JP, Chhim V, Sorn S, bound Emerg Dis 64(5):1633–1640
Guitian J, Drew TW (2015) Emergence of 26. Looi LM, Chua KB (2007) Lessons from the
highly pathogenic porcine reproductive and Nipah virus outbreak in Malaysia. Malays J
respiratory syndrome (HP-PRRS) in medium- Pathol 29(2):63–67
scale swine farms in southeastern Cambodia. 27. Acland HM, Littlejohns IR (1975) Encephalo-
Prev Vet Med 118(1):93–103 myocarditis virus infection of pigs. 1. An out-
17. Puranaveja S, Poolperm P, break in New South Wales. Aust Vet J 51(9):
Lertwatcharasarakul P, Kesdaengsakonwut S, 409–415. https://doi.org/10.1111/j.
Boonsoongnern A, Urairong K, Kitikoon P, 1751-0813.1975.tb15789.x
Choojai P, Kedkovid R, Teankum K, Thana- 28. Maurice H, Nielen M, Vyt P, Frankena K, Koe-
wongnuwech R (2009) Chinese-like strain of nen F (2007) Factors related to the incidence
porcine epidemic diarrhea virus, Thailand. of clinical encephalomyocarditis virus (EMCV)
Emerg Infect Dis 15(7):1112–1115 infection on Belgian pig farms. Prev Vet Med
18. Duy DT, Toan NT, Puranaveja S, Thanawong- 78(1):24–34. https://doi.org/10.1016/j.pre
nuwech R (2011) Genetic characterization of vetmed.2006.09.002
porcine epidemic diarrhea virus (PEDV) iso- 29. Vansteenkiste K, Van Limbergen T,
lates from southern Vietnam during 2009- Decaluwé R, Tignon M, Cay B, Maes D
2010 outbreaks. Thai J Vet Med 41(1):55–64 (2016) Clinical problems due to encephalo-
19. Wang D, Fang L, Xiao S (2016) Porcine epi- myocarditis virus infections in two pig herds.
demic diarrhea in China. Virus Res 226:7–13 Porcine Health Manag 2(1):1–8. https://doi.
20. Bora M, Bora DP, Manu M, Barman NN, org/10.1186/s40813-016-0036-z
Dutta LJ, Kumar PP, Poovathikkal S, Suresh 30. Paton DJ, Simpson V, Done SH (1992) Infec-
KP, Nimmanapalli R (2020) Assessment of risk tion of pigs and cattle with bovine viral diar-
factors of African swine fever in India: perspec- rhoea virus on a farm in England. Vet Rec 131:
tives on future outbreaks and control strate- 185–188
gies. Pathogens 9(12):1044. https://doi.org/ 31. Chenais E, Depner K, Guberti V, Dietze K,
10.3390/pathogens9121044 Viltrop A, Ståhl K (2019a) Epidemiological
21. Ciarello FP, Capucchio MT, Ippolito D, considerations on African swine fever in Eur-
Colombino E, Gibelli LRM, Fiasconaro M, ope 2014–2018. Porcine Health Manag 5(1):
Moreno Martin AM, Di Marco Lo Presti V 1–10. https://doi.org/10.1186/s40813-018-
(2020) First report of a severe outbreak of 0109-2
Aujeszky’s disease in cattle in Sicily (Italy). 32. Chenais E, Lewerin SS, Boqvist S, Ståhl K,
Pathogens 9(11):954 Alike S, Nokorach B, Emanuelson U (2019b)
22. Janke BH, Paul PS, Landgraf JG, Halbur PG, Smallholders’ perceptions on biosecurity and
Huinker CD (2001) Paramyxovirus infection disease control in relation to African swine
in pigs with interstitial pneumonia and
fever in an endemically infected area in North- surveillance and control strategies. Sci Rep
ern Uganda. BMC Vet Res 15(1):1–13 6(1):1–11
33. Valeeva NI, Van Asseldonk MAPM, Backus 41. Office International des Épizooties (OIE)
GBC (2011) Perceived risk and strategy effi- (2018) Terrestrial Animal Health Code
cacy as motivators of risk management strategy (2018). http://www.oie.int/standard-
adoption to prevent animal diseases in pig setting/terrestrial-code/access-online/
farming. Prev Vet Med 102(4):284–295 42. Rodrı́guez-Prieto V, Vicente-Rubiano M, Sán-
34. Food and Agriculture Organization of the chez-Matamoros A, Rubio-Guerri C,
United Nations/World Organisation for Ani- Melero M, Martı́nez-López B, Martı́nez-
mal Health/World Bank (2010) Good prac- Avilés M, Hoinville L, Vergne T, Comin A,
tices for biosecurity in the pig sector – issues Schauer B (2015) Systematic review of surveil-
and options in developing and transition lance systems and methods for early detection
countries. In: FAO animal production and of exotic, new and re-emerging diseases in ani-
health paper no. 169. FAO, Rome mal populations. Epidemiol Infect 143(10):
35. Beltran-Alcrudo D, Falco JR, Raizman E, 2018–2042
Dietze K (2019) Transboundary spread of pig 43. OIE South-East Asia and China Foot-and-
diseases: the role of international trade and Mouth Disease (SEACFMD) Campaign
travel. BMC Vet Res 15(1):1–14. https://doi. (2018) A field manual for animal disease out-
org/10.1186/s12917-019-1800-5 break investigation and management. OIE
36. Meng XJ (2012) Emerging and re-emerging Sub-Regional Representation for South-East
swine viruses. Transbound Emerg Dis 59: Asia, Department of Livestock Development,
85–102 69/1 Phaya Thai Road, Ratchathewi, Bangkok
37. Li Y, Ji G, Wang J, Tan F, Zhuang J, Li X, Tian 10400, Thailand
K (2016) Complete genome sequence of an 44. World Health Organization (2018) Managing
NADC30-like porcine reproductive and respi- epidemics: key facts about major deadly dis-
ratory syndrome virus characterized by recom- eases. World Health Organization
bination with other strains. Genome Announc 45. Centers for Disease Control and Prevention
4(3):e00330–e00316. https://doi.org/10. (2016) Investigating an outbreak. https://
1128/genomeA.00330-16 www.cdc.gov/csels/dsepd/ss1978/lesson6/
38. Miller RS, Sweeney SJ, Slootmaker C, Grear section2.html
DA, Di Salvo PA, Kiser D, Shwiff SA (2017) 46. World Organisation for Animal Health (OIE)
Cross-species transmission potential between 2014 Guidelines for animal disease control –
wild pigs, livestock, poultry, wildlife, and OIE. https://www.oie.int/fileadmin/Home/
humans: implications for disease risk manage- eng/Our_scientific_expertise/docs/pdf/A_
ment in North America. Sci Rep 7(1):1–14. Guidelines_for_Animal_Disease_Control_
https://doi.org/10.1038/s41598-017- final.pdf)
07336-z 47. Dee S, Neill C, Singrey A, Clement T,
39. Balkhy HH, Abolfotouh MA, Al-Hathlool RH, Cochrane R, Jones C, Patterson G, Spronk G,
Al-Jumah MA (2010) Awareness, attitudes, Christopher-Hennings J, Nelson E (2016)
and practices related to the swine influenza Modeling the transboundary risk of feed ingre-
pandemic among the Saudi public. BMC Infect dients contaminated with porcine epidemic
Dis 10(1):1–7. https://doi.org/10.1186/ diarrhea virus. BMC Vet Res 12(1):51
1471-2334-10-42 48. Stoian AM, Zimmerman J, Ji J, Hefley TJ,
40. Guinat C, Relun A, Wall B, Morris A, Dixon L, Dee S, Diel DG, Rowland RR, Niederwerder
Pfeiffer DU (2016) Exploring pig trade pat- MC (2019) Half-life of African swine fever
terns to inform the design of risk-based disease virus in shipped feed. Emerg Infect Dis
25(12):2261
Chapter 2
Collection of Samples, Their Preservation

and Transportation
Ashok Kumar, Kaushal Kishor Rajak, Ajay Kumar Yadav, Vishal Rai,
Mukesh Bhatt, R. P. Singh, and R. K. Singh
Abstract
Pigs are an important livestock species raised for meat, and their products play a significant role in the
livelihood of people in the country’s north-eastern states. Detection of diseased porcine in the field is critical
for disease treatment and control. Pigs, such as other livestock, were subjected to a slew of contagious
existing, emerging, and re-emerging viral diseases, necessitating the use of a diagnostic laboratory and
research organization globally. The diagnosis of viral diseases is fundamentally dependent on time and
precise management. Collecting whole blood and tissues from multiple febrile or recently deceased animals
is the preferred method for detecting herds early in infection. Particular tissues should be collected as
aseptically as possible. Preferably, two or three humanely euthanized pigs in the early stages of disease
displaying typical clinical signs and necropsied immediately will yield the most reliable diagnostic data. A
detailed history of the disease outbreak, as well as a preliminary diagnosis based on clinical evaluation and
necropsy findings, should be included. Animal selection, sample selection, sample handling, sample proces-
sing, necropsy technique, specimen collection media, and adequate storage all have a direct impact on the
accuracy and effectiveness of laboratory results in assisting in the resolution of health problems. Existing
and emerging swine pathogens, particularly those of a transboundary nature, must be closely monitored,
and appropriate health interventions must be developed on a priority basis. With limited vaccine availability,
the emergence of new diseases such as African swine fever (ASF) and porcine reproductive and respiratory
syndrome (PRRS) are threats to pig production in India.
Key words Contagious, Emerging, Specimen, Pathogens, Necropsy, Equipment, Viral transport
media (VTM), Evaluation, Backyard and Microscopy
1 Background
Pig is even-toed ungulate animal hail from genus Sus within family
Suidae of the modern classification of the kingdom. Among the
livestock species, the pig is one of the imperative species for meat
production, which engages in recreation a pivotal role to provide
livelihood support to the landless farmers and pitiable sections of
society, especially in the North-Eastern region of the country.
21
22 Ashok Kumar et al.
Porcine species is closely related to humans in the mean of an

anatomical, physiological, and biological system that why inten-
sively used in the research system. According to Michael Swindle,
author of “Swine in the Laboratory”. If works in the pig, then it has
a high possibility of working in the human. The pig industry is
growing day by day, due to increased demand for pork and its
products, due to the urbanization of society. The pig is considered
as the only species bearing the highest litter size among meat-
producing animals and the most efficient feed converting species
with the shortest generation interval and the fastest growth rate.
Pigs are mostly maintained in a poor sanitary condition and mostly
by backyard and internal sector producers which lead to economic
losses due to various viral diseases. Molecular assays for detection of
nucleic acids in biologic specimens are valuable diagnostic tools
supporting clinical diagnoses and therapeutic decisions.
2 Introduction
Pig is an important livestock species, which plays a crucial role in the

livelihood in north-eastern states of the country. Pig is the most
efficient feed converting animal species. Among meat-producing
animals it is the only litter bearing animal having shortest genera-
tion interval and fastest growth rate. Pigs are mostly maintained in
poor sanitary condition and mostly by backyard and internal sector
producers. Pigs are susceptible to many viral diseases such as Clas-
sical Swine Fever (CSF), Foot and Mouth Disease (FMD), Swine-
pox, Transmissible Gastroenteritis, Calicivirus, Porcine circovirus
2, Influenza, Pseudorabies, and Adenoviruses although some
emerging porcine diseases such as Porcine reproductive and respi-
ratory syndrome virus (PRRSV), African swine fever, and Porcine
respiratory coronavirus (PRCoV).
Pathogens causing significant respiratory, GIT, and skin disease
in growing pigs and development of viraemia and the body distri-
bution of susceptible macrophages lead to the shedding of patho-
gen [1]. The veterinary officer of various states has primary
responsibility for provide tentative diagnoses of disease and final
decision concerning management of swine health problems for
practice [2]. The veterinary diagnostic laboratory and research
institute can be an important supporting arms of the veterinary
practice by different ways such as (1) providing a consultation
service, (2) providing technical assistance in performing laboratory
testing, and (3) collecting and disseminating information on current
research.
Modern diagnostic laboratories have facilities and personnel
highly trained in various areas of laboratory such as virology, clinical
pathology, immunology, biochemistry, theriogenology, biotech-
nology, and microbiology. Highly sophisticated equipments for
Collection of Samples, Their Preservation and Transportation 23
laboratory such as real time polymerase chain reaction (PCR),

scintillation counters, automated cell counters, autoanalyzers,
atomic absorption spectrophotometers, Mass spectrometers, high
pressure liquid and gas chromatographic equipment, next genera-
tion sequencing, elaborate equipment for culture of virus and
electron microscopes are routinely used in many diagnostic labora-
tories today. The basic approaches for laboratory viral diagnosis are
the isolation of the virus, demonstration of the virus or some viral
product in clinical specimens (direct methods), and detection and
measurement of viral-specific antibodies (indirect methods). Each
approach has its merits, but direct demonstration of the virus
and/or viral products is the most effective and useful approach
for routine diagnosis [3].
Each organization is geared toward the problems that occur in
the areas in which they serve. Depends upon severity of cases may
referred to specialised laboratories for more details analyses if
required. The technical capabilities of the organization can be
used for monitoring herd health programs as well as for facilitating
diagnosis in disease outbreaks. Accuracy of laboratory results and
their effectiveness in helping to resolve the health problems are
highly dependent on clinical evaluations of the cases by the profes-
sional practitioner and also dependent on selection, collection of
specimen, processing, packaging, preservation, and transport of
specimens to be submitted for the analysis.
General guidelines for the collection and submission of speci-
mens are presented in Table 1. Most laboratories supply a specimen
submission form that should be completed with the available perti-
nent information. In the absence of a form, the veterinarian should
supply as complete a history as possible. Veterinarians should con-
tact the diagnostic laboratory if they have any questions.
Complete history is essential for performing the appropriate
tests and for proper interpretation of laboratory results. Organiza-
tion should communicate with trained laboratory person in such a
manner that all requests are fulfilled and steps are taken to obtain
the information recorded.
3 Suitable Criteria for Collection and Transport and Preservation of Samples
The following criteria are suitable for proper selection of specimen

based on viral infection like type of animals, types of specimens,
type of organisms as listed in Table 1. Whenever possible, animals
should be submitted directly to the diagnostic laboratory for com-
plete necropsy examination. If a herd problem exists, more than
one animal should be submitted. Bus and courier service may be
used to ship small animals, provided they are packaged in leak-proof
insulated containers with sufficient ice or cold packs. Do not freeze
animals submitted for necropsy. For tissue specimen to minimize
Table 1
Selection site of specimen for laboratory inspection and diagnosis
S.
No. System Selection site of cases
1 External examination Appearance of hair coat, skin lesion location and description,
conjunctiva, eyes, ears, feet, hooves, nostrils, mouths, anus, and
vulva
2 Respiratory system Nasal cavity, larynx, bronchi, lungs, and pleura
3 Circulatory system Heart, pericardium, and blood vessels
4 Digestive system Oral cavity, teeth, oesophagus, stomach, duodenum, jejunum, ileum,
colon, liver, gall bladder, and pancreas
5 Urinary system Kidney, ureters, and urinary bladder
6 Genital system Ovaries, uterus, vagina, testicles, spermatic cord, and male accessory
sex glands
7 Endocrine system Thyroids, parathyroid, adrenals, and pituitary
8 Lymphoreticular and Lymph node, spleen, thymus tonsil, and bone marrow
hematopoietic system
9 Musculoskeletal system Muscles, bones, cartilages, and tendons
10 Nerves system Brain, spinal cord, peripheral nerves, and meninges
contamination during necropsy, it is best to collect a routine set of

tissues prior to thorough examination. Recommended tissues are
lung, kidney, liver, spleen, small intestine, large intestine, and
mesenteric lymph nodes. Brain tissue or head should also be col-
lected if central nervous system disease is suspected [3]. Other
tissues containing abnormalities noted during the thorough exam-
ination should also be collected. A portion of these tissues should
be placed in leak-proof plastic bags and placed under refrigeration.
While it is recommended that each tissue be placed in a separate
bag, it is absolutely essential that intestine be separated from other
tissues; otherwise, biological examinations will be compromised.
Tissues should be brought directly to the laboratory or shipped
under refrigeration by over-night mail, bus, or courier service.
Tissues collected during the latter part of the week should be frozen
and shipped on Monday [4].
Since many viruses produce characteristic microscopic lesions,
small pieces (1/4 inch thick) of each tissue should be placed in 10%
buffered formalin for histopathologic examination. An entire lon-
gitudinal half of the brain should be submitted. These samples
should not be frozen. The feces type specimen feces should be
collected from acutely ill animals and placed in leak-proof
containers [5]. While well-saturated swabs are adequate for many
individual virologic examinations, several milliliters or grams of
feces permit a more complete diagnostic work-up including
bacteriologic and parasitologic examinations. Samples should be

submitted to the laboratory using cold packs as coolant. According
to WHO the swabs specimens nasal and ocular swabs are useful for
isolating viruses from animals with upper respiratory-tract infec-
tions [6]. Genital infections may also be diagnosed by examining
swabs collected from the reproductive tract (vagina and penis
mucosa). These swabs should be collected from acutely ill animals
and placed directly into screw-capped tubes containing a viral
transport medium. The sampling of several animals in different
stages of the illness increases the likelihood of isolating the causative
agent. Swabs are also useful for the sampling of vesicular lesions.
Fresh vesicles should be ruptured and the swab saturated with the
exuding fluid. Two swabs should be collected, one for virus isola-
tion and one for electron microscopy. Major viral diseases of por-
cine for specimen collection and transport media to laboratory
diagnosis are listed in Table 2.
The swab for virus isolation should be placed in viral transport
medium and the swab for electron microscopy should be placed in a
screw-capped tube containing one or two drops of distilled water.
Scab material from the more advanced lesions should also be sub-
mitted. There are several commercially available viral transport
media that help maintain the viability of viruses during shipment
to the laboratory. Most of these transport media are balanced salt
solutions containing high protein content and antibiotics to pre-
vent bacterial overgrowth. Many diagnostic laboratories provide
their own version of transport medium to practicing veterinarians
upon request. For direct examination of specimen slide preparation
needed at fresh specimens a number of infectious diseases can be
diagnosed by examining slides prepared from blood and tissues.
Conjunctival scrapings are particularly useful for diagnosing her-
pesvirus infections in pigs [7]. Imprints made from liver, spleen,
and lungs are especially useful for diagnosing flavivirus and herpes-
virus infections of swine [8, 9]. Slides should have sufficient cells to
allow thorough examination but should not be so thick as to cause
difficulty in staining. A conjunctival scraper or some other device
(blunt end of scalpel blade) should be used to scrape the conjunc-
tiva; cotton swabs are not adequate. Matted eyes should be cleaned
and flushed prior to scraping the conjunctiva. Tissue imprints
should be made by lightly touching the microscope slide with
fresh cuts of tissue previously blotted with a paper towel to absorb
some of the blood [10]. Slides should be air-dried and sent to the
laboratory in slide holders to prevent breakage. Several slides per-
mit a more thorough diagnostic work-up, including cytologic
examinations. For the serological examination blood samples
should be collected in sterile tubes containing no anticoagulants.
These should be submitted to the laboratory in specially designed
Styrofoam holders to avoid breakage [11]. Blood samples should
not be frozen or allowed to overheat. If samples cannot be delivered
Table 2
Major viral diseases of porcine for specimen collection and transport media to laboratory diagnosis
Specimens for
Isolation and of
Primary System Screening of Transport
Viral disease involved to disease specimen conditions Transport media
Influenza Respiratory, digestive Nasal swab, lung 4 C/ Transport medium
tissue (PM) Ambient 199, PBS-glycerol
Nasal swab, lung and temp/a transport
tracheal swab medium.
(PM), serum, Culturette
lymph node Leibovitz CVTM
Veal or tryptose
broth
Transmissible Digestive, respiratory Jejunum, blood, Ambient PBS-glycerol
gastroenteritis serum and temp/a transport
(TGE) intestinal contents medium,
(small and large), Leibovitz CVTM,
feces, lungs, lymph RPMI, and
node, serum EMEM
Rotavirus Digestive Intestinal contents, Ambient PBS-glycerol and
(Reoviruses) feces, serum temp/a EMEM
Coronavirus Digestive Intestinal contents, Ambient PBS-glycerol
feces, tonsil, lungs, temp/a transport
stomach, small medium, RPMI,
intestine, brain, and EMEM
spinal cord, serum
Calicivirus Digestive Intestinal contents, Ambient PBS-glycerol and
feces, serum temp/a EMEM
Adenovirus Digestive, respiratory Intestinal contents, Ambient PBS-glycerol and
feces, lungs, lymph temp/a EMEM
node, serum
Astroviruses Digestive Intestinal contents, Ambient PBS-glycerol and
feces, serum temp/a EMEM
Pseudorabies Respiratory system Lungs, lymph node, Ambient PBS-glycerol and
and nerves system tonsils, serum, temp/a EMEM
brain
Vesicular stomatits Digestive and Vesicular fluid, saliva, Ambient PBS-glycerol and
musculoskeletal and affected temp/a EMEM
mucous
membranes
collected early in
the disease
Parvovirus Digestive, Mummified or Ambient PBS-glycerol
reproductive, and aborted fetuses, temp/a transport
musculoskeletal placenta, fluids, and medium, RPMI,
skin lesions and EMEM
(continued)
Table 2
(continued)
Specimens for
Isolation and of
Primary System Screening of Transport
Viral disease involved to disease specimen conditions Transport media
Picornavirus Respiratory, digestive, Vesicular fluid, Ambient PBS-glycerol
(SMEDI, FMD, reproductive, and affected skin and temp/a transport
enteroviruses) musculoskeletal mucous medium, RPMI,
membranes, blood and EMEM
with anticoagulant,
and serum
Porcine Reproductive and Bronchoalveolar Ambient PBS-glycerol
reproductive and respiratory lavage (BAL), temp/a transport
respiratory serum, lung, lymph medium, RPMI,
syndrome nodes, tonsil, and and EMEM
spleen
Aborted and
mummified fetus
Swinepox Musculoskeletal and Vesicular fluid, scabs, Ambient PBS-glycerol and
skin and scrapings from temp/a EMEM
lesions
Hog cholera, japans Digestive, Kidney, spleen, tonsil, Ambient PBS-glycerol
encephalitis reproductive, lymph nodes, brain, temp/a transport
(Flavivirus) nervous, and whole blood, and medium, RPMI,
urogenital serum and EMEM
African swine fever Respiratory and Blood, spleen, tonsil, Ambient PBS-glycerol
lymphoreticular and lymph nodes temp/a transport
medium, RPMI,
and EMEM
a
As per specimen required temperature
to the laboratory within a reasonable time, serum should be

removed and refrigerated or frozen.
4 Collection and Transport and Preservation of Samples
Sample selected for laboratory analyses depend, of course, on the

nature of the problem. A live untreated pig with representative
clinical signs is usually the sample of choice for laboratory analysis.
Specimen must be taken as early as possible in the acute phase of
sickness [12]. To check the bacterial contamination all the speci-
men including blood should be collected aseptically. Swab and
other specimen should be placed in virus transport medium
Table 3
Equipment and supplies for use in performing necropsies and collecting laboratory samples
Equipment Supplies
Knife Swabs
Steel Blood vials
Stone Plastic bags
Foreceps Sterile syringe
Scissors Sterile needles
Saw Wide mouth container with 10% formalin
Cleaver pH paper
Gloves Shipping container
Coveralls Refrigerant
Boots Marking pen or pencil notepad
Pail brush Disinfectant
(VTM). VTM should be prepared sterile and distributed in 2 ml

quantities in suitable sterile glass/plastic container [13]. Therefore
it may be more expedient for the practitioner to collect samples
during a field necropsy or from live animals and send them to the
laboratory via an acceptable mode of transportation. Certain equip-
ment and supplies are designed for the use in performing necropsies
and collecting laboratory samples (Table 3) [14].
Transmission from infected to susceptible animals via aerosols
has been demonstrated under experimental conditions for porcine
reproductive and respiratory syndrome virus (PRRSV) influenza
virus porcine respiratory coronavirus (PRCoV) [4, 9]. Viral trans-
port media for use in collecting nasal swab and throat swab are as
follows (1) Add 10 g veal infusion broth and 2 g bovine albumin
fraction to sterile distilled water (to 400 ml) (2) Add 0.8 ml Gen-
tamycin sulfate solution (50/ml) and 3.2 ml amphotericin B
(250 μg/ml) sterilized by 0.22 μm filtration. Another viral trans-
port media PBS-Glycerol transport medium (1) Phosphate-
buffered saline (PBS):—NaCl 8 g—KCl 0.2 g—Na2HPO4
1.44 g—KH2PO4 0.24 g—Distilled water to make 1 l (2) Autoclave
PBS and mix 1:1 with sterile glycerol to make 1 l (3) To 1 l
PBS/glycerol add:—benzylpenicillin (2 106 IU/l)—streptomy-
cin (200 mg/liter)—polymyxin B (2 106 IU/l)—gentamicin
(250 mg/liter)—nystatin (0.5 106 IU/l)—ofloxacin hydrochlo-
ride (60 mg/l), and—sulfamethoxazole (0.2 g/l) Dispense
1.0–2.0 ml of transport medium into sterile plastic screw-cap vials
(Cryovials). It is best to store these vials at 20 C until used.
However, they can be stored at +4 C for 48–96 h (optimally less
than 48 h) or at room temperature for short periods of 1–2 days.
After collecting specimens in VTM, it is to be packed properly

before sending to a suitable laboratory. Packaging has three main
aims to maintain the specimen viability, to prevent it leaking outside
the package and to prevent cross contamination [11]. Biological
materials survive better at low temp. So it is adviced to maintain the
low temperature during transport. Low temperature may be
achieved by using solid CO2, wet ice or frozen pads. Specimens in
VTM can be sent to a nearby laboratory for diagnosis by keeping it
on ice through messenger, mail, freight, and courier.
5 Conclusion
The disease can be diagnosed in laboratory by histopathology,

electron microscopy (EM), detection of viral antigens by immuno-
fluorescence and polymerase chain reaction (PCR) with suitable
and sufficient specimens. Sampling carefully to include acutely
affected pigs will be the greatest help to the producer, the patholo-
gist, and the veterinarian seeking treatment or prevention strate-
gies. As a result of several intense research efforts the disease
etiology, its pathogenesis, pathology, immunology were well stud-
ied. With the use of this knowledge a wider development in exten-
sion activities, anti-viral therapeutics and vaccine strategy for both
humans and susceptible animals is needed to eradicate viral disease
from our globe.
References
1. Murphy F, Fauquet C, Bishop D, Gharial S, 7. The Pig Site (2008) Handling and restraining
Jarvis A, Martelli G, Mayo M, Summers M pigs. The pig site, Sheffield, England. http://
(1995) Virus taxonomy: classification and w w w. t h e p i g s i t e . c o m / a r t i c l e s / 2 3 9 2 /
nomenclature of viruses. Sixth report of the handling-and-restraining-pigs
international committee on taxonomy of 8. Gloster J, Donaldson AI, Hough MN (1984)
viruses. Springer-Verlag, New York, pp 79–91 Analysis of a series of outbreaks of Aujeszky’s
2. Grandien M (1988) Paramyxoviridae: the para- disease in Yorkshire in 1981–1982: the possi-
influenza viruses. In: Lennette EH, Halonen P, bility of airborne disease spread. Vet Rec 114:
Murphy FA (eds) Laboratory diagnosis of 234–239
infectious diseases, principles and practice, vol 9. Juozaitis A, Willeke K, Grinshpun SA, Don-
2. Springer-Verlag, New York, pp 490–491 nelly J (1994) Impactiononto a glass slide or
3. Johnson FB (1990) Transport of viral speci- agar versus impingement into a liquid forthe
mens. Clin Microbiol Rev 3(2):120–131 collection and recovery of airborne microor-
4. Cavanagh D (1997) Nidovirales: a new order ganisms. Appl Environ Microbiol 60:861–870
comprising Coronaviridae and Arteriviridae. 10. Lin X, Willeke K, Ulevicius V, Grinshpun SA
Arch Virol 142:629–633 (1997) Effect of sampling time on the collec-
5. World Organisation for Animal Health (2019) tion efficiency of all-glass impingers. Am Ind
Terrestrial animal health code. OIE, Paris Hyg Assoc J 58:480–488
6. Kwon HM (1996) Analysis of the spike glyco- 11. Pirbright Institute (2015) Requirements for
protein gene and nonstructural protein gene of packaging and dispatch of biological materials
transmissible gastroenteritis virus using PCR to the Pirbright Institute, Reference Labora-
and RFLP analysis. Kor J Vet Res 36 tories, Pirbright, U.K. http://www.pirbright.
(3):627–633 ac.uk/ref_Labs/Default.aspx
12. Meinkoth JH, Cowell RL (2002) Sample col- respiratory syndrome virus and swine influenza
lection and preparation in cytology: increasing virus. Appl Environ Microbiol 72:4811–4818
diagnostic yield. Vet Clin North Am Small 14. Madeley CF, Lennette DA, Halonen P (1988)
Anim Pract 32(6):1187–1207 Specime collection and transport. In: Lennette
13. Hermann JR, Hoff SJ, Yoon KJ, Burkhardt EH, Halonen P, Murphy FA (eds) Laboratory
AC, Evans RB, Zimmerman JJ (2006) Optimi- diagnosis of infectious diseases, principles and
zation of a sampling system for recovery and practice, vol 2. Springer- Verlag, New York, pp
detection of airborne porcine reproductive and 7–11
Chapter 3
Methods for Quantification of Viruses

Mukesh Bhatt, Chris Einstein, Kiran, Arfa Fayaz, Vishal Rai, Monu Karki,
Ashok Kumar, Ajay Kumar Yadav, and Kaushal Kishor Rajak
Abstract
Virus quantification is widely practised in both commercial and academic laboratories involved in research
or production of viral vaccines, recombinant proteins, viral antigens, or antiviral agents. For this, the cell
culture-based endpoint dilution assays are the most widely used methods. However, these infectivity assays
are laborious, time consuming, and susceptible to failures due to the contamination of cells. With the
advancement in science, a number of other methods based on chemical or physical principles have been
developed for determining the viral load in a given sample. These methods include electron microscopy,
hemagglutination assay, qPCR, flow cytometry, and serological assays such as ELISA. However, all of these
methods have their own limitations and advantages associated with them and therefore one must be careful
while selecting an appropriate method to determine the virus titer and interpretation of results. Here, we
describe the theory and practical aspects of the most commonly used methods for virus quantification and
their practical utility in the field of virology.
Key words Virus titer, Infectivity assays, Quantal assays, Endpoint dilution
1 Introduction
Since the recognition of viruses as important pathogen for human

and animals, the search for ideal diagnostic tests has continued.
Most of the viral diagnostic assays developed till date focus primar-
ily on the detection of the causative agent rather than actually
quantifying the number of virions. Virus quantification involves
counting the number of viruses in a specific volume to determine
the virus concentration. Although the procedure for virus quantifi-
cation is complex and is not useful in clinical practice, it is utilized in
both research and development (R&D) in commercial and aca-
demic laboratories as well as production situations where the quan-
tity of virus at various steps is an important variable [1].
Methods quantifying the viruses fall into two discrete cate-
gories; infectivity assays and the tests that measures specific virion
components such as specific viral protein or the viral genome
31
32 Mukesh Bhatt et al.
[2]. Infectivity assays are the tests that measure virions that can
successfully infect a cell to produce infectious progeny. The inacti-
vated or non-infectious virion are not counted in such assays. The
second type of virus quantification assays is based on the principle of
chemical/physical measurement of virus particles and includes
serologic assays, polymerase chain reaction (PCR), and hemagglu-
tination assays (HA). Such methods are not able to differentiate the
infective and non-infective viral particles and thus give the result
even if the sample does not contain a single infective virus [3].
2 Infectivity Assays
In infectivity assays, viruses are inoculated onto a monolayer of

susceptible host cells, embryonated eggs, or animals. An infectivity
assay measures the virus particles capable of replicating in a particu-
lar cell type or animal. These assays can further be divided into two
types: (1) quantitative assays (e. g.: plaque assay) and (2) quantal
assays or end-point dilution assays.
2.1 Quantitative Plaque assay is the standard method that has long been used to
Assay (Plaque Assay) determine the virus titer (i.e., infectious dose). It determines the
number of plaque-forming units (pfu) in a sample and the titer of a
virus stock is represented by plaque-forming units per milliliter
(pfu/mL). Typically, tenfold serial dilutions of the virus stock are
inoculated into each well of six-well plate or animals in replicates
and after incubation periods extending from few days to weeks,
infectious particles produce visible zones of infected cells called
plaques (Fig. 1). The inoculated cell cultures are laid with a semi-
solid media (overlay medium) to localize the spread of infection to
the immediate vicinity of originally infected cell. The commonly
used overlay media include agar, methyl cellulose, tragacanth, and
starch gel [4]. Agar is the most commonly used overlay media;
however, it is inhibitory to some viruses. Methyl cellulose which is
overlaid at 37 C or lower temperature is preferred for the thermo-
labile viruses [4]. The pfu/mL result represents the number of
infectious particles in the sample, assuming that each plaque is
caused by a single infectious virus particle. Plaques are easily seen
following staining of monolayers either by incorporating neutral
red in overlay media or direct staining with methylene blue or
crystal violet [4, 5]. The important points to be taken into account
while performing plaque assay include host cellular compatibility
with the virus in question, appropriate viral growth conditions,
sufficient dilution ranges in order to clearly differentiate plaques,
and correct overlay selection followed by staining for the cells and
virus in question [6].
Methods for Quantification of Viruses 33
Fig. 1 Plaque assay using foot and mouth disease virus in BHK-21 cells
Viruses that are non-cytocidal and do not produce plaques can

be titrated by counting other types of infective centers. Haemad-
sorbing viruses can be titrated by counting the number of focal
areas to which RBCs are adsorbed. Groups of infected cells are
called foci and the assay is termed as focus-forming assays (FFA)
[2]. Although the FFA is a variation of the plaque assay, it employs
immunostaining techniques using fluorescently labelled antibodies
specific for a viral antigen to detect infected host cells and infectious
virus particles before an actual plaque is formed. The FFA delivers
the results in lesser time as compared to other methods of virus
titration and the virus titer is expressed as FFU/mL [7].
Plaque assay protocol [5] for FMD virus:
1. Culture the BHK-21 cells in growth medium (GMEM+10%
FBS) in a six-well plate to give 90–100% confluence by the
following days.
2. Dilute the virus suspension tenfold serially from 1:101 to 1:
106.
3. Discard the growth medium and wash the cells with serum-
free GMEM.
4. Infect the confluent monolayers of BHK-21 cells with 100 μL
volume of diluted virus (each dilution per well) and 400 μL
volume of GMEM (total 500 μL volume/well) keeping a well
seeded with cells in 500 μL of serum-free GMEM as control.
5. Incubate the plates at 37 C under 5% CO2 tension for 1 h for
virus adsorption with a gentle shaking at 30 min.
6. Discard the unadsorbed virus after 1 h and overlay the mono-
layer with 3 mL of autoclaved agar overlay (at about 40 C).
After the gel solidifies, incubate the plates at 37 C for up to
48 h.
7. After 48 h, remove the agar overlay from wells and for fixation
of cells, add 10% formalin to each well of plate followed by
incubation at 37 C for 1 h. After one hour, stain the plates
with 0.1% crystal violet in 10% formalin for 15 min (1 mL/
well) followed by washing with distilled water and air dry at
37 C.
8. Count the plaques and record the morphology.

9. Calculate the virus titer using the following formulas:
Pfu=mL ¼ Average no:of plaques per dilution=ðD V Þ:
where; D: Dilution factor (for dilution 102; D is 0.01 and for
dilution 104; D is 0.0001).
V: Volume of virus added per well (in mL; 0.1 mL in the
above protocol).
2.2 Quantal Assay or Many animal viruses do not form plaques on the cell monolayer but
End-Point Dilution induce a visible cytopathic effect (CPE). These morphological
Assay changes that are observable under microscope can be exploited
for virus quantification in “end-point dilution assays.” These assays
are called “quantal assay” as the end-point titer is based on the
outcome of infection and does not represent the absolute number
of virus particles in a sample. Consequently, the unit of infectivity
measured by this method may require more than one infectious
particle [8]. There are different ways of expressing the titer such as
TCID50, LD50, and EID50, depending on the host system that is
used. When end-point titration assay is performed in cultured cells,
the titer of the virus is recorded as TCID50 or median tissue culture
infectious dose. EID50 or median egg infectious dose is the titer
when embryonated eggs are inoculated with the virus for the
determination of titer. The titer obtained is referred to as LD50 or
median lethal dose if the virus can cause the death in 50% of the
animals. ID50 or median infectious dose is the titer of virus that
would infect 50% of the test animals [8, 9].
Due to distinct differences in assay methods and principles,
TCID50 and pfu/mL are not equivalent. According to the Poisson
distribution, which describes the number of random events (virus
particles) occurring at a known average rate (virus titer) in a fixed
space (the amount of virus medium in a well); the theoretical
relationship between TCID50 and PFU is approximately 0.70
PFU ¼ 1 TCID50 (thneedle; http://www.protocol-online.org/
biology-forums/posts/1664.html). However, a study using two
different strains of Enterovirus demonstrated that it is difficult to
establish the relationship between PFU and TCID50 and it was
found to be different for two different strains [10].
There are three methods to determine the end-point titer
including a more recent method given by Ramakrishnan [11] that
has been proposed to be used along with the existing methods:
(a) Reed–Muench method.
(b) Improved Karber Method.
(c) Ramakrishnan method.
Protocol for determining end-point titer:
Fig. 2 Preparing ten-fold serial dilution of virus sample
Virus 1 Virus 2
1 2 3 4 5 6 7 8 9 10 11 12
A 10-1 10-1 10-1 10-1 10-1 CC 10-1 10-1 10-1 10-1 10-1 CC
B 10-2 10-2 10-2 10-2 10-2 CC 10-2 10-2 10-2 10-2 10-2 CC
-3 -3 -3 -3 -3 -3 -3 -3 -3 -3
C 10 10 10 10 10 CC 10 10 10 10 10 CC
-4 -4 -4 -4 -4 -4 -4 -4 -4 -4
D 10 10 10 10 10 CC 10 10 10 10 10 CC
-5 -5 -5 -5 -5 -5 -5 -5 -5 -5
E 10 10 10 10 10 CC 10 10 10 10 10 CC
F 10-6 10-6 10-6 10-6 10-6 CC 10-6 10-6 10-6 10-6 10-6 CC
G 10-7 10-7 10-7 10-7 10-7 CC 10-7 10-7 10-7 10-7 10-7 CC
H CC CC CC CC CC CC CC CC CC CC CC CC
Fig. 3 Microtitration plate format for determining end-point titer (Note: 101 to 106; serial virus dilutions and
CC; uninfected cell control)
1. Dilute the virus sample (tenfold dilution; Fig. 2) in growth

medium for titration on a single 96-well cell culture plate as
mentioned in the following. Change tips between the dilutions
and use a fresh tip for each transfer.
2. Dispense 0.1 mL virus suspension to each well (Fig. 3) keeping
five replicates per dilution (101 to 106).
3. Using a multichannel micropipette add 0.1 mL of cell suspen-
sion first into all the control wells (without virus) and then to
virus wells.
4. Incubate the plates in 5% CO2 atmosphere at 37 C.
5. Carefully change the medium every second day. For this, the
microplate is held upside down and the contents are discarded
by gentle thrust downwards once or twice. Then, put two
drops of maintenance media in each of the wells using a
10 mL serological pipette.
6. Observe for cytopathic effect (CPE) and record the results.
1 2 3 4 5 6 7 8 9 10 11 12
A + + + + +
B + + + + +
C + - + + +
D - + + - +
E - + + - -
F - - - + -
G - - - - -
H - - - - -
Fig. 4 Microtitration plate reading indicating presence or absence of CPE in respective wells
3 Calculation of End-Point Titer
3.1 Reed–Muench Let us take an example for the determination of the end-point titer.
Method The positive (+) sign in Fig. 4 indicates the presence of CPE and the
minus () sign indicates no CPE.
The above results (Fig. 4) can be summarized in the tabular
form as given in Table 1.
Accumulated values for the total number of wells showing CPE
are obtained by adding in the direction of lowest to the highest
values. The accumulated infected ratios and the percentage infected
for each dilution are calculated.
In the example depicted in the Table 1 it can be seen that
infectivity in the dilution 104, is higher than 50% (67) and in the
next higher dilution, 105 it is only 33%. Therefore, to find the 50%
endpoint dilution, which obviously lies between these two dilutions
(104 to 105) first, proportionate distance (PD) is required to be
calculated using a simple formula.
%Infection above 50% 50%
PD ¼
%Infection above 50% %Infection below 50%
67 50 17
PD ¼ ¼ ¼ 0:50
67 33 34
The proportionate distance obtained, thus, has to be corrected

to find out exactly 50% infectivity by the dilution factor, also called
exponential of dilution (ED). The exponential of dilution of exactly
50% infectivity ¼ PD (ED next below 50% ED next above
50%) + ED next above 50%.
Log10 TCID50 ¼ PD ðED next below 50% ED next above 50%Þ þ ED next above 50%
¼ 0:50 ½ð5Þ ð4Þ þ ð4Þ
¼ 4:50
Table 1
Tissue culture infectivity data for determination of 50% end-point by Reed–Muench method
Test results Accumulative value
Virus No. of culture No. of culture not Not Ratio %

dilution infected infected Infected infected infected infected
101 5 0 20 0 20/20 100
2
10 5 0 15 0 15/15 100
3
10 4 1 10 1 10/11 91
4
10 3 2 6 3 6/9 67
5
10 2 3 3 6 3/9 33
6
10 1 4 1 10 1/11 9
7
10 0 5 0 15 0/15 0
Table 2
Tissue culture infectivity data for determination of 50% end-point by Spearman–Karber method
Virus dilution Ratio infected Proportion infected

101 5/5 1.0
2
10 5/5 1.0
3
10 4/5 0.80
4
10 3/5 0.60
5
10 2/5 0.40
6
10 1/5 0.20
7
10 0/5 0
Hence, the titer of the virus is: 104.50 TCID50/0.1 mL or

5.50
10 TCID50/mL.
3.2 Improved Karber For determining end-point using the Karber method, the infectiv-
Method ity assay results given in Fig. 4 can be summarized as seen in
Table 2.
TCID50 can be calculated by Karber method using the follow-
ing formula:
Log10 TCID50 ¼ L d ðs 0:5Þ
where L ¼ log 10 of the most concentrated virus dilution tested,
d ¼ log dilution factor, s ¼ sum of the proportion
(1.0 + 1.0 + 0.80 + 0.60 + 0.40 + 0.20 + 0 ¼ 4.0).
Table 3
Tissue culture infectivity data for determination of 50 % end-point by Ramakrishnan method (based
on Fig. 4)
Virus dilution Died/infected Inoculated/replicates per dilution Death score

1
10 5 5 5/5 ¼ 1.0
2
10 5 5 5/5 ¼ 1.0
3
10 4 5 4/5 ¼ 0.80
4
10 3 5 3/5 ¼ 0.60
5
10 2 5 2/5 ¼ 0.40
6
10 1 5 1/5 ¼ 0.20
7
10 0 5 0/5 ¼ 0
Therefore, Log10 TCID50 ¼ ð1Þ ð1Þ½4 0:5 ¼ 4:50

Hence, the titer of the virus would be ¼ 104.50 TCID50/
0.1 mL, or 105.50 TCID50/mL.
3.3 Ramakrishnan Ramakrishnan [11] has recently proposed two formulas for deter-
Method mining the virus titer using the end-point dilution method which
can be used to quantify the virus in a given sample in addition to the
existing methods, but not exclusively. The virus titer can be calcu-
lated as follows (Table 3).
Formula 1:

total no:of animals died
Log10 50%end‐point dilution ¼ þ 0:5
number of animals inoculated per dilution
log dilution factor:
Log10 50%end‐point dilution ¼ ½4 þ 0:5 1

¼ 4:50
Therefore, titer of the virus would be ¼ 104.50 TCID50/
0.1 mL, or 105.50 TCID50/mL.
Formula 2 (if any accidental death occurred):
Log10 50%end‐point dilution ¼ ðtotal death score þ 0:5Þ log dilution factor
¼ ð4 þ 0:5Þ 1 :
¼ 4:50
Hence, the titer of the virus would be ¼ 104.50 TCID50/
0.1 mL, or 105.50 TCID50/mL.
The determination of virus titer as calculated using the three
formulae gave the same results for virus quantification assay.
RBCs
Viral proteins
Virus
Indicator cells/PBMCs
Fig. 5 Schematic representation of haemadsorption phenomenon
4 Haemadsorption Assay-Based Infectivity Assay for African Swine Fever Virus

(ASFV)
The haemadsorption (HAD) test [12] is based on the characteristic

of ASFV infected monocytes to form a rosette of erythrocytes
around the infected cell. A positive result in the HAD test is defini-
tive for ASF diagnosis and is represented as rosette formation
around the infected leukocytes (Fig. 5).
The end-point titer is determined by any of the methods as
described earlier and is represented as HAD units yielding a 50% of
cumulative infection (HADU50) per milliliter. The test is semi-
quantitative in nature and the only limitation of this assay is the
maintenance of primary cells which can be more difficult to manage
than established cell lines [12].
Briefly, the HAD test is performed in Microtest I plates (Fal-
con) with 60 wells per plate, which is inoculated with five serial
tenfold dilutions of a virus sample (12 wells per dilution). The
indicator cell must be an ASFV-sensitive cell capable of adhering
erythrocytes on its surface membrane when infected with the virus,
as peripheral blood monocytes or alveolar macrophages, prepared
from swine are used for ASFV in this assay. The detailed protocol
for haemadsorption assay for ASFV has been described by Carras-
cosa and colleagues [13].
5 Chemical/Physical Methods of Virus Quantitation
Chemical/physical methods of virus quantitation measure the

amount (or relative amount) of a viral protein, genome, or enzyme
in a sample. Although these assays provide no information about
the amount of infectious virus in a sample, they are often conve-
nient, quick, and quite reproducible and can often be correlated
back to infectivity assays as a quick way to estimate the infectivity of
a sample. Few examples of such methods are as follows:
1. Direct visualization of virions by electron microscopy (EM).

2. Hemagglutination (HA) assay.
3. Genome quantification by PCR.
4. Serological assays.
5. Flow cytometry or flow virometry.
5.1 Direct With the advancements in electron microscopy, it has become

Visualization of Virions possible to determine the virus titer [14]. However, it has some
by EM limitations like the cost of the procedure, the expertise required and
the limited sensitivity (at least l06 particles/mL must be present)
[15]. It is reported that the titer obtained by quantitative transmis-
sion electron microscopy (TEM) are often higher than the results
from other assays as all particles, regardless of infectivity are quan-
tified. Because of high instrument cost and the amount of space and
support facilities needed, TEM equipment is available in a limited
number of facilities and hence this method has limited utility in
virus quantification.
5.2 Hemaggluti- The viruses that have the ability to agglutinate the RBCs
nation (HA) Assay (e.g. influenza viruses) can be quantified by hemagglutination
(HA) assay and the results are expressed in terms of hemagglutina-
tion units (HAU). It relies on the fact that hemagglutinin, a surface
protein of influenza viruses, agglutinates red blood cells and causes
red blood cells to clump together. The assay takes shorter time of
around 1–2 hrs to complete and is based on the technical expertise
of the operator. A haemagglutination assay has been developed for
the detection of porcine circovirus 2 (PCV2) and it was found that
the assay could detect 104.09 TCID50/mL of PCV2 [16]. The
detection limit was found to be even lower than immunocapture
ELISA for PCV2 which could detect as low as 400 TCID50/mL of
PCV2 [17]. Similarly, HA properties have also been reported for
other porcine viruses such as porcine Deltacoronavirus [18],
PRRSV [19], and porcine haemagglutinating encephalomyelitis
virus [20].
5.3 Genome Quantitative real-time PCR (qPCR) is a reliable, rapid, highly

Quantification by PCR sensitive, and specific assay for nucleic acid quantification. Due to
its ability to detect and measure minute amount of nucleic acid in a
wide range of samples, it has become a yardstick in the field of
molecular biology with its wide array of applications including
microbial quantification, gene expression analysis, microarray veri-
fication, identification of transgenes, etc. Conventional quantitative
PCR has already proven its utility in the monitoring of viral load as a
useful marker of disease progression and as a component to study
the effect of antiviral compounds [21–23]. The severity of a num-
ber of diseases has been linked to the viral load and thus real-time
PCR quantification can be used as an important tool to estimate

viral load and to study its role in disease progression, latency, and
reactivation [24–30].
Real-time polymerase chain reaction (PCR) or quantitative
PCR (qPCR) is used in both ways; qualitative test for diagnosis of
viral infections and as a quantitative test for quantifying the virus
load. Quantitative detection is based on the fluorescence detection
activity either by using fluorescent dyes such as SYBER Green or
using sequence-specific probes tagged with a fluorophore attached
to one end. The results of qPCR for virus quantification are
expressed as genome copies/mL. The viral load can be quantified
by two methods: relative quantification and absolute quantifica-
tion. Relative quantification is based on the comparison between
the expression of a target gene versus a reference/internal/endog-
enous control gene. Generally, relative quantification is sufficient
on most occasions and is simpler to develop [28]. However, abso-
lute quantification is useful when the results are required to be
provided in terms of units or a sufficient number of reference/
controls are not available or the quantification is required to access
the progression or recovery from virus infection. Absolute quanti-
fication employs a standard curve to interpolate the copy number in
a given sample. The standard curve is obtained by plotting Ct values
(PCR cycles that show statistically significant increases in the prod-
uct) obtained by testing a sample of known concentration against
log-transformed concentrations of serial tenfold dilutions of the
target nucleic acid.
In comparison to other methods of virus quantification, real-
time PCR is far more convenient, reliable, and better suited to
quick decision making in a clinical situation [30, 31]. In addition,
qPCR can also provide information related to strain/genotype of
virus along with the early diagnosis and quantification of virus
which may help in adopting a timely treatment regimen for a
specific viral infection. Other advantages of using qPCR include
higher reproducibility and less inter and intra-assay variability as
compared to other methods [28]. However, it is noteworthy that
the quantitative estimates of qPCR are generally higher than other
methods of virus quantification. This is due to the reason that
qPCR amplifies all the target nucleic acids including infectious
and non-infectious particles, defective interfering particles, and
free nucleic acids in sample, while in other methods, either the
virus particles (electron microscopy) or infective units are counted
(end-point dilution assays).
Table 4
Common enzymes and substrate combinations used in ELISA
Wavelength for detection

Enzyme Complementary substrate (nm)
Alkaline phosphatase (AP) pNPP: p-nitrophenyl-phosphate 405
ß-galactosidase (ß-gal) ONPG: Ortho- 405
Nitrophenyl- -galactoside
Horseradish peroxidase OPD: o-phenylenediamine 492
(HRP) dihydrochloride 450
TMB: 3,30 ,5,50 -tetramethylbenzidine
5.4 Serological Quantitative serological assays such as ELISA are based on the
Assays or Enzyme antigen antibody interaction which is measured by enzyme’s ability
Linked to convert a reagent to a detectable signal that can be used to
Immunosorbent Assay calculate the concentration of the antigen in the sample [32]. The
(ELISA) common enzymes and substrate combinations used in different
ELISA formats are given in Table 4. ELISA can be used to detect
and quantify both antigen and antibody in different formats and is
comparatively less time consuming and high throughput method
for simultaneous detection and quantification of protein antigens
and antibody. Antigen detection ELISA can be performed in three
formats which include direct ELISA, sandwich ELISA, and com-
petitive ELISA. For quantification of viral antigen in ELISA test,
the absorbance (OD values) of the test samples is compared with
the standard curve obtained by parallel testing of sample containing
known concentration of target protein. However, as these assays do
not provide the results in absolute units, they cannot be used for
absolute virus quantification.
Cell-ELISA can be used in conjunction with end-point dilution
methods to determine virus titer in terms of TCID50/mL. This
strategy can be utilized in non-cytopathic viruses that do not cause
visible CPE. In such cases, the cell monolayer is first infected with
different dilutions of viruses as described in end-point dilution
method and then after the incubation period is over, the monolayer
is fixed with fixative agents such as paraformaldehyde followed by
staining with antigen specific antibody conjugated to enzymes. The
color development is then measured in ELISA plate reader after
addition of substrate specific for the particular enzyme. The posi-
tive or negative results are recorded based on the OD values
obtained for the non-infected control wells.
5.5 Flow Cytometry Flow cytometry and its derivative fluorescence-activated cell sorting
or Flow Virometry (FACS) have been methods of choice since the 1970s to analyze
and purify individual cells. In early days, the quantification of
viruses through flow cytometry was challenging due to the very
small size of virus particle in contrast to the resolution limit of

standard flow cytometers, which was supposed to be about
300–500 nm [33]. Although flow cytometry has been used since
long for virus characterization and enumeration [34] and the term
“flow virometry” was given by Grivel et al. in 2013, it has been used
for a wide array of viruses only recently [35].
In flow cytometry the number of intact virus particles is quan-
tified when the fluorescent tagged virus particles are passed through
a laser beam. The analysis of samples requires careful preparation of
the samples, fixing, labeling, and heating to promote the pene-
trance of the dye [36, 37]. Dyes such as SYBR Green-I or SYBR
Gold at 80 C are used to stain the viral nucleic acids. For RNA
viruses, potential RNA nucleic acid stains such as Styryl-TO
TOTO-1, SYTO 12, and 14, SYTO RNASelect, and LDS 751 are
employed [35]. The buffers in which the viruses are suspended
previous to flow cytometric analysis are also important. Out of
different buffers used for virus suspension which include tris-
EDTA buffer, 5% sucrose-NTE buffer, PBS or 0.1–1% PFA,
EDTA containing buffers have been reported to prevent the aggre-
gate formation and allow the distribution of viruses during flow
cytometric analysis [37–40]. To achieve the optimum results, final
viral suspension should be filtered several times to remove the
particulate matter and contaminants.
The results of flow cytometric analysis are represented in virus
particles per mililitre (vp/mL). However, the quantification results
are higher than that of end-point dilution methods and are compa-
rable to electron microscopic assays (TEM). This is due to the
reason that flow cytometry counts the infectious and
non-infectious particles while end-point dilution methods only
take into account the infectious virus particles only [41]. Bonar
and colleagues showed that flow virometry is a much more sensitive
technique when compared to ELISA and has similar detection of
viruses to qPCR assays [42]. Overall although flow virometry
requires sophisticated equipment and sample preparation, it is a
very sensitive method for virus quantification with a linear working
range of 105–109 vp/mL and an analysis time of ~10 min with a
short sample preparation time.
6 Applications of Virus Quantification
6.1 Vaccine Determination of virus load in vaccines is an important step to

Production avoid any harmful effects related to vaccines. Generally, the amount
of viral antigen in inactivated or live attenuated vaccines is repre-
sented in terms of TCID50/mL. The virus quantification is also
vital in the process of vaccine development and production as
determination of ideal multiplicity of infection (moi) is very crucial
to grow the virus in high titer in production facilities.
6.2 Antiviral Before recommending the antivirals for treatment of a particular

Development viral infection, a number of trials are conducted in vitro and in
animal models to determine the safety and therapeutic limit of the
concerned drug [43]). The specific antiviral activity is measured
using a quantitative assay to measure virus replication in the pres-
ence of increasing concentrations of the product compared to
replication in the absence of product. The effective concentration
(EC50) is the concentration of product at which virus replication is
inhibited by 50%. The EC50 value is then used to calculate the
therapeutic index (i.e., CC50 value/EC50 value) of an antiviral
and it is desirable to have a high therapeutic index giving maximum
antiviral activity with minimal cell toxicity.
6.3 Viral Virus quantification is also applied as a tool to determine the

Therapeutics response of the animals or individual to the treatment protocol.
Besides this, quantification of virus titer, viral antigen or viral RNA
in the patient can also be employed for management of viral dis-
eases and determination of infectivity (transmissibility) of an indi-
vidual to susceptible population [1].
6.4 Routine Virus titer determination is a routine procedure in laboratories

Virological Assays conducting the research in the field of virology. Virus quantification
is performed and used as a guiding stone in number of assays such
as virus neutralization test (VNT), growth kinetics studies, optimi-
zation of different assays such as ELISA and PCR. Besides this,
virus titer is also taken into account during experimental animal
studies for the purpose of raising hyper-immune sera and to deter-
mine the expression of certain proteins in host system of in vitro in
response to different concentration of virus antigens.
7 Conclusion
Virus quantification is of prime importance for commercial and

academic laboratories involved in the research or production of
viral vaccines, recombinant proteins, viral antigens, or antiviral
agents. The most common and accurate methods for determining
the virus titer include plaque assay and 50% tissue culture infectious
dose (TCID50). These tests can be used to determine the infectious
virus titer in a sample and there is no replacement for these tests.
However, these tests are time consuming and need utmost caution
as they are susceptible to failures due to contamination of cell
culture system. Although with the advancement in science a num-
ber of other methods have been used for virus quantification
including transmission electron microscopy (TEM), qPCR,
ELISA, and flow virometry, there remain significant drawbacks
associated with all of these assays. Although the modern methods
are rapid and provide results in a single day or span of hours, they
require costly equipment and expertise for performing these assays.
In addition, these assays are at backfoot when the infectious virus
titer with high precision is required for sensitive virological proce-
dures such as vaccine production and antiviral optimization. In
general, there is still a need for new analytical methods that can
rapidly quantify viral concentration to reduce costs and alleviate
bottlenecks associated with current assays.
References
1. Berger A, Preiser W (2002) Viral genome journal of virology 23(3):303–310. https://

quantification as a tool for improving patient doi.org/10.1007/s13337-012-0105-0
management: the example of HIV, HBV, HCV 11. Ramakrishnan MA (2016) Determination of
and CMV. J Antimicrob Chemother 49 50% endpoint titer using a simple formula.
(5):713–721. https://doi.org/10.1093/jac/ World J Virol 5(2):85–86. https://doi.org/
dkf050 10.5501/wjv.v5.i2.85
2. Payne S (2017) Methods to study viruses. In: 12. de León P, Bustos MJ, Carrascosa AL (2013)
Viruses. Academic Press, London, pp 37–52 Laboratory methods to study African swine
3. Fox JC, Kidd IM, Griffiths PD, Sweny P, fever virus. Virus Res 173(1):168–179.
Emery VC (1995) Longitudinal analysis of https://doi.org/10.1016/j.virusres.2012.
cytomegalovirus load in renal transplant recipi- 09.013
ents using a quantitative polymerase chain reac- 13. Carrascosa AL, Bustos MJ, de Leon P (2011)
tion: correlation with disease. J Gen Virol 76 Methods for growing and titrating African
(Pt 2):309–319. https://doi.org/10.1099/ swine fever virus: field and laboratory samples.
0022-1317-76-2-309 Curr Protoc Cell Biol Chapter 26:Unit 26.14
4. Mohanty SB, Dutta SK (1981) Cultivation and 14. Malenovska H (2013) Virus quantitation by
assay of viruses. In: Veterinary virology. Lea & transmission electron microscopy, TCID50,
Febiger, Philadelphia, PA and the role of timing virus harvesting: a case
5. Bhatt M, Mohapatra JK, Pandey LK, Mohanty study of three animal viruses. J Virol Methods
NN, Das B, Prusty BR, Pattnaik B (2018) 191(2):136–140
Mutational analysis of foot and mouth disease 15. Fox JC, Emery VC (1992) Quantification of
virus nonstructural polyprotein 3AB-coding viruses in clinical samples. Rev Med Virol 2:
region to design a negative marker virus. 195–203
Virus Res 243:36–43. https://doi.org/10. 16. Cheng H, Yang L, Cai Z, Qiao X, Du L, Hou J,
1016/j.virusres.2017.10.010 Chen J, Zheng Q (2020) Development of hae-
6. Baer A, Kehn-Hall K (2014) Viral concentra- magglutination assay for titration of porcine
tion determination through plaque assays: circovirus type 2. Anal Biochem 598:113706
using traditional and novel overlay systems. J 17. Huang L, Wei Y, Xia D, Liu D, Zhu H, Wu H,
Vis Exp (93):e52065. https://doi.org/10. Li F, Liu C (2019) A broad spectrum mono-
3791/52065 clonal antibody against porcine circovirus type
7. Flint SJ, Enquist W, Racaniello VR, Skalka AM 2 for antigen and antibody detection. Appl
(2009) Virological methods. In: Principles of Microbiol Biotechnol 103:3453–3464.
virology. ASM Press, Hoboken, NJ https://doi.org/10.1007/s00253-019-
8. Condit CR (2013) Principles of virology. In: 09715-0
fields virology, 6th edn. Wolters Kluwer 18. Zhang Y, Han L, Xia L, Yuan Y, Hu H (2020)
Health/Lippincott Williams & Wilkins, Assessment of hemagglutination activity of
Philadelphia porcine deltacoronavirus. J Vet Sci 21(1):e12.
9. MacLachlan NJ, Dubovi EJ (2017) Virus rep- https://doi.org/10.4142/jvs.2020.21.e12
lication. In: Fenner’s veterinary virology, 5th 19. Jusa ER, Inaba Y, Kohno M, Mashimo H, Hir-
edn. Academic Press, London ose O (1996) Hemagglutination with porcine
10. Pourianfar HR, Javadi A, Grollo L (2012) A reproductive and respiratory syndrome virus. J
colorimetric-based accurate method for the Vet Med Sci 58(6):521–527. https://doi.org/
determination of enterovirus 71 titer. Indian 10.1292/jvms.58.521
20. Mora-Dı́az JC, Piñeyro PE, Houston E, 30. Tanaka N, Kimura H, Iida K, Saito Y, Tsuge I,
Zimmerman J, Giménez-Lirola LG (2019) Yoshimi A, Matsuyama T, Morishima T (2000)
Porcine Hemagglutinating encephalomyelitis Quantitative analysis of cytomegalovirus load
virus: a review. Front Vet Sci 6:53. https:// using a real-time PCR assay. J Med Virol 60
doi.org/10.3389/fvets.2019.00053 (4):455–462. https://doi.org/10.1002/(sici)
21. Clementi M (2000) Quantitative molecular 1 0 9 6 - 9 0 7 1 ( 2 0 0 0 0 4 ) 6 0 : 4 <4 5 5 : : a i d -
analysis of virus expression and replication. J jmv14>3.0.co;2-q
Clin Microbiol 38:2030–2036 31. Locatelli G, Santoro F, Veglia F, Gobbi A,
22. Holodniy M, Katzenstein D, Sengupta S, Lusso P, Malnati MS (2000) Real-time quanti-
Wang AM, Casipit C, Schwartz DH, tative PCR for human herpesvirus 6 DNA. J
Konrad M, Groves E, Merigan TC (1991) Clin Microbiol 38(11):4042–4048
Detection and quantification of human immu- 32. Kemeny DM, Challacombe SJ (1988) ELISA
nodeficiency virus RNA in patient serum by use and other solid phase immunoassays: theoreti-
of the polymerase chain reaction. J Infect Dis cal and practical aspects. Wiley, Chichester
163:862–866 33. Chandler WL (2016) Measurement of micro-
23. Menzo S, Bagnarelli P, Giacca M, Manzin A, vesicle levels in human blood using flow cyto-
Varaldo PE, Clementi M (1992) Absolute metry. Cytometry B Clin Cytom 90:326–336.
quantitation of viremia in human immunodefi- https://doi.org/10.1002/cyto.b.21343
ciency virus infection by competitive reverse 34. Marie D, Brussaard CPD, Thyrhaug R,
transcription and polymerase chain reaction. J Bratbak G, Vaulot D (1999) Enumeration of
Clin Microbiol 30(7):1752–1757. https://doi. marine viruses in culture and natural samples by
org/10.1128/jcm.30.7.1752-1757.1992 flow cytometry. Appl Environ Microbiol 65
24. Kearns AM, Turner AJL, Taylor CE, George (1):45–52. https://doi.org/10.1128/AEM.
PW, Freeman R, Gennery AR (2001) 65.1.45-52.1999
LightCycler-based quantitative PCR for rapid 35. Lippé R (2018) Flow Virometry: a powerful
detection of human herpesvirus 6 DNA in clin- tool to functionally characterize viruses. J
ical material. J Clin Microbiol 39:3020–3021 Virol 92(3):e01765–e01717. https://doi.
25. Kimura H, Morita M, Yabuta Y, Kuzushima K, org/10.1128/JVI.01765-17
Kato K, Kojima S, Matsuyama T, Morishima T 36. Khalil JY, Langlois T, Andreani J, Sorraing JM,
(1999) Quantitative analysis of Epstein–Barr Raoult D, Camoin L, La Scola B (2016) Flow
virus load by using a real-time PCR assay. J cytometry sorting to separate viable giant
Clin Microbiol 37(1):132–136. https://doi. viruses from amoeba co-culture supernatants.
org/10.1128/JCM.37.1.132-136.1999 Front Cell Infect Microbiol 6:202. https://doi.
26. Lallemand F, Desire N, Rozenbaum W, Nicolas org/10.3389/fcimb.2016.00202
JC, Marechal V (2000) Quantitative analysis of 37. Brussaard CP, Marie D, Bratbak G (2000)
human herpesvirus 8 viral load using a real- Flow cytometric detection of viruses. J Virol
time PCR assay. J Clin Microbiol 38 Methods 85(1-2):175–182. https://doi.org/
(4):1404–1408. https://doi.org/10.1128/ 10.1016/s0166-0934(99)00167-6
JCM.38.4.1404-1408.2000 38. Chen F, Lu JR, Binder BJ, Liu YC, Hodson RE
27. Laue T, Emmerich P, Schmitz H (1999) (2001) Application of digital image analysis
Detection of dengue virus RNA inpatients and flow cytometry to enumerate marine
after primary or secondary dengue infection viruses stained with SYBR gold. Appl Environ
by using the TaqMan automated amplification Microbiol 67(2):539–545. https://doi.org/
system. J Clin Microbiol 37(8):2543–2547. 10.1128/AEM.67.2.539-545.2001
https://doi.org/10.1128/JCM.37.8.2543- 39. Ferris MM, McCabe MO, Doan LG, Rowlen
2547.1999 KL (2002) Rapid enumeration of respiratory
28. Mackay IM, Arden KE, Nitsche A (2002) Real- viruses. Anal Chem 74(8):1849–1856.
time PCR in virology. Nucleic Acids Res 30 https://doi.org/10.1021/ac011183q
(6):1292–1305. https://doi.org/10.1093/ 40. Landowski M, Dabundo J, Liu Q, Nicola AV,
nar/30.6.1292 Aguilar HC (2014) Nipah virion entry kinetics,
29. Ohyashiki JK, Suzuki A, Aritaki K, Nagate A, composition, and conformational changes
Shoji N, Ohyashiki K, Ojima T, Abe K, Yama- determined by enzymatic virus-like particles
moto K (2000) Use of real-time PCR to moni- and new flow virometry tools. J Virol 88
tor human herpesvirus 6 reactivation after (24):14197–14206. https://doi.org/10.
allogeneic bone marrow transplantation. Int J 1128/JVI.01632-14
Mol Med 6(4):427–432. https://doi.org/10. 41. Shen CF, Meghrous J, Kamen A (2002) Quan-
3892/ijmm.6.4.427 titation of baculovirus particles by flow
cytometry. J Virol Methods 105(2):321–330. submitting virology studies to the agency. Cen-
https://doi.org/10.1016/s0166-0934(02) ter for Drug Evaluation and Research (CDER)
00128-3 at the Food and Drug Administration,
42. Bonar MM, Tilton JC (2017) High sensitivity Rockville, MD
detection and sorting of infectious human 44. Brussaard CP (2004) Optimization of proce-
immunodeficiency virus (HIV-1) particles by dures for counting viruses by flow cytometry.
flow virometry. Virology 505:80–90. https:// Appl Environ Microbiol 70:1506–1513.
doi.org/10.1016/j.virol.2017.02.016 https://doi.org/10.1128/AEM.70.3.1506-
43. CDER (2006) Guidance for industry antiviral 1513.2004
product development — conducting and
Chapter 4
Protocols for Isolation of Genetic Materials from RNA

Viruses
Nihar Nalini Mohanty, Vikas Gupta, Laxmi Narayan Sarangi, Rohini Bhat,
and Sathish B. Shivachandra
Abstract
The isolation of viral RNA with purity and integrity is a critical element for the overall success of viral
diagnosis. The era of classical virology has transcended way beyond the labor-intensive manual method of
RNA extraction to the modern-age efficient and simpler protocols. With an aim to obtain a RNA material
free from carry over contaminants such as protein, unwarranted cellular genome, and chemicals, etc. there
are three major techniques followed worldwide such as organic extraction viz phenol-guanidine isothiocy-
anate (GITC)-based solutions, silica-membrane-based spin column technology, and paramagnetic particle
technology. The method of extraction and the flow of processes within a particular method would vary with
the type of material being handled. The major considerations while extracting RNA from tissue sample
would be eliminating endogenous RNase that would compromise RNA integrity. The final step of RNA
extraction is the storage of the isolated genome, which solely depend upon the purpose with which the
extraction was carried out. If the sample is not intended for immediate application, then several commer-
cially available formulations such as FORMAzol and RNA stable have been found suitable for long-term
storage.
Key words RNA, Phenol-Guanidine, Silica-membrane, Paramagnetic particle
1 Introduction
The isolation of viral RNA with purity and integrity is a critical

element for the overall success of viral diagnosis and RNA-based
assays [1]. A low quality RNA may compromise the results of
downstream applications which are often labor-intensive, time-con-
suming, and very expensive [2]. To ensure acceptable viral RNA
quality, the RNA extraction procedure must fulfill a number of
requirements with the final preparation must be free from protein,
genomic DNA, nucleases, and carryover phenol or alcohol [3, 4],
Buckingham,2007]. Structurally viruses could be divided into two
major types (a) enveloped viruses and (b) non-enveloped viruses. In
enveloped viruses, the nucleocapsid core is housed within a
49
50 Nihar Nalini Mohanty et al.
glycolipid shell or envelope, whereas in case of the non-enveloped

or naked viruses the nucleocapsid lacks the glycolipid cover. The
presence and absence of shell though not remarkable but alters the
accessibility to the viral RNA. The viral RNA encased within the
capsid are broadly classified in two types single-stranded RNA and
double-stranded RNA.
The extraction techniques vary significantly with the type of
genetic material under study. There are three major techniques
extensively used for RNA extraction: organic extraction, such as
phenol-guanidine isothiocyanate (GITC)-based solutions, silica-
membrane-based spin column technology, and paramagnetic parti-
cle technology. The method used for RNA isolation is very similar
to those described for DNA; however, special precautions need to
be taken during RNA isolation. RNA molecules are relatively short
and single stranded, therefore easily damaged by mechanical shear-
ing during the process of sample preparation. Furthermore, RNA is
very vulnerable to digestion by ribonucleases (RNase) which are
present endogenously in various concentrations in certain cell types
as well as exogenously on hands and surfaces [5]. The extraction
tactics must be formulated in such a way, so as to include sufficient
management of endogenous RNase activity. Failure to eliminate
potential sources of RNase contamination is likely to yield a
degraded RNA sample for utility. Even traces of carry over contam-
ination could compromise the RNA integrity. Since, the RNA have
a short half-life, experiments involving RNA are most judiciously
planned in close proximity of RNA isolation [6]. It is always impor-
tant to use RNAse-free solution during extraction procedure as well
as RNAse-free pipette tips and glassware/plastic wares.
Chemically and biologically, the RNA is more labile than DNA,
particularly at elevated temperatures and in the presence of alkali.
The activity of a variety of resilient ribonucleases (RNase), further
adds to the handling difficulties. Therefore, proper collection, ship-
ment, and processing of the sample specimens are crucial for diag-
nosis of diseases caused by RNA viruses. Proper maintenance of
cold chain, use of virus transport medium, and addition of glycerol
to the transport media are suggested to preserve the integrity of the
infecting virus during sample transport. However, if the virion
rupture, the RNA comes out to the solution and the endogenous
nucleases degrades the RNA thereby resulting in false negative test
results. In order to overcome such scenarios, many researchers are
using solutions such as RNAlater which is a storage reagent that
stabilizes the RNA thereby preserving the RNA integrity. It can be
used both for preserving the RNA in the tissue and clinical samples
but as far as the question of its utility, in storing extracted RNA
is concered, it is debatable. Furthermore, the importance of ultra-
low temperature shipment of the specimen has always been exigent.
Protocols for Isolation of Genetic Materials from RNA Viruses 51
Efficient methodologies have been designed for isolation of

RNA, which in due course should be refined and optimized as per
use. The protocol for isolation of viral RNA depends upon the base
material which could be anything starting from cell pellet to,
blood, tissue, cell culture supernatant, etc. It is important to con-
sider the source and type of specimen during selection of the
extraction procedure to be adopted; for example, the method/kit
used for extraction of RNA directly from clinical specimen may not
be suitable for extraction of RNA from virus infected cell culture
supernatant. Irrespective of the source, the primary aim involves
the release of the viral RNA from its protein and glycolipid housing
and the cellular structures encompassing them. Protocols for the
isolation of RNA begin with lysis mediated by buffers that typically
consists of harsh chaotropic agents such as one of the guanidinium
salts (guanidinium thiocyanate, guanidinium hydrochloride)
sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LiDS),
N-laurylsarcosine (sarcosyl), urea, phenol, or chloroform. All
these agents act by disrupting the cellular and subcellular structures
and help in releasing the viral RNA while maintaining an RNase-
free environment. Appropriate steps must be taken to break
through the cell wall in order to access the cellular contents. It is
worth noting that an endless list of permutations on fundamental
RNA extraction techniques exists; for example, some techniques
support isolation of poly(A) material directly from a cellular lysate
without prior purification of total RNA. As far as which RNA
extraction procedure is to be used always depends on the investiga-
tor. It always helps in devising the proper extraction protocol and
further downstream applications [6].
2 Important Considerations for Purification of the RNA from Clinical Specimen
Any worthwhile strategy for RNA purification must achieve specific

objectives, if data derived from the final RNA preparation are to be
meaningful and applicable for downstream processes. There are
many procedures for isolating viral RNA from various biological
sources, and each procedure in some way accomplishes these tar-
gets. One should follow few core concepts that accompanies most
of the RNA extraction procedures, such as:
1. Selection of an appropriate method of membrane
solubilization.
2. Ensuring total inhibition of nuclease activity.
3. Selection of a method for deproteinization of the sample.
4. Selection of a method for nucleic acid concentration.
5. Selection of proper storage conditions for purified RNA.
The first consideration towards viral RNA isolation strategy is

the method of cellular disruption to release the viral RNA from its
protein housing. The method of lysis will determine the extent of
subcellular disruption of the sample and the subsequent access to
the viral RNA. For example, a lysis buffer that is used successfully
with tissue culture cells may be entirely inappropriate for whole
tissue samples. The method by which membrane solubilization is
accomplished will dictate the requirement of any additional steps
that may be required in viral RNA extraction.
The second one is to inhibit nuclease activity. Some lysis
reagents do act as strong nuclease inhibitor while others require
additional nuclease inhibitors to safeguard the RNA during the
isolation procedure.
The third factor is complete deproteinization of the sample.
The complete removal of protein from a cellular lysate is of para-
mount importance in the isolation of both DNA and RNA. Pro-
teinase K/Protease can be used to digest the proteins whereas
RNAse-free DNAse can be added to degrade the contaminating
DNA. Organic extraction using phenol and chloroform or dissol-
ving the sample in guanidium salt buffers are also used for removal
of the proteins.
Concentration of RNA is very important in the purification
schemes. The most versatile method for concentrating nucleic
acids is precipitation using various combinations of salt and buffer.
For example, one common method is to add 0.1 vol of 3 M sodium
acetate (pH 5.2) to a nucleic acid sample, followed by the addition
of 2.5 vol of 95–100% ethanol. Addition of carrier RNA (usually
poly(A)-homopolymers or other molecules that mimic nucleic
acid) is another concept used by many researchers for concentration
of RNA, especially for purification of RNA at low concentrations.
To form a precipitate, a large number of molecules need to aggre-
gate. In cell free sample specimens, viz. cerebrospinal fluid, synovial
fluids, nasal secretions, etc. the concentration of RNA/DNA is low
and addition of carrier RNA increases the yield of the RNA. Fur-
thermore, it also protects the target RNA by reducing the damage
caused by RNAses. It may not be required when sample material
contains large quantity of DNA/RNAs such as blood, stool,
Semen, etc., [6].
Because of the naturally labile character of RNA, improper
storage of excellent RNA samples will often result in degradation
in a relatively short time. There are many opinions as to the proper
temperature, buffer, and storage for the purified viral RNA.
3 Lysis Buffer
There are a number of formulations available for lysis buffers which

depends on the degree of cellular disruption required and RNase
inhibition. There is no one right way to extract RNA from cells and
tissues the only thumb rule is to avoid RNase activity. Gentle lysis
buffers such as NP-40 are normally not preferred due to their
inability to inhibit the RNase activity and with the rupture of cell
membrane the sequestered RNase are suddenly liberated
compromising the RNA integrity.
3.1 Chaotropic Lysis The best suited way to deal with stubborn RNases is to disrupt cells
Buffers in guanidinium lysis buffer [7]. Guanidinium buffers efficiently
denature and solubilize proteins, including RNase. It is not neces-
sary to add additional RNase inhibitors to such lysis buffers, and
even the RNA isolation procedures can be performed with room
temperature reagents. RNA lysis buffers that contain guanidinium
thiocyanate or guanidinium-HCl reproducibly yield very high-
quality RNA samples, which is because of the extremely chaotropic
nature of these chemicals. They have been reported to be the most
effective protein denaturants [8–10]. The efficiency of protein
denaturation, including disruption of RNases, may be enhanced
by the inclusion of β-mercaptoethanol (β-ME) which acts to break
intramolecular protein disulfide bonds. However common reduc-
ing reagent, dithiothreitol (DTT) should be avoided in this partic-
ular application because of its chemical reactiveness with
guanidinium.
In this approach, the salient chemical differences between
RNA, protein, and DNA are exploited by creating an acidic pH
environment and judiciously blending organic solvents. As a result
of the ease of this approach, numerous products and methodolo-
gies have been developed for the rapid, efficient purification of both
RNA and DNA (and protein) from the same biological source
[11, 12]. The principal drawback of all of these procedures is that
it is very difficult to discriminate between cytoplasmic and nuclear
RNA. Again the isolated RNA becomes susceptible to nuclease
degradation when the denaturants have been removed. Therefore,
it is necessary to guard against the RNase peril consistently.
3.2 Guanidinium This method of extraction is a modification of the procedure of

Acid-Phenol Extraction Chomczynski and Sacchi [13]. The highest quality RNA indisput-
ably results from the extraction of RNA mediated by chaotropic
lysis buffers, and guanidinium-containing buffers are among the
most effective. Chomczynski and Sacchi [13] eased the purification
of undegraded RNA by treatment of cells with guanidinium
thiocyanate-containing lysis buffers, without subsequent CsCl
ultracentrifugation. In subsequent modifications, the RNA is
isolated in a very short time by extraction of a guanidinium cell or
tissue lysate with an acidic phenol solution, followed by the addi-
tion of chloroform to facilitate partitioning of the aqueous and
organic material. Now a days several formulations of guanidinium
thiocyanate and phenol are readily available commercially [e.g.,
TRI Reagent (Molecular Research Center), TRIzol (Thermo

Fisher Scientific)]. Upon phase separation, RNA is retained in the
aqueous phase, while DNA and proteins partition into the organic
phase. RNA is then recovered by precipitation with isopropanol and
collected by centrifugation. These commercial formulations have
become an inseparable partner to systems such as spin column or in
magnetic bead format for ease in processing and to maximize purity
[e.g., Direct-zol (Zymo Research) and QIAzol 1 RNeasy
(Qiagen)]. In general, RNA can be efficiently isolated from less
than 1 mg of tissue or 105 cells, usually in less than 1 h, using these
procedures. A protocol by Farrell [6] is hereby exampled below for
extraction of viral RNA and as such protocols are subject to differ
from different labs. However, the overall principle of extraction
remains fundamentally consistent.
1. Wear gloves and appropriate protective wear.
2. Harvest cells by centrifugation and re-suspend cell pellets in
100 μL of the following formulation (solution D) per 106 cells:
Solution D:
(a) 4 M guanidinium thiocyanate
(b) 25 mM sodium citrate, pH 7.0
(c) 0.5% sarcosyl
(d) 100 mM β-ME.
Pipette up and down or gently vortex in order to break up
the cell pellet.
3. Transfer the lysate to a centrifuge tube. For each 1 mL of
solution D lysis buffer used in step 2, add:
(a) 0.1 mL 2 M sodium acetate, pH 5.2,
(b) 1 mL water-saturated phenol (molecular biology grade),
(c) 0.2 mL chloroform:isoamyl alcohol (49:1).
Cap the tube and mix carefully and thoroughly by inversion
following the addition of each reagent and invert vigorously for
an additional 30 s after all reagents have been added.
4. Cool sample on ice for a minimum of 15 min; centrifuge at
4 C at high speed to separate the phases.
5. Transfer aqueous (upper) phase containing the RNA to a fresh
tube and mix with 0.75 vol of ice-cold isopropanol. Store at
20 C for at least 1 h to precipitate RNA.
6. Collect precipitate by centrifugation at 10,000 g for 20 min
at 4 C. Carefully decant and discard supernatant.
Caution: Do not exceed the recommended maximum
g-force for any of the tubes used in this protocol.
7. Completely dissolve RNA pellet in 300 μL of solution D (see

step 2) and then transfer to a RNase-free 1.7 mL
microfuge tube.
8. Reprecipitate the RNA by the addition of 0.75 vol of ice-cold
isopropanol and store at 20 C for 1 h.
9. Collect precipitate at 12,000 g in a microcentrifuge for
10 min at 4 C. Carefully decant and discard supernatant.
10. Wash pellet three times with 500 μL 70% ethanol per wash,
followed by a final wash with 500 μL 95% ethanol. If the RNA
does not dislodge during these washes, there is no need to
re-centrifuge. Allow tubes to air-dry to remove residual etha-
nol. Store the RNA as an ethanol precipitate until it is to
be used.
Note: The final wash with 95% ethanol will accelerate the
drying process.
11. Re-dissolve RNA in the smallest possible volume of TE (Tris
EDTA)buffer or nuclease-free water. Incubation at 65 C for
10 min may facilitate solubilization, though this is unnecessary
if the RNA did not dry out completely following the ethanol
washes. Following the determination of concentration, store
the RNA in suitable aliquots at 80 C. Avoid repeated freez-
ing and thawing.
4 Density Gradient Centrifugation
The several procedures for the isolation of viral RNA from

biological sources enriched in RNase employs cell and tissue dis-
ruption with guanidinium buffer. The resulting intermixing of
subcellular components mandates the separation of these biochem-
ical macromolecules from each other, with particular regard to the
removal of DNA from RNA preparations. This is attributable to the
measurable differences in the densities of DNA, RNA, and protein
which allow partitioning by banding or pelleting them in a density
gradient. Isopycnic centrifugation, a type of density gradient cen-
trifugation, is a technique in which macromolecules move through
a density gradient until they find a density equal to their own.
Macromolecules therefore accumulate at that position in the gradi-
ent, floating there until the end of the centrifugation run. In some
cases, RNA is of greater density than any position in the gradient
and accumulates as a pellet at the bottom of the tube. The classical
gradients used for this type of separation are cesium chloride
(CsCl), cesium trifluro acetate (CsTFA), and cesium sulfate
(Cs2SO4). Other materials such as glycerol, ficoll, metrizamide,
and sucrose also have specific applications; they are readily available
at low cost and high purity. Nucleic acid resolution based on
Fig. 1 Nucleic acid resolution based on differences in density
differences in density is best performed using one of the cesium

salts (see Fig. 1).
CsTFA (GE Healthcare Life Sciences) is an excellent CsCl
alternative for density gradient separation of RNA and DNA by
isopycnic centrifugation. At ultracentrifugation RCFs, CsTFA will
likewise self-form a gradient. The CsTFA has the advantage of easy
dissociation of proteins from nucleic acids, banding of RNA, simul-
taneous recovery of DNA, RNA, and protein from the same gradi-
ent, greater yield of RNA and efficient inhibition of RNase activity.
However, the requirement for a very expensive ultracentrifuge, the
relatively long duration of centrifugation, the labor-intensive clean-
up of gradient-purified RNA, and fewer samples at a time has
replaced isopycnic centrifugation with newer reagents and kits [6].
A protocol, as described by [14], has been highlighted below
for extraction by isopycnic extraction of RNA.
l Extraction buffer.
– 100 mM Tris–HCl, pH 7.5.
– 50 mM EDTA, pH 8.0.
– 500 mM NaCl.
– 5% β-Mercaptoethanol.
l CsCl Cushion.
– 96% CsCl.
– 100 mM EDTA, pH 8.0.
l 14% Sarcosyl.
l 1 M NaCl.
4.1 Procedure The following method is based on 0.5 g of starting material but it
can be applied to larger tissue samples by increasing buffer volumes
and the size of the ultracentrifuge tube.
1. Homogenize 0.5 g of tissue in liquid nitrogen and transfer the
resulting powder to a 15 mL centrifuge tube.
2. Immediately add 3.33 mL of extraction buffer and 1 mL of
14% Sarcocyl, vortex briefly.
3. Add 0.65 g of CsCl powder, mix well, and incubate at 65 C for
15 min.
4. Centrifuge at 4 C for 20 min at 9500 rpm/9383 g (C1015
in GS-15/Allegra21/Allegra X-22 Series rotor).
5. Filter the supernatant through Miracloth (Calbiochem) and
store on ice while preparing the CsCl cushion.
6. Add 1.6 mL of CsCl to a 5 mL ultracentrifuge tube.
7. Apply 3.4 mL of the filtered supernatant carefully to the top of
the cushion.
8. Centrifuge at 20 C for 20 h at 40,000 rpm/2,84,061 g
(SW40Ti rotor).
9. The centrifugation results in two phases. Genomic DNA is
located at the interface, whereas RNA is located at the bottom
of the tube below the cushion phase. Remove 1/3 of the upper
phase and carefully add a similar volume of DEPC(Diethylpyr-
ocarbonate) water to the tube (referred to subsequently as a
wash step).
10. Repeat this washing step 2 times removing more of the upper
phase each time without disturbing the cushion. Make sure to
remove the genomic DNA at the interface is completely.
11. Remove ½ of the cushion and wash with DEPC water.
12. Remove the second half of the cushion, re-suspend the RNA in
133 μL DEPC water, and transfer to a 1.5 mL
microcentrifuge tube.
13. Add 2.5 vol absolute ethanol (EtOH), mix, and incubate at
20 C for a minimum of 2 h.
14. Centrifuge at 4 C for 30 min at 1000 rpm/10,732 g
(S0410 rotor) in a microcentrifuge.
15. Wash the RNA pellet in 0.5 mL 70% ethanol. Repeat the
centrifugation and re-suspend the pellet in 133 μL DEPC
water.
16. Precipitate overnight by adding 1/10 vol of 1 M NaCl and 2.5
vol of absolute EtOH.
17. Centrifuge at 4 C for 30 min at 10,000 rpm, wash with

0.5 mL 70% EtOH, air-dry at room temperature, and
re-suspend the pellet in 30–50 μL of DEPC water.
18. Store the RNA at 80 C.
The concentration and quality of the RNA is determined by
measuring the absorbance at both 260 nm and 280 nm. The purity
of RNA is estimated by the ratio of absorbance at 260–280 nm with
RNA absorbing at 260 nm and proteins absorbing at 280 nm. A
ratio of 1.8–2.0 indicates high purity.
Molecule Density (g/mL)

Protein 1.2–1.5
DNA 1.5–1.7
RNA 1.8–2.0
5 Silica Technology
Currently, the use of glass fiber filters (a.k.a. silica binding technol-
ogy) is the most popular method for small-scale RNA isolation and
is used in conjunction with guanidinium-based cell lysis. The silica
filter columns that are small enough for use with a standard micro-
centrifuge. These filters consist of glass microfibers that are posi-
tioned in the bottom of small plastic column that fits inside a
standard 1.5 mL microcentrifuge tube. The nucleic acid purifica-
tion and clean-up procedure is efficient and is performed in a
remarkably short time. In general, RNA (or DNA) binds to silica
in a high-salt, chaotropic environment. Such affinity-based nucleic
acid isolation is referred to as solid-state extraction. Following a
series of washes, the purified material is eluted from the silica matrix
under very low-salt conditions and in a very small elute (see Fig. 2).
Care should be taken to avoid any of the ethanol-containing wash
buffer in the eluted RNA, which will make the sample completely
useless for applications such as electrophoresis (the sample will float
out of the well, even after the addition loading buffer) and for any
enzymatic manipulation (due to enzyme inhibition).
For example, a protocol from a commercial kit (Thermo Scien-
tific GeneJET Viral DNA and RNA Purification Kit #K0821) for
extraction of viral RNA is detailed. This is a sample protocol for
RNA purification from 200 μL of EDTA- or citrate-treated plasma,
serum, blood or milk samples. Similar such protocols are available
from other manufacturers which could be referred for the use in
one laboratory.
Fig. 2 Silica-based purification
5.1 Step Procedure 1. Add 50 μL of column preparation liquid to the center of spin
column membrane, so that the membrane is entirely
moistened.
Notes:
(a) Before starting the procedure, each new spin column must
be prepared by treating it with Column Preparation Liq-
uid. Column treatment maximizes binding of the nucleic
acids to the membrane, resulting in more consistent
yields.
(b) Do not centrifuge the prepared column. The prepared
column should be stored at room temperature until it is
used for sample processing.
2. Load 200 μL of sample to an empty 1.5 mL
Add 200 μL of Lysis Solution (supplemented with Carrier
RNA) and 50 μL of Proteinase K, mix thoroughly by vortexing
or pipetting.
3. Incubate the sample for 15 min at 56 C in a thermomixer/
water bath. Leave thermomixer turned on for eluent preheat-
ing during later steps of the procedure.
4. Centrifuge for 3–5 s at full speed to collect any sample solution

from the inside of the lid.
Note: Supplement Lysis Solution with Carrier RNA prior
to use.
5. Add 300 μL of ethanol (96–100%) and mix by pipetting or
vortexing. Incubate the sample at room temperature for 3 min.
6. Centrifuge for 3–5 s at full speed to collect drops from the
inside of the lid.
7. Transfer the lysate to the prepared spin column preassembled
within the wash tube.
8. Centrifuge the column for 1 min at 6000 g.
9. Discard the wash tube containing flow-through.
10. Place the spin column into a new 2 mL wash tube.
Notes:
(a) Ensure that a new spin column has been prepared as
described in step 1.
(b) Close the bag with spin columns tightly after each use.
11. Add 700 μL of wash buffer 1 supplemented with ethanol to the
spin column.
spin column.
Note: Supplement concentrated wash buffer 2 with ethanol
prior to the first use.
spin column.
23. Centrifuge the column for 3 min at 16,000 g.
24. Discard the wash tube containing remaining flow-through.
25. Place the spin column into a new 1.5 mL elution tube.
26. Add 50 μL of eluent preheated to 56 C to the center of spin
column membrane.
27. Incubate for 2 min at room temperature.
28. Centrifuge the column for 1 min at 13,000 g.

29. Discard the spin column.
Note: Lower volume of eluent (30–40 μL) can be used in
order to concentrate eluted nucleic acids. Larger elution
volumes (up to 100 μL) can also be used but may result in
the dilution of the viral nucleic acid sample. Keep the elution
tube containing pure viral nucleic acids. Use the purified
nucleic acids immediately or store at 20 C or 70 C.
6 Affinity Matrices
In addition to the approaches described above for the isolation of

RNA using silica technology, there are products available which
capture polyadenylated transcripts/RNA. For example, mRNA iso-
lation kits with tracts of oligo(dT) covalently linked to a solid
support. In such cases the polyadenylated transcripts are captured
through canonical base-pairing between the poly(A) tail and the
oligo(dT) in a high-salt environment. The linkage material could be
cellulose, polystyrene, and/or latex beads. An older type of affinity
selection in which poly(A)1 mRNA was affinity-captured using a
column packed with poly(U) linked to sepharose beads [15] is no
longer favored because of the relatively poor binding capacity of
poly(U) matrices and the fact that quantitative recovery of RNA
from a poly(U) matrix usually required formamide-based elution
buffers. The nucleic acid species on the column is often referred to
as the bait, while the target that can bind to it are known as the prey.
7 Purification Using Magnetic Beads
Magnetic beads are made up of tiny (20–30 nm) particles of iron

oxides, such as magnetite (Fe3O4), which gives them super para-
magnetic properties. Super paramagnetic beads are different to
ferromagnets in a manner that they exhibit magnetic behavior
only in the presence of an external magnetic field, which enables
the beads to be separated in suspension, along with the target they
are bound to. Since they do not attract each other outside of a
magnetic field, they can be used without any concern about
unwanted clumping. The types of magnetic beads are based on
the different surface coatings and chemistries which give each
type of bead its own binding properties.
This extraction approach relies on using magnetic beads with a
coating that can bind nucleic acids reversibly by just adjusting
buffer conditions. After binding target nucleic acid, an external
magnetic field attracts the beads to the outer edge of the tube,
thus immobilizing them. While the beads are immobilized, the
Fig. 3 Magnetic bead-based purification
bead-bound nucleic acid is retained during the washing steps.

Adding elution buffer, and removing the magnetic field then
releases the nucleic acid as a purified sample, ready for quantifica-
tion and analysis.
This approach removes the need for vacuum or centrifugation,
which minimizes stress or shearing forces on the target molecules,
requires fewer steps and reagents than other extraction protocols.
An illustration is provided in Fig. 3.
8 Separation of Double-Stranded RNA from Single-Stranded RNA
There are viruses which houses double-stranded RNA such as

viruses belonging to family Reoviridae. Double-stranded RNA
can be isolated from cells and tissues by several different methods.
The use of conventional techniques for extraction and purification
of dsRNA from the infected cell culture is tedious and time-
consuming. Hence, double-stranded RNA is first isolated and pur-
ified with phenol-chloroform extraction followed by differential
lithium chloride (LiCl) precipitation to remove the single-stranded
RNA (ssRNA). This involves several sequential steps and takes
around 30–48 h to purify the dsRNA bluetongue virus (BTV) [16].
The procedure is based on differential solubility of different
types of nucleic acid in LiCl. LiCl offers major advantages over
other RNA precipitation methods in that it does not precipitate
DNA, protein or carbohydrate [17]. LiCl precipitation also
removes inhibitor of translation or cDNA synthesis from RNA
preparations [18].
A much quicker method, which requires only 24 h for purifica-

tion of dsRNA, has been described by Attoui et al. [19]. This
method is currently in vogue for purification of dsRNA of BTV
and other dsRNA viruses [20–23]. In this method the total RNA
pellet is dissolved in 100 μL of RNase-free water, mixed with an
equal volume of 4 M LiCl solution and incubated overnight at
4 C. This is followed by centrifugation at 18,000 g for 5 min
to precipitate ssRNA. The dsRNA in the supernatant is precipitated
at 20 C for 2 h by addition of 200 μL of isopropanol and 50 μL
of 7.5 M ammonium acetate, and pelleted by centrifugation at
18,000 g. the pellet is finally washed with 75% ethanol.
The dsRNA can also be purified by CF11 cellulose chromatog-
raphy [24]. This is a rapid method but requires column conditions
to be strictly followed and has not been in wide use.
Several authors have used nuclease such as RNaseT1for degra-
dation of ssRNA from a mixed population of ssRNA and DsRNA.
Several nucleases are available commercially which are used which
have been used as per suitability for differentiation, characteriza-
tion, and purification of RNA.
A protocol for extraction of double-stranded RNA [25] is
briefed as follows:
1. Infected cells were pelleted by centrifugation at 500 g for
10 min at 4 C.
2. Pellet was re-suspended in 1 mL of lysis buffer (1% SDS, 0.1 M
sodium acetate, pH 5.0) and incubated at 37 C for 30 min.
3. 100 μg/mL proteinase K (Final concentration) was added to it
and incubated at 56 C for 1 h.
4. The mixture was then centrifuged twice (12,000 g for
15 min at 4 C) with an equal volume of 3:2 phenol-
chloroform suspensions.
Note: phenol guanidinium method or commercially avail-
able combinations such as Trizol could also be used.
5. The aqueous phase was removed and mixed with two volumes
of ethanol and 0.3 M sodium acetate, pH 5.0 (final
concentration).
6. Precipitated at 70 C for 2 h.
7. Total RNA was recovered by centrifugation (12,000 g for
15 min at 4 C) and dissolved in 0.5 mL Tris-EDTA (TE,
10 mM Tris–HCl, pH 8.0, 1 mM EDTA).
8. Single-stranded RNA (ssRNA) was removed by precipitation at
4 C for 8 h with 2 M LiCl (Final concentration).
9. The supernatant, which contained dsRNA was precipitated
with 4 M LiCl (Final concentration) at 4 C for 8 h. The
dsRNA was recovered by centrifugation (12,000 g for
15 min at 4 C).
10. The pellet was washed with 75% ethanol and re-suspended in
20 μL TE by heating at 60 C for a few minutes.
9 Filter Paper Matrix Cards
Detection and characterization of viral pathogens from field sam-

ples owing to the instability of RNA molecules has always remained
challenging. Further the need of appropriate transport temperature
and media in resource poor settings mostly compromise the test
results. In recent developments filter paper-based system in speci-
men collection and transport is gaining momentum as it maintains
the integrity of the nucleic acid during sample transport and storage
[26]. In this process the test samples (blood, plasma, serum, cere-
brospinal fluid, tongue epithelium, etc.) is spotted onto the
specialized filter paper and allowed to dry. These specialized filter
papers are impregnated with chemical mixture that lyse the cells and
organelles and also inactivate the pathogenic virus upon contact.
The nucleic acid is entangled in the matrix and remains tightly
bound. The proteins including the ribonucleses are denatured
and thus allowing the long storage of the nucleic acid for longer
period. Literatures have cited several researches that have evaluated
these cards by storing the card at temperature ranging from 20 to
37 C (median 25 C) for period varying from 1 to 8 months
(median 30 days). At the laboratory, the viral RNA is retrieved
from the matrix depending upon the type of filter paper used.
The purified RNA is then used for molecular detection assays.
These specialized cards have been successfully used for molecular
detection of various RNA pathogens such as porcine reproductive
and respiratory syndrome virus (+ssRNA), avian influenza virus
(-ssRNA), measles (-ssRNA), foot and mouth disease virus
(+ssRNA), infectious bronchitis virus (+ssRNA), infectious bursal
disease virus (ds RNA), rabies virus (-ssRNA), west nile virus
(+ssRNA), etc. (Reviewed by in [27]). Several such filer papers
could be sourced from FTA® [(Flinders Technology Associates)
cards, GE Healthcare Life Sciences] which are most commonly
used [27]. The other filter paper matrix that has been used for
detection of infectious disease diagnosis include ADVANTEC
chromatography paper No. 526, ADVANTEC filter paper
No. 2, Whatman filter paper 903.
10 Storage of Purified RNA
The correct storage conditions for the purified viral RNA samples
have remained pivot for any study. Improper storage, over a period
of merely a few hours or as long as several months, is likely to have a
profound negative impact on the probable utility of purified RNA.
The key considerations that have to be taken into account is the

source of RNA and the timeline within which the RNA sample is to
be used and the solution/buffer in which the RNA has been
dissolved.
Purified RNA is most stable when stored as an ethanol precipi-
tate at 80 C. Under these conditions RNA can be stored confi-
dently for several months or even longer.
RNA species also show enhanced stability when stored for
weeks in hydrated form at 20 C. If the sample is to be used
within the week of purification, the RNA may be stored stably at
20 C, again as an ethanol precipitate.
The “clock begins ticking” when a purified sample of RNA is
dissolved in aqueous buffer, either sterile water or modified TE
buffer (10 mM Tris; 0.1 mM EDTA; overall pH 7.5). TE buffer has
the advantage of being able to chelate the magnesium ions that are
often carried over from certain isolation procedures, and the low
EDTA concentration is unlikely to cause problems in downstream
PCR procedures. The low concentration of EDTA can prevent
magnesium-induced strand breakage in later steps of an isolation
procedure, particularly at elevated temperatures. The most suited
action is to determine the RNA concentration and then store the
remaining RNA sample in suitable aliquots at 80 C.
Conventionally, RNA has also been stored in varying concen-
trations of SDS, VDR, and ultrapure formamide. However, the
incomplete removal of these “protectant” compounds will reduce
the utility of the RNA in most downstream applications. Other
options for long-term storage of RNA include highly purified
100% formamide [28], a commercially available stabilized form of
formamide known as FORMAzol (Molecular Research Center,
Cincinnati, OH). Some authors have found the use of FORMAzol
very promising for preserving RNA and have successfully demon-
strated the stability up to 2 years at 20 C. RNAstable (Biomatrica,
San Diego, CA) is suitable for long-term storage or shipping of
purified samples of RNA. The manufacturers claim that RNA stable
can preserve the total RNA/ mRNA/ miRNA for 12 years at
ambient temperature and thereby they can eliminate sample loss
due to freeze/thaw cycles [29].
References
1. Wink M (2006) An introduction to molecular methods in biology and medicine, 2nd edn.
biotechnology: molecular fundamentals, meth- CRC Press, Boca Raton, FL
ods and application in modern biotechnology. 4. Buckingham L, Flaws ML (2007) Molecular
Wiley-VCH, Weinheim diagnostics: fundamentals, methods, & clinical
2. Tan SC, Yiap BC (2009) DNA, RNA, and applications. F.A. Davis, Philadelphia, PA
protein extraction: the past and the present. J 5. Amani P, Hofmann A (2018) Basic
Biomed Biotechnol principles. In: Hofmann A, Clokie S (eds) Wil-
3. Cseke LJ, Kaufman PB, Podila GK, Tsai C-J son and Walker’s principles and techniques of
(2004) Handbook of molecular and cellular biochemistry and molecular biology.
Cambridge University Press, Cambridge, pp method for isolation of intact, translationally

8 – 3 9 . h t t p s : // d o i . o r g / 1 0 . 1 0 1 7 / active ribonucleic acid. DNA 2:329–335
9781316677056.004 19. Attoui H, Billoir F, Cantaloube JF, Biagini P,
6. Farrell RE (2017) RNA isolation strategies. In: de Micco P, de Lamballerie X (2000) Strategies
RNA methodologies, 5th edn. Academic Press, for the sequence determination of viral dsRNA
pp 75–115. https://doi.org/10.1016/B978- genomes. J Virol Methods 89(1–2):147–158
0-12-804678-4.00003-8 20. Potgieter AC, Steele AD, Van Dijk AA (2002)
7. Chirgwin JM, Przybyla AE, MacDonald RJ, Cloning of complete genome sets of six dsRNA
Rutter WJ (1979) Isolation of biologically viruses using an improved cloning method for
active ribonucleic acid from sources enriched large dsRNA genes. J Gen Virol 83(9):
in ribonuclease. Biochemistry 18:5294–5299 2215–2223
8. Cox RA (1968) The use of guanidinium chlo- 21. Singh KP, Maan S, Samuel AR, Rao S, Meyer
ride in the isolation of nucleic acids. In: AJ, Mertens PPC (2004) Phylogenetic analysis
Grossman L, Moldave K (eds) Methods in of bluetongue virus genome segment 6 (encod-
enzymology, vol 12. Academic Press, Orlando, ing VP5) from different serotypes. Vet Ital
FL, pp 120–129, part B 40(4):479–483
9. Gordon JA (1972) Denaturation of globular 22. Potgieter AC, Page NA, Liebenberg J, Wright
proteins. Interaction of guanidinium salts IM, Landt O, Van Dijk AA (2009) Improved
with three proteins. Biochemistry 11: strategies for sequence-independent amplifica-
1862–1970 tion and sequencing of viral double-stranded
10. Nozaki Y, Tanford C (1970) The solubility of RNA genomes. J Gen Virol 90(6):1423–1432
amino acids, diglycine, and triglycine in aque- 23. Maan S, Rao S, Maan NS, Anthony SJ, Attoui
ous guanidinium hydrochloride solutions. J H, Samuel AR, Mertens PP (2007) Rapid
Biol Chem 245:1648–1652 cDNA synthesis and sequencing techniques
11. Chomczynski P (1993) A reagent for the for the genetic study of bluetongue and other
single-step simultaneous isolation of RNA, dsRNA viruses. J Virol Methods 143
DNA, and proteins from cell and tissue sam- (2):132–139
ples. BioTechniques 15:532–536 24. Herring AJ et al (1982) Rapid diagnosis of
12. Majumdar D, Avissar YJ, Wyche JH (1991) rotavirus infection by direct detection of viral
Simultaneous and rapid isolation of bacterial nucleic acid in silver-stained polyacrylamide
and eukaryotic DNA and RNA: a new approach gels. J Clin Microbiol 16(3):473–477
for isolating DNA. BioTechniques 11(1): 25. Chomczynski P (1992) Solubilization in form-
94–101 amide protects RNA from degradation.
13. Chomczynski P, Sacchi N (1987) Single-step Nucleic Acids Res 20:3791–3792
method of RNA isolation by acid guanidinium 26. Biswas SK, Chand K, Pandey AB, Mondal B
thiocyanate-phenol-chloroform extraction. (2016) Experimental protocol for quick extrac-
Anal Biochem 162:156–159 tion of double-stranded rna (dsRNA) of blue-
14. Ott T, Krusell L, Udvardi M (2005) RNA iso- tongue virus (BTV) from infected BHK-21
lation using CsCl gradients. In: Lotus japoni- cells with ribonuclease T1 (RNAse T1) at dif-
cus handbook. Springer, Dordrecht, pp ferent concentrations. Anim Sci 10(1)
125–128 27. Smith LM, Burgoyne LA (2004) Collecting,
15. Lindberg U, Persson T (1974) Messenger archiving and processing DNA from wildlife
RNA isolation with poly(U) agarose. Methods samples using FTA® databasing paper. BMC
Enzymol 34:496–499 Ecol. 4, 4
16. Squire KRE, Chuang RY, Osburn BI, Knudson 28. Cardona-Ospina JA, Villalba-Miranda MF,
DL, Doi RH (1983) Rapid methods for com- Palechor-Ocampo LA, Mancilla LI, Sepúl-
paring the double-stranded RNA genome pro- veda-Arias JC (2019) A systematic review of
files of bluetongue virus. Vet Microbiol 8(6): FTA cards® as a tool for viral RNA preserva-
543–553 tion in fieldwork: Are they safe and effective?.
17. Barlow JJ, Mathias AP, Williamson R, Gam- Prev Vet Med 172:104772
mack DB (1963) A simple method for the 29. Stevenson HS, Wang Y, Muller R, Edelman
quantitative isolation of undegraded high DC (2015) Long-term stability of total RNA
molecular weight ribonucleic acid. Biochem in RNAstable® as evaluated by expression
Biophys Res Commun 13:61–66 microarray. Biopreserv Biobank 13(2):
18. Cathala G, Savouret J, Mendez B, West BL, 114–122. https://doi.org/10.1089/bio.
Karin M, Martial JA, Baxter JD (1983) A 2014.0068
Chapter 5
Multiplex PCR for Diagnosis of Porcine Diseases

Manjisha Choudhury, Ajay Kumar Yadav, Seema Rani Pegu,
Rajib Deb, and Swaraj Rajkhowa
Abstract
Molecular diagnostics have revolutionized the efficiency of diagnosis of infectious diseases. An accurate
diagnosis of a disease would prevent further infestation of infections in the healthy population. Although
several laboratory techniques exists such as cell-culture, serological, and molecular based methods that were
developed in the past decades for highlighting these diseases, however, because of their robustness, high
sensitivity, specificity, rapidness, and suitability of the range of types of samples that can be analyzed, nucleic
acid amplification tests are suitable for the diagnosis of pathogenic infections. Since the development of
polymerase chain reaction (PCR), this molecular technique has been widely utilized as a nucleic acid
amplification tool and besides being utilized as a laboratory tool, it has proved to have an exceptional
potential in clinical applications, including detection of specific or broad spectrum pathogens, evaluation of
emerging novel infections, disease surveillance, early threat of bio-threat agents, and antimicrobial resis-
tance profiling. The multiplex PCR is the technique where we can simultaneously detect more than one
organism in a single reaction. The multiplexing may be done either with conventional PCR or with real time
PCR. This technique provides detection of mixed infection including both viruses and bacteria, time saving
and cost effective.
Key words Molecular diagnostics, Polymerase chain reaction (PCR), Multiplex PCR, Multiplex
qPCR, Mixed infections, Rapidness
1 Introduction
Molecular diagnostics have revolutionized the efficiency of diagno-

sis of infectious diseases. An accurate diagnosis of a disease would
prevent further infestation of infections in the healthy population.
Although several laboratory techniques exists such as cell-culture,
serological, and molecular based methods that were developed in
the past decades for highlighting these diseases, however, because
of their robustness, high sensitivity, specificity, rapidness, and suit-
ability of the range of types of samples that can be analyzed, nucleic
acid amplification tests (NAATs) are suitable for the diagnosis of
pathogenic infections. Since the development of polymerase chain
67
68 Manjisha Choudhury et al.
reaction (PCR) in 1985, this molecular technique has been widely

utilized as a nucleic acid amplification tool and besides being uti-
lized as a laboratory tool, it has proved to have an exceptional
potential in clinical applications, including detection of specific or
broad spectrum pathogens, evaluation of emerging novel infec-
tions, disease surveillance, early threat of bio-threat agents and
antimicrobial resistance profiling. The initial application of PCR
for diagnosis of porcine diseases dates back to 1989, reporting the
wide applicability of PCR in detection of Shiga-like toxin produc-
ing Escherichia coli (E. coli) [1], verocytotoxin-producing E. coli in
diseased pigs [2], pseudorabies virus from acutely diseased as well as
latently infected pigs [3, 4], Porcine Haemophilus pleuropneumonia
infection [5], Porcine respiratory Coronavirus infection [6], foot
and mouth disease (FMD) virus in porcine tissues [7], Porcine
Parvovirus (PPV) infections [8], porcine rotavirus G types [9],
porcine group A rotavirus [10], porcine reproductive and respira-
tory syndrome virus (PRRSV) infections [11].
Although it has been three decades since its development, PCR
is regarded as a gold standard and continues to be the technique of
choice for nucleic acid amplification both as analytical and as a
diagnostic tool. However, a number of modifications of the con-
ventional PCR technique have been developed; one of the variant
of this classic technique is the multiplex PCR. The first report of
application of multiplex PCR in diagnosis of inherited genetic
disease was described in 1988 for detection of majority of deletions
in the Duchenne muscular dystrophy (DMD) gene in humans [12].
This chapter will highlight the parameters critical in developing
a multiplex PCR, troubleshooting of commonly encountered pro-
blems during optimization, strategic actions to overcome such
problems and highlight application of multiplex PCR in diagnosis
of various porcine diseases.
2 Principle of Multiplex PCR
Multiplex PCR (M-PCR) is the technique of simultaneous amplifi-

cation of more than two target sequences in a single reaction using
more than two primer pairs.
A multiplex PCR is a modification of the conventional single-
plex PCR. In a conventional singleplex PCR, one single target
sequence is amplified in a single reaction tube, whereas a multiplex
PCR facilitates the simultaneous amplification of multiple target
sequences in a single reaction tube (Figs. 1 and 2 illustrate the
technique of conventional PCR and multiplex PCR).
There are two variants of multiplex PCR. The first variant of
multiplex PCR includes a single template PCR reaction. This tech-
nique utilizes a single template which could be a genomic DNA or a
complementary DNA (cDNA) in a single reaction tube alongwith
Multiplex PCR for Diagnosis of Porcine Diseases 69
Fig. 1 Conventional PCR
multiple primer pairs to amplify multiple targets within the tem-

plate (Fig. 2a). An example of a report described the rapid detection
of enteric pathogens by multiplex PCR in porcine feces from a
single DNA template extracted from porcine feces [13]. Fig. 3
illustrates a multiplex PCR developed by [13], designed to amplify
a 655 base pair (bp) portion of the Lawsonia intracellularis 16S
rRNA gene, a 354 bp portion of the Brachyspira hyodysenteriae
NADH oxidase gene, and a 823 bp portion of the Brachyspira
pilosicoli 16S rRNA gene.
The second variant of multiplex PCR includes multiple tem-
plate PCR reaction. This technique utilizes multiple templates in a
single reaction tube alongwith multiple primer pairs to amplify
specific targets in the template (Fig. 2b). An example of a report
that described the application of multiplex PCR in clinical applica-
tion for detection of major swine viruses in pigs with multiple
infections, considered a range of samples, viz. diarrheic stool sam-
ples, aborted fetus organs, and principal organs collected from
post-weaning multisystemic wasting (PMWS)—suspected pigs
[14]. Fig. 4 illustrates a multiplex PCR developed by [14] detecting
Fig. 2 Multiplex PCR

Fig. 3 Multiplex PCR for rapid detection of enteric pathogens in porcine feces in
agarose gel showing the relative sizes of PCR products from three bacterial
pathogens: P—Brachyspira pilosicoli, L—Lowsonia intracellularis, H—Brachy-
spira hyodysenteriae, M—molecular mass markers, Lane 1—negative control,
Lane 2—B.hyodysenteriae strain B78, Lane 3—B. pilosicoli strain P43/6/78,
Lane 4—L. intracellularis strain 2189/94, Lanes 5–10—field samples
Fig. 4 Multiplex PCR for detection of six swine viruses in agarose gel showing the
relative sizes of PCR products in lane 1
six viruses PRRSV, Japanese encephalitis virus (JEV), Getah virus

(GETV), transmissible gastroenteritis virus (TGEV), porcine epi-
demic diarrhea virus (PEDV), and Porcine Rotavirus A (PoRV-A).
The most important criteria for amplification of more than two
targets in a single reaction is dependent on the compatibility of the
primer pairs used in the reaction. An essential requirement for all
the primers in a multiplex PCR is to have similar melting tempera-
tures (Tm) for efficient annealing and subsequent dissociation of the
primers from their complementary target sequences at approxi-
mately the same temperature, permitting amplification of each
target to occur at that particular temperature and time. For exam-
ple, a multiplex will not at all work in case one pair of primer anneals
to the target sequence at that instant when another pair of primer is

dissociating from its respective target sequence. It is, therefore, due
to this reason that designing of all the primer pairs for a multiplex
PCR follows the criteria of having difference of their Tms between
few degrees to each other.
Another important aspect of designing a multiplex PCR is such
that relative sizes of the target amplicons are visually identifiable
and distinguishable from each other on a gel electrophoresis sys-
tem. For example, two amplicons of a multiplex PCR having a
minimum difference of approximately 43–50 bp would be easily
distinguishable on an agarose gel. [14] developed a multiplex PCR
for major porcine viruses, reporting an amplicon size of 160 bp for
GETV detection and 203 bp for PPV detection on agarose gel.
3 Optimization of a Multiplex PCR
Most of the parameters and strategies used for developing a con-

ventional singleplex PCR [15] would be helpful for developing a
multiplex PCR assay. However, some additional parameters are to
be considered that play a critical role in optimizing a multiplex PCR
for efficient detection of multiple targets.
The primary challenge while optimizing a multiplex PCR is the
elimination of the formation of primer dimers. Primer dimers are
basically short undesirable products that are generally formed at the
room temperature prior to the first round of PCR amplification.
The formation of these primer dimers are due to annealing between
primers that possess 30 end complementary overlappings of two or
more nucleotides and the abundant availability of the polymerase
enzyme prior to the first round of PCR amplification aids in the
extension of these annealed primers to amplify such short untar-
geted amplicons. Therefore, the main requirement to eliminate
such primer dimers is due to the fact that these short untargeted
products are efficiently amplified and they effectively and equally
compete with the amplification of the desired targets thereby
impacting the overall efficiency of a multiplex PCR. A point to be
noted here is that with the increase in the number of targets of a
multiplex PCR assay, the chances of accumulation of primer dimers
also increases as the chances of a number of primers to possess
complementary overlappings of two or more nucleotides at their
30 ends also significantly increases.
Another challenge while optimizing a multiplex PCR is the
reproducibility of the assay in maintaining the target specific ampli-
fications and obtaining equivalent yields of each of the targets.
A detailed analysis of each of the parameters to be considered
has been discussed in the following sections that will aid in optimiz-
ing a multiplex PCR assay.
3.1 Designing of Ideally it is a pre-requirement that all the primers designed for a
Primers multiplex PCR assay should have nearly similar Tms or a maximum
Tm difference of 5 to 6.5 degrees among the primers so that a
single annealing temperature would be optimal for all the primer
pairs to amplify their specific targets within a single reaction. For
example, Table 1 lists five pairs of primers used in a bacterial
multiplex PCR for detection of virulence associated genes of Pas-
teurella multocida from pigs developed in our laboratory [16]. The
Tm of the primers designed has a range of 49.9–56 degrees with a
maximum difference of 6.4 degrees between them and a minimum
difference of 0 degrees between them. Most of the primers
designed have Tm in and around 55–56 degrees facilitating the
optimization of the annealing temperature of the assay efficiently at
56 degrees.
The primer pairs of a multiplex PCR should have nearly similar
amplification efficiencies for their respective targets. This could be
achieved when the designed primers have an optimal length of
18–30 bp, a GC content of 35–60% and do not possess any signifi-
cant homology either internally or among one another. For exam-
ple, the primers listed in Table 1 show an uniformity in their length
of 20 bases, maintaining a GC content of 35–50% and 50% in
majority of the primers and do not show presence of any secondary
structure among them.
In order to prevent preferential amplification of shorter pro-
ducts over larger ones, the amplicon size range between the smal-
lest and the largest amplicons should not exceed 500–600 bp.
Another example showing the properties of primer pairs of two
independent multiplex PCRs developed in our laboratory for
one-step multiplex PCR for rapid and simultaneous detection of
three porcine viruses, viz. Porcine Circovirus type 2, Porcine Par-
vovirus, and Classical Swine Fever virus and multiplex PCR for
simultaneous detection of N, M, and GP5 genes for diagnosis of
PRRSV have been listed in Tables 2 and 3, respectively.
While designing the primers, the probable candidate sequences
can be evaluated using an online Oligo analyzer tool (e.g. OligodT
analyzer), to screen presence or absence of any secondary structures
such as hairpin formations within the candidate sequences and
probability of formation of any dimers. In addition, a thorough
homology search (e.g. BLAST) of the candidate sequences would
ensure prevention of any non-specific primer annealing to pseudo-
genes or partially homologous nucleotide sequences.
3.2 Reaction Every component of a PCR reaction mix contributes to successful

Component amplification of the targets. Therefore, optimization of these indi-
Optimization vidual components may be needed for improvement in sensitivity
or specificity of the assay.
(a) Concentration of Magnesium chloride (MgCl2)
Table 1
Properties of primers used in a bacterial multiplex PCR assay for rapid detection of virulence
associated genes of Pasteurella multocida from pigs [16]
GC
Target Length content Melting Amplicon

genes Primer Sequence (bp) % temperature (Tm) C size (bp)
ompH F: CTGGTTTAGCGCTTGGTG 20 50 56 242
TT
R: TCTACCCCAAGCTGCTT 20 50 56.3
CAA
ompA F: AGCGCGTAGATTACA 20 50 55.9 350
GACCA
R: GTGACCTGTTGCGCTGA 20 55 56
TAG
plpB F: CCAAAATTGCGAAG 20 35 49.9 443
GAAAAA
R: CGCGAAATCGACAT 20 45 52.4
CATCTA
hgbA F: AAGTCGCTAAAATCGC 20 40 52.8 548
GAAA
R: ATCCCAAAATGGCGTAA 20 45 53.3
CAG
pfhA F: TTTAGCGGGGAGTT 20 50 55.7 753
CAGCTA
R: GTGACATCGCCGG 20 50 55.4
TAACTTT
Table 2
Properties of primers used in one-step multiplex PCR for rapid and simultaneous detection of three
porcine viruses

Sl. no. Target viruses Length (bp) GC content % Tm C Amplicon size (bp)
1. PCV-2 forward 20 45 54.1 553
PCV-2 reverse 20 45 53
2. PPV forward 20 55 57.4 326
PPV reverse 21 52.4 56.9
3. CSFV forward 20 50 55.1 171
CSFV reverse 20 55 55.4
The concentration of magnesium chloride in the reaction

mix and the annealing temperature of the primers play a vital
role in specificity and yield of the products. 1.5 mM MgCl2 is
the recommended concentration for a standard PCR
Table 3
Properties of primers used in a multiplex PCR for simultaneous detection of three genes for diagnosis
of PRRSV

Sl. no. Target genes Length (bp) GC content % Tm C Amplicon size (bp)
1. N—Forward 24 48 58.9 372
N—Reverse 22 49.1 59.6
2. M—Forward 21 52 59.2 525
M—Reverse 26 53.9 52.7
3. ORF5—Forward 21 57 57.9 603
ORF5—Reverse 22 59 57.9
Table 4
Detailed concentration of each reaction components optimized for most of the multiplex PCR
Reaction Component (Amount/concentration) per 25 μl reaction

10 PCR buffer 2.5 μl
MgCl2 2 mmol/l
Each dNTP 0.2 mmol/l
Taq DNA polymerase 1U
Forward primers 20 pmol
Reverse primers 20 pmol
Template DNA 3 μl
[17]. However, it has been reported that the efficiency of

certain multiplex amplifications was found to be enhanced
from increased concentration of MgCl2 in the range of
3–10 mM [18] and 1.8–10.8 mM [17]. Therefore, an optimal
concentration of MgCl2 for a reaction needs to be standar-
dized since an addition of suboptimal concentrations of
MgCl2 may result in high levels of non-specific amplification
and reduced product yield. Table 4 may be referred for
detailed concentration of each reaction components opti-
mized for most of the multiplex PCR working efficiently in
our laboratory.
(b) Concentration of Deoxynucleoside Triphosphates (dNTPs)
The concentration of dNTPs in a reaction is of significance
since dNTPs bind to divalent cations (magnesium ions) quan-
titatively, therefore variation in the concentration of dNTPs
would affect the optimal concentration of MgCl2 as well.
DNA Polymerases also require free magnesium ions besides
Mg2+ ions bound to dNTPs. Therefore, a suboptimal increase
Table 5
Three examples of polymerases used in multiplex PCRs
DNA polymerase Concentration of buffer pH of buffer Concentration of KCl salt

AmpliTaq gold 15 mM Tris–HCl 8.0 50 mM KCL
Stoffel CM 10 mM Tris–HCl 8.0 10 mM KCL
Taq polymerase (Fermentas) 20 mM Tris–HCl 8.4 50 mM KCL
in the concentration of dNTPs in a reaction would inhibit any

amplification of the targets. For example, for a standard PCR
reaction, the recommended concentration of MgCl2 is
1.5 mM at dNTP concentrations of around 200 μM each.
(c) Buffer and Salt
The optimal concentration of buffer and its pH and the
concentration of KCl is dependent on the DNA Polymerase
being used in the reaction. Table 5 lists three examples of
polymerases used in multiplex PCRs, showing optimal activity
in their respective buffer and salt concentrations.
It has been reported that generally buffers with lower salt
concentrations work better for primer pairs amplifying longer
targets and buffers with higher salt concentrations work better
for primer pairs amplifying shorter targets [17].
(d) Concentration of Primer
The concentration of each primers used in multiplex PCR
reactions generally ranges from 100 to 400 nM but these
concentrations may vary between targets due to their differ-
ence in priming efficiencies (Fig. 5). While developing multi-
plex PCRs in our laboratory, initially an equimolar
concentration of each primer is used in the range
100–200 nM under multiplex conditions to determine equiv-
alent yields of all the specific targets. In certain instances, we
have also encountered uneven amplification with respect to
one or two targets of a multiplex assay and such targets
showed faint amplifications in comparison to other targets of
the same multiplex assay under the same optimized cycling
conditions. In such cases, altering the proportions of various
primers of a reaction, by increasing the concentrations of the
weakly amplifying primers and decreasing the concentration of
the strongly amplifying primers was observed to overcome
such problems. Another strategy to overcome such problem
is by doubling the concentration of the weakly amplifying
primers of a reaction alone.
(e) DNA Polymerase
The enzymes used more often for multiplex PCR are
recombinant thermostable DNA polymerase from Thermus
Fig. 5 Effect of concentration of primer variation on a multiplex PCR assay. Lane

1 depicts no amplification due to non-optimal primer concentration, Lane 2–4
depicts effect of varying concentrations of primer pairs
aquaticus (e.g. AmpliTaq DNA Polymerase), Stoffel fragment

of Taq DNA polymerase (e.g. Stoffel CM), chemically mod-
ified version of AmpliTaq DNA polymerase, (e.g. AmpliTaq
Gold), DreamTaq DNA Polymerase (Fermentas), Hot start
polymerase (e.g. SapphireAmp Fast PCR master mix), Phu-
sion high-fidelty master mix, Q5 Hot Start High Fidelty DNA
polymerase, Multiplex PCR enzymes (Takara Bio.).
The concentration of the enzyme used for optimization of
multiplex PCRs is 5–10 units per 100 μl reaction. The most
effective concentrations of the enzyme has been reported to be
0.4 μl or 2 U/25 μl reaction [17], 0.25 μl/50 μl reaction [14],
1 U/25 μl reaction [16].
(f) Amount of template
A template concentration of 100 ng of bacterial or viral
DNA or cDNA is more than sufficient for initial optimization
of multiplex PCR assays. A common error that is committed in
optimizing is addition of excess amount of template to a
reaction which can inhibit a PCR, resulting in a smear-like
pattern when visualized on an agarose gel or show non-specific
amplification (Fig. 6). An assay can be optimized to such an
extent to be efficient enough to amplify targeted products
from a minimum amount of 450 pg of viral genomic DNA
or RNA as a starting template [19].
Fig. 6 Gel photo of excess addition of templates. Lane 3 depicts smear-like

patter due to excess amount of template added in PCR reaction
(g) Reaction volume

The total volume of a multiplex PCR reaction could range
from a minimum of 10 μl to a maximum volume of 100 μl.
The choice of the reaction volume depends on number of
pathogens or genes targeted for a multiplex PCR assay and
the optimization based on the cost of the reagents and the
DNA polymerase consumed.
(h) Use of adjuvants
Certain additives although optional to use in a PCR reac-
tion mix has been recommended in various reports [17, 20,
21] in order to improve the efficiency of amplification, sensi-
tivity, and specificity of a multiplex PCR assay. Table 6 lists the
examples of such adjuvants and the concentrations used for in
multiplex PCR assay.
For example, Fig. 7 reveals a comparative difference in the
detection of three genes of PRRSV on using 5% DMSO while
developing a multiplex PCR in our laboratory.
There are also ample of commercially available PCR mas-
ter mixes that are widely used in the development of multiplex
PCRs thus, saving time and reducing the cumbersome energy
and effort for optimization of each and every reaction compo-
nents of a PCR mix.
Table 6
Adjuvants and the concentrations used for in multiplex PCR assay
Adjuvants Concentration
Dimethylsulfoxide (DMSO) 5%
Glycerol 5%
Bovine serum albumin (BSA) 0.8 μg/μl
Betaine 1M
Fig. 7 Effect of 5% DMSO in detection of three genes of PRRSV. Lane 1 depicts

multiplex PCR products without addition of DMSO, Lane 2 depicts the effect of
adding 5% DMSO resulting in three distinct products of multiplex PCR
4 Multiplex PCR Troubleshooting
There are many problems or difficulties that could be encountered

while optimizing and developing a multiplex PCR.
1. There could be preferential amplification of certain specific
targets in comparison to the other targets of the multiplex
assay.
2. The assay could be poorly sensitive or specific.
3. There could be spurious amplified products due to the forma-
tion of primer dimers.
4. The primer pairs of the multiplex assay may not have similar
amplification efficiencies for their respective targets.
5. There could be inhibitors of Taq polymerase enzyme present in

the clinical samples that would prevent the detection of specific
targets.
Optimizing the multiplex PCR with the primer pairs may yield
multiple non-specific products which may mask the amplification of
the desired products, impacting the overall sensitivity and specific-
ity of the assay. Such non-specific PCR products can be strategically
eliminated partially or completely by increasing the annealing tem-
perature of the primer pairs which would increase the efficiency of
annealing of primer to its target and prevent the formation of
primer dimers. Following that, it may be necessary to increase or
decrease the concentration of magnesium chloride (MgCl2) in the
reaction mix, or to lower the concentration of primer pairs in the
reaction mix, or to add certain additives to the reaction mix, such as
DMSO, BSA, betaine, or glycerol, if the annealing temperature is
increased. The temperature at which the primer pairs are annealed
does not remove non-specific products.
Certain substances may be present in the clinical samples which
act as inhibitors of Taq polymerase enzyme, thereby inhibiting the
detection of pathogens by PCR. These substances are generally
proteinaceous in nature and stall the activity of the enzyme by
modifying the quaternary structure of the enzyme or by blocking
the active site of the enzyme [21]. For example, salts such as
oxalates and urea, heparin, urine, fecal, semen, hair, and iron-
containing compounds such as hemoglobin in blood, serum, or
plasma can inhibit the activity of the enzyme. Therefore, these
samples require prior treatment of deproteination with organic
solvents such as phenol or chloroform.
Preferential strategies such as “hot-start” or use of uracil
N-glycosylase alone or combination of both is known to enhance
the specificity of the reaction. The hot-start protocol is basically
increasing the reaction temperature to a temperature (above
80 degrees or at 95 degrees) higher than the Tm of the primers
and then subsequently adding the Taq polymerase enzyme or pri-
mers to the reaction tube prior to the initiation of the multiplex
PCR program, to enhance annealing of primers to its specific target
sequences in the template(s) and this would eliminate non-specific
amplification and primer dimers. The use of enzymatic method
such as uracil N-glycosylase has been reported to minimize PCR
product carryover and enhance the sensitivity of the PCR [22].
During troubleshooting, an initial strategy would be to ensure
that the corresponding singleplex PCRs of the targets have been
successfully working fine but it is the multiplex reaction that is not
successfully giving the desired specificity, results and/or yield.
Based on our laboratory experiences while developing multiplex
PCR and literature reports [18, 21], Table 7, therefore, lists the
commonly encountered problems after visualization on gel electro-
pheresis while developing a multiplex PCR assay, the possible
causes of these problems and the recommended strategies and
solutions to overcome these problems.
5 Key Pointers for Multiplex PCR Development
1. The developed multiplex PCR should be robust, sensitive, and

rational for detection of specific pathogens or targets in the
assay.
2. The primer pairs designed should efficiently and accurately
detect the targeted pathogens without raising any ambiguity
of presence or absence of the pathogens and the amplicons
should be easily interpretable in the detection system.
3. The sensitivity and specificity aspect of the multiplex assay
needs to be evaluated prior to any clinical application as a
diagnostic tool and should be supported with a well-
documented comparison of their optimization with their
corresponding singleplex PCRs by serial dilutions of the target
template(s) as well as clinical sample(s).
4. Strategies and precautions should to be considered to prevent
false negative results due to reaction failure.
5. Validation of the assay should be confirmed using external or
internal control known targets in addition to the unknown
samples being screened to indicate reaction failure.
6 Evaluation as a Potential Diagnostic Tool
Any developed assay to be proposed as a potential diagnostic tool

involves comparison of its performance to a gold standard tech-
nique in terms of its percentage sensitivity and specificity. Analytical
specificity is the ability of the assay to accurately distinguish
between targeted and non-targeted pathogens or genes. Analytical
sensitivity refers to the minimum concentration of a pathogens or
targets that can efficiently amplified by the assay. The sensitivity of
an assay is expressed as pg or fg of template or as copy numbers of
template by performing serial ten-fold dilutions of the template(s).
Multiplex PCRs having sensitivity up to 0.1 fg template or 1–5
copies of template detection have been reported [23, 24].
Table 7
82
Troubleshooting of Multiplex PCR
Sl.
no. Common problem Possible causes Recommended solutions
1. Problems due to carryover contamination Residual or carryover templates or reaction The routine use of autoclaved filter tips while
observed components may be accumulated on pipettes setting up PCR reactions is mandatory
Pipettes needs to be UV sterilized prior to use
and after use
Pre- and post-sterilization of work benches and
glasswares needs to be followed
Specimen handling, PCR setup and amplicon The usage of four separate designated
Manjisha Choudhury et al.
detection performed in the same work bench laboratory/rooms/areas needs to be

and in the same room followed
1. Sample or template handling and
preparation
2. Setting up of PCR mix
3. Addition of template(s)
4. Amplicon detection
2. PCR assay initially was working successfully with The routine use of both positive and negative
the designed primer pairs but suddenly shows controls needs to be followed to ensure the
non-specific with a different set of the same assay is valid
primer pairs or with a new batch of the same
primer pairs
3. No PCR products observed at all Enzyme activation is not complete The cycling parameters and pre-PCR heat
activation of chemically modified enzymes
needs to be checked
One or more component(s) have been left out of A new reaction mix needs to be prepared
the reaction
Inhibitors may be present in the template(s) Alternate extraction procedure needs to be
implemented or extraction of template
(s) needs to be repeated on a sterile work
bench
Product may be degraded due to UNG activity The PCR product needs to be kept on ice
immediately after completion of PCR
4. Low yield of all products observed Incomplete amplification may have occurred Pre-PCR enzyme activation time needs to be
increased
Cycle number of PCR needs to be increased
Concentration of enzyme needs to be increased
Concentration of MgCl2 needs to be increased
pH of Tris–HCl buffer may be too high pH of Tris–HCl buffer needs to be decreased to
(sub-optimal activation of the chemically 8.0
modified enzymes)
Concentration of the primers may be too low Concentration of the primers needs to be
increased
The annealing temperature may be too high The annealing temperature needs to be
decreased
The template(s) may be insufficient for the The concentration and the quality of the
reaction or have been degraded template to be used in the reaction needs to
be checked
5. Low yield of one of the products is observed The concentration of the primer pair specific to The concentration of the primer pair specific to
the low yield PCR product may be too low the low yield PCR product needs to be
increased
The primer pair specific to the low yield PCR The primer pair needs to be redesigned in case
product may be poorly designed of necessity
Secondary structure may be present in the Alternate enzymes may be utilized
template(s) (e.g. DreamTaq, sapphire, Phusion)
dUTP may be incorporated inefficiently Some TTP may be added to the reaction
The concentration of the enzyme needs to be
increased
The annealing temperature may be too high The annealing temperature needs to be
decreased
6. Low yield or loss of one or more large fragments There could be preferential amplification of The primer concentration of the large PCR
may be observed smaller products products needs to be increased
The concentration of the enzyme may be too low The concentration of the enzyme may be
increased
The extension time may be too short The extension time may be increased
Multiplex PCR for Diagnosis of Porcine Diseases
The template may be degraded The quality of the template needs to be checked
The concentration of the template may be too The concentration of the template needs to be
low checked
83
(continued)
84
Table 7
(continued)
Sl.
no. Common problem Possible causes Recommended solutions
7. High background and non-specific PCR The concentration of the enzyme may be too The concentration of the enzyme needs to be
products observed high decreased
Manjisha Choudhury et al.
Presence of too much enzyme at early cycles Hot-start protocols needs to be preferred (hot
start manually or use of simplified hot-start
enzymes)
The pre-PCR heat cycle may be too long The pre-PCR heat activation of chemically
modified enzymes needs to be decreased
The concentration of MgCl2 may be too high The concentration of MgCl2 needs to be
decreased
The primers may be poorly designed The primers needs to be redesigned
There could be sub-optimal amount of template The concentration of the template to be used in
used in the reaction the reaction needs to be checked
The annealing temperature could be too low The annealing temperature needs to be
increased
Certain additives such as DMSO, glycerol, BSA,
betaine is needed to be added to avoid
non-specific amplification
Preparation of reaction mix needs to be
performed in ice to prevent annealing of
primers to non-complementary sequences at
room temperature
7 Advantages of Multiplex PCR
1. Analysis of multiple targets in a single assay

Rather than performing multiple number of PCR amplifi-
cations for detecting multiple regions of a gene or a pathogen
or detecting multiple viruses or bacteria in an infected sample,
for example, respiratory tract secretions, it would be rational to
amplify all targets of interest simultaneously within a single
assay.
2. Possess high degree of sensitivity
The assay is highly sensitive in detecting both
non-cultivable virus and neutralized virus present in antigen-
antibody complexes.
3. Internal controls
False negatives in comparison to singleplex PCRs can
always be revealed within an assay since every amplicon may
serve as an internal control for all the other amplified
amplicons.
8 Application of Multiplex PCR in Porcine Diseases
(a) Detection of bacterial and viral pathogens in clinical and epi-

demiological studies.
(b) Routine screening of individual pathogens, symptom-
associated pathogens and evaluating various pathogens asso-
ciated with diseases.
(c) Typing and sub-typing of strains of various pathogens in
epidemiological studies.
(d) Detection of co-infections of pathogens during outbreaks.
Typing of PRRSV by a multiplex PCR assay, developed by
[25] was one of the early reports of application of multiplex
PCR in detection of porcine diseases. Another exceptional
utilization of multiplex PCR is reported by Ogawa et al. in
2009 for detection of major swine DNA and RNA viruses in
pigs with multiple infections. This report includes detection of
nine viruses, viz. PCV-2, PPV, PRRSV, JEV, PoRV-A, PEDV,
TGEV, Getahvirus, suid herpesvirus 1 within a single assay.
There has been an extensive use of multiplex PCRs in detec-
tion of porcine diseases irrespective of the type of template
(s) that has been utilized. For example.
1. Multiplex PCR for detection of porcine DNA virus(s)
There are reports of multiplex PCR for detection of the
DNA contained emergent disease agents such as African swine
fever virus (ASFV), Aujesky disease and PCV [26]. Yang et al.
in 2019 [27] developed a multiplex assay to detect and discrim-

inate porcine circoviruses such PCV-1, PCV-2, and PCV-3 in
clinical specimens.
2. Multiplex PCR for detection of porcine RNA virus(s)
There are reports of multiplex RT-PCR for simultaneous
detection of North American Genotype of PRRSV, Swine
Influenza virus (SIV), and Japanese encephalitis virus (JEV)
[28]. Fujii et al. in 2019 [29] developed a semi-nested multi-
plex PCR for genotyping of Rotavirus. We have developed a
multiplex PCR assay for detection of three genes of PRRSV in
our laboratory (Table 3).
3. Multiplex PCR for detection of porcine DNA and RNA viruses
There are reports of multiplex PCR for simultaneous
detection of six swine DNA and RNA viruses, viz. Classical
Swine Fever Virus (CSFV), PRRSV, JEV, PCV-2, PPV, and
Porcine Pseudorabies virus (PRV) [19]. Hu et al. in 2015
[30] had developed a multiplex PCR for simultaneous detec-
tion of CSFV, ASFV, PRRSV, and porcine pseudorabies virus.
We have developed a multiplex PCR assay for simultaneous
detection of PCV-2, PPV, and CSFV in our laboratory
(Table 2).
4. Multiplex PCR for detection of bacterial DNA pathogens in
swine
Phillips et al. in 2009 [31] had developed a multiplex PCR
to detect Lawsonia intracellularis, Brachyspira hyodysenteriae,
and Brachyspira pilosicoli in feral pigs. Elder et al. in 1997 [32]
had developed a multiplex PCR for simultaneous detection of
Lawsonia intracellularis, Serpulina hyodysenteriae, and Salmo-
nellae in porcine intestinal specimens. A rapid multiplex PCR
for detection of virulence associated genes of Pasteurella mul-
tocida from pigs [16] and a multiplex PCR assay for simulta-
neous detection of three important pathotypes of Escherichia
coli from diarrheic piglets Rajkhowa et al. [33] has been devel-
oped in our laboratory.
8.1 Porcine Porcine respiratory disease complex, commonly known as PRDC, is

Respiratory Diseases one of the major concerns for pig rearers and involve multiple viral
and bacterial pathogens. Lung et al. in 2017 [34] had reported a
multiplex PCR assay for detection of bacteria and viruses associated
in swine respiratory diseases such as PRRSV, Influenza A virus,
PCV-2, porcine Coronavirus, Mycoplasma hyopneumoniae, Pasteur-
ella multocida, Salmonella, and Streptococcus suis.
8.2 Porcine Major economic losses are faced in the piggery sector due to
Reproductive Diseases reproductive problems such as stillbirths, mummified fetus, embry-
onic death, and infertility. Such problems are due to infection by
opportunistic bacteria, viruses, and sometimes fungi and protozoa
that are often endemic in herds. PRRSV, Pseudorabies virus

(Aujeszsky’s disease), PCV-2, PPV, CSFV, Swine Influenza virus
(SIV), Porcine enterovirus, Brucella suis, Leptospira pomona, Lep-
tospira bratislava, Erysipelotrix rhusiopathiae are some of the patho-
gens associated with reproductive diseases of pigs. There are
numerous reports on utilization of multiplex PCRs for diagnosis
of such pathogens associated with reproductive diseases. In 2003,
Kim and Chae [35] developed a multiplex nested PCR for differen-
tiation of PCVs and PPV from pigs with Post-weaning multisyste-
mic wasting syndrome (PMWS). Pan et al. in 2005 [36] reported
multiplex PCR for rapid detection of pseudorabies virus, PPV, and
PCV-2.
8.3 Clostridial The disease is caused by Clostridium perfringens, which is an inhab-

Enteritis itant of the large intestine. It usually occurs when the piglet has had
insufficient intake of colostrums and is one of the causes of mortal-
ity in neonates. Meer and Songer in 1997 [37] reported a multiplex
PCR assay for genotyping of the major toxins of Clostridium
perfringens. Kanakaraj et al. in 1998 [38] had reported a multiplex
assay for detection of Clostridium perfringens in feces and intestinal
contents of pigs and in swine feed.
8.4 Porcine Diarrhea Diarrhea or scours in piglets are common at both neonatal and
post-weaning stage and is a common cause of mortality in piglets.
E. coli, TGEV, clostridial diseases, coccidiosis, and PoRV-A are
some of the pathogens associated with pre-weaning diarrhea. The
potential pathogens causing diarrhea in post-weaning piglets
include E. coli, PoRV-A, TGEV, Salmonellosis, and Campylobacter.
Seungtae et al. in 2014 [39] had reported a diagnostic test for
enteric diarrhea in pigs utilizing an efficient multiplex PCR. In
2019, Liu et al. [40] had reported detection and differentiation
of five diarrhea related pig viruses such as PEDV, TGEV, PoRV-A,
PoRV-C, and PCV-2 utilizing a multiplex PCR assay. A multiplex
PCR assay for simultaneous detection of three important patho-
types of Escherichia coli from diarrheic piglets was reported by
Rajkhowa et al. [33]. Ding et al. in 2019 [41] had reported a
multiplex RT-PCR for detection of major enteric RNA viruses in
pigs such as PEDV, TGEV, PoRV-A, porcine kobuvirus (PKV),
porcine sapovirus (PSaV), and porcine deltacoronavirus (PDCoV).
8.5 Porcine Parasitic The diagnosis of parasites in pig management is also emphasized as
Diseases the parasites can infect them and produce a wide range of clinical
manifestations. Beck et al. in 2009 [42] reported the utilization of a
multiplex PCR in detecting Trichinella pseudospiralis in muscle
tissue of domestic pig in Croatia. Lin et al. in 2008 [43] had
reported the utilization of multiplex PCR for differentiation of
two porcine nodule worms, viz. Oesophagostomum dentatum and
Oesophagostomum quadrispinulatum. Sato et al. in 2006 [44] had
reported utilization of a multiplex PCR in identifying Afro-

American genotype of Taenia solium in cysts samples obtained
from pigs. The report revealed occurrence of porcine cysticercosis
in Brazilian pigs.
9 Conclusion
Multiplex PCRs have proved to be robust, rapid, precise, and a

comprehensive diagnostic tool in effective management of pigs and
the diseases associated with them. The potential of this technique
to detect multiple pathogens infecting the pig population is itself
significant in routine screening of individual pathogens, symptom-
associated pathogens, evaluating various pathogens associated with
diseases, detection of co-infections, assessment of status of a dis-
ease, and identification of carriers of infection(s) in order to reduce
morbidity and mortality. Although this technique can sometimes
be cumbersome in terms of its optimization but once developed,
the efficiency of multiplex PCRs in minimizing the need of addi-
tional confirmatory diagnostic tests, possessing characteristic fea-
ture of high sensitivity, and specificity in identification of infectious
agents, reducing the detection time and cost involved over other
traditional cultivation methods in identification of pathogens from
clinical samples and its rapidity in implementation of effective
therapies, promotes this technique to have an excellent utility in
clinical diagnosis of porcine diseases.
References
1. Karch H, Meyer T (1989) Single primer pair pleuropneumonia infection in China. Chinese
for amplifying segments of distinct Shiga-like J Anim Vet Sci
toxin genes by polymerase chain reaction. J 6. Rasschaert D, Duarte M, Laurde H (1990)
Clin Microbiol 27(12):2751–2757 Porcine respiratory coronavirus differs from
2. Meyer T, Bitzan M, Sandkamp O, Karch H transmissible gastroenteritis virus by a few
(1989) Synthetic oligodeoxyribonucleotide genomic deletions. J Gen Virol 71
probes to detect verocytptoxin – producing (11):2599–2607
Escherichia coli in diseased pigs. FEMS Micro- 7. Meyer RF, Brown CC, House C, House JA,
biol Lett 57(2):247–252 Molitor TW (1991) Rapid and sensitive detec-
3. Belak S, Ballagi-Pordany A, Flensburg J, Virta- tion of foot-and-mouth disease virus in tissues
nen A (1989) Detection of pseudorabies virus by enzymatic RNA amplification of the poly-
DNA sequences by polymerase chain reaction. merase gene. J Virol Methods 34(2):161–172
Arch Virol 108(3–4):279–286 8. Molitor TW, Oraveerakul K, Zhang QQ, Choi
4. Jestin A, Foulon T, Pertuiset B, Blanchard P, CS, Ludemann LR (1991) Polymerase chain
Labourdet M (1990) Rapid detection of pseu- amplification (PCR) amplification for detection
dorabies virus genome sequences in biological of porcine parvovirus. J Virol Methods 32
samples from infected pigs using polymerase (2–3):201–211
chain reaction DNA amplification. Vet Micro- 9. Gouvea V, Santos N, Timenetsky MDC
biol 1(4):317–328 (1994a) Identification of bovine and porcine
5. Xufu Y, Faquan P (1990) Confirmation and rotavirus G types by PCR. J Clin Microbiol
diagnosis of Porcine Haemophilus 32(5):1338–1340
10. Gouvea V, Santos N, Timenetsky MDC Molecular methods for virus detection. Aca-
(1994b) VP4 typing of bovine and porcine demic Press, San Diego, pp 219–236
group A rotaviruses by PCR. J Clin Microbiol 22. Loewy ZG, Mecca J, Diaco R (1994) Enhance-
32(5):1333–1337 ment of Borrelia burgdorferi PCR by uracil N-
11. Suarez P, Zardoya R, Prieto C, Solana A, glycosylase. J Clin Microbiol 32(1):135–138
Tabares E, Bautista JM, Castro JM (1994) 23. Erlich H, Gelfand D, Sninsky J (1991) Recent
Direct detection of the porcine reproductive advances in the polymerase chain reaction. Sci-
and respiratory syndrome (PRRS) virus reverse ence 252:1643–1651
polymerase chain reaction (RT-PCR). Arch 24. Mahony J, Luinstra K, Sellors J, Chernesky M
Virol 135:89–99 (1993) Comparison of plasmid and chromo-
12. Chamberlain JS, Gibbs RA, Ranier JE, Nguyen some based polymerase chain reaction assays
PN, Caskey CT (1988) Deletion screening of for detecting Chlamydia trachomatis nucleic
the Duchenne muscular dystrophy locus via acids. J Clin Microbiol 9:2241–2245
multiplex DNA amplification. Nucleic Acids 25. Gilbert SA, Larochelle R, Magar R, Cho HJ,
Res 16:11141–11156 Deregt D (1997) Typing of porcine reproduc-
13. La T, Collins AM, Phillips ND, Oksa A, Hamp- tive and respiratory syndrome viruses by a mul-
son DJ (2006) Development of multiplex PCR tiplex PCR assay. J Clin Microbiol 35
for rapid detection of enteric pathogens Law- (1):264–267
sonia intracellularis, Brachyspira hyodysenter- 26. Gerilovych A, Stegniy B, Golovko V,
iae, and Brachyspira pilosicoli in porcine Solodiankin O, Gerilovych I, Smolyaninova Y,
faeces. Lett Appl Microbiol 42(3):284–288 Rudova N (2015) Development of the multi-
14. Ogawa H, Taira O, Hirai T, Takeuchi H, plex PCR for detection of the DNA contained
Nagao A, Ishikawa Y, Tuchiya K, Nunoya T, emergent diseases agents in pigs (African swine
Ueda S (2009) Multiplex PCR and multiplex fever, Aujeszky disease, Circoviral disease).
RT-PCR for inclusive detection of major swine Online J Public Health Inform 7(1):e131
DNA and RNA viruses in pigs with multiple 27. Yang K, Jiao Z, Zhou D, Guo R, Duan Z, Tian
infections. J Virol Methods 160:210–214 Y (2019) Development of a multiplex PCR to
15. Saiki RK (1989) The design and optimization detect and discriminate porcine circoviruses in
of the PCR. In: PCR technology, principles and clinical specimens. BMC Infect Dis 19:778
applications for DNA amplification. Stockton 28. Chen HY, Wei ZY, Zhang HY, Lu XL, Zheng
Press, New York, pp 7–16 LI, Cui CA, Liu J, Zhu QL, Wang ZX (2010)
16. Rajkhowa S (2015) Development of a novel Use of multiplex RT-PCR assay for simulta-
multiplex PCR assay for rapid detection of vir- neous detection of North American genotype
ulence associated genes of Pasteurella multo- PRRSV, swine influenza virus and Japanese
cida from pigs. Lett Appl Microbiol 61 encephalitis virus. Agric Sci China 9
(3):293–298 (7):1050–1057
17. Henegariu O, Heerema NA, Dlouhy SR, Vance 29. Fujii Y, Doan YH, Wahyuni RM, Lusida MI,
GH, Vogt PH (1997) Multiplex PCR: critical Utsumi T, Shoji I, Kazuhiko K (2019)
parameters and step-by-step protocol. Bio- Improvement of rotavirus genotyping method
Techniques 23:504–511 by using the semi-nested multiplex PCR with
18. Zangenberg G, Saiki R, Reynolds R (1999) new primer set. Front Microbiol 10:647
Multiplex PCR: Optimization Guidelines. In: 30. Hu L, Lin XY, Yang ZX, Yao XP, Li GL, Peng
Chapter 6, PCR applications protocols for SZ, Wang Y (2015) A multiplex PCR for simul-
functional genomics. Academic Press, pp taneous detection of classical swine fever virus,
73–94 African swine fever virus, highly pathogenic
19. Xu XG, Chen GD, Huang Y, Ding L, Li ZC, porcine reproductive and respiratory syndrome
Chang CD, Wang CY, Tong DW, Liu HJ virus, porcine reproductive and respiratory syn-
(2012) Development of multiplex PCR for drome viruses and pseudorabies in swine. Pol J
simultaneous detection of six swine DNA and Vet Sci 18(4):715–723
RNA viruses. J Virol Methods 183(1):69–74 31. Phillips ND, La T, Adams PJ, Harland BL,
20. Apte A, Daniel S (2003) PCR primer design. Fenwick SG, Hampson DJ (2009) Detection
In: Chapter 7, PCR Primer A laboratory man- of Brachyspira hyodysenteriae, Lowsonia intra-
ual, 2nd edn. Cold Spring Harbor Laboratory cellularis, and Brachyspira pilosicoli in feral pigs.
Press, Cold Spring Harbour, NY, pp 61–74 Vet Microbiol 134(3–4):294–299
21. Mahony JB, Chernesky MA (1995) Multiplex 32. Elder RO, Duhamel GE, Mathiesen MR,
polymerase chain reaction. In: Chapter 10, Erickson ED, Gebhart CJ, Oberst RD (1997)
Multiplex polymerase chain reaction for
simultaneous detection of Lowsonia intracellu- contents of pigs and in swine feed. Vet Micro-
laris, Serpulina hyodysenteriae and salmonellae biol 63(1):29–38
in porcine intestinal specimens. J Vet Diagn 39. Seungtae K, Sharma N, Seog KH, Young MG,
Investig 9(3):281–286 Kyoo OS, Yi PS, Yeun LJ, Jeong DK (2014)
33. Rajkhowa S, Sarma D, Shakuntala I, Sahoo N Establishment of diagnostic test for enteric
(2015) Multiplex PCR assay for simultaneous diarrhea in pigs using efficient multiplex poly-
detection of three important pathotypes of merase chain reaction. Indian J Anim Sci 84
Escherichia coli from diarrhoeic piglets. Indian (11):1157–1162
J Anim Sci 85(11):1202–1204 40. Liu G, Jiang Y, Opriessnig T, Gu K, Zhang H,
34. Lung O, Adjei SO, Buchanan C, Joseph T, Yang Z (2019) Detection and differentiation of
King R, Erickson A, Detmer S, Ambagala A five diarrhea related pig viruses utilizing a mul-
(2017) Multiplex PCR and microarray for tiplex PCR assay. J Virol Methods 263:32–37
detection of swine respiratory pathogens. 41. Ding G, Fu Y, Li B, Chen J, Wang J, Yin B,
Transbound Emerg Dis 64(3):834–848 Sha W, Liu G (2019) Development of a multi-
35. Kim J, Chae C (2003) Multiplex nested PCR plex RT-PCR for the detection of major diar-
compared with insitu hybridization for the dif- rhoeal viruses in pig herds in China.
ferentiation of porcine circoviruses and porcine Transbound Emerg Dis 67(2):678–685
parvovirus from pigs with postweaning multi- 42. Beck R, Beck A, Lucinger S, Florijancic T,
systemic wasting syndrome. Can J Vet Res 67 Boskovic I, Marinculic A (2009) Vet Parasitol
(2):133–137 159(3–4):304–307
36. Pan QX, Chen D, He KW, Huang KH (2005) 43. Lin RQ, Ai L, Zou FC, Verweij JJ, Jiang Q, Li
Multiplex PCR for rapid detection of pseu- MW, Song HQ, Zhu XQ (2008) A multiplex
dorabies virus, PPV and PCV-2. Virol Sin 20 PCR tool for the specific identification of Oeso-
(6):603–606 phagostomum spp. from pigs. Parasitol Res 103
37. Meer RR, Songer JG (1997) Multiplex poly- (4):993–997
merase chain reaction assay for genotyping 44. Sato MO, Cavalcante TV, Sako Y, Nakao M,
Clostridium perferingens. Am J Vet Res 58 Yamasaki H, Yatsuda AP, Nakaya K, Ito A
(7):702–705 (2006) Evidence and potential for transmission
38. Kanakaraj R, Harris DL, Songer JG, Bosworth of human and swine Taenia solium Cysticerco-
B (1998) Multiplex PCR assay for detection of sis in the Piracuruca region Piaui, Brazil. Am J
Clostridium perferingens in feces and intestinal Trop Med Hyg 75(5):933–935
Chapter 6
Protocols for Isolation of Plasmid DNA

Vinod Kumar Singh, Vikas Gupta, and Chayanika Das
Abstract
Plasmid DNA isolation is indivisible step in the development of diagnostic assays based on recombinant
proteins and other molecular biology experiments. There are hundreds of protocols published for isolation
and purification of plasmid DNA and still the search is on for the cost and time saving methods. Each
protocol published has its own advantages and limitation and the plasmid DNA obtained by different
protocols vary in purity and yield. Many of these are the modification of the classical alkaline lysis method
while the available rapid isolation methods employ different solid phase minicolumns. To discuss all the
methods of plasmid isolation will require volumes of book space hence in this chapter the most common
and trusted protocols used invariably in diffferent laboratories around the world are described.
Key words Plasmid, Isolation, Methods, Molecular assays
1 Introduction
Among the various signature molecules in a pathogen cell, nucleic

acid or genetic material is one of the important targets for diag-
nostics assays. The nucleic acids content of a cell comprise of
genomic or chromosomal DNA, plasmid DNA, and the different
types of RNAs. The chromosomes are simply arrays of genetic
material which translates into a single DNA molecule per chromo-
some at molecular level but structurally, it is a complicated structure
involving proteins as well [1]. In most of the bacteria, single copy of
closed circular chromosome is present that encodes all the essential
functions. However, in higher organism the genetic information is
distributed among number of chromosomes [2]. Furthermore, the
plasmids are double-stranded circular DNA molecules independent
of chromosomal DNA and are found in most of the bacteria
[3]. These self-replicating genetic elements have also been reported
in some of the lower eukaryotic organisms such as yeasts [4]. Escher-
ichia coli (E. coli) fertility factor F was the first plasmid discovered
for its ability to mediate transfer of chromosome markers from one
strain to another [5]. However, until the discovery of plasmids
91
92 Vinod Kumar Singh et al.
encoded resistance to antibiotics in epidemic strains of Shigella in

late 1950s in Japan, plasmids research did not receive much impor-
tance [6]. Thereafter, in the next two decades, much research was
centered on plasmids imparting drug resistance, particularly on the
mechanism of resistance. It also became apparent during this period
that the plasmids were not just responsible for fertility or drug
resistance but they confer much greater variety of host properties.
Apart from the defined benefits plasmids specify to the bacterium,
the naturally occurring plasmids have also been tailored to produce
new plasmids, which are being used as cloning vectors in recombi-
nant DNA research [7]. While the presence of a plasmid can be
detected genetically as a change in the phenotype of the bacterial
cell, often the isolation of plasmid is required to carry out studies in
different scientific contexts, including restriction enzyme mapping,
as factors in the spread of antibiotic resistance, determination of
size and number of plasmids, nucleotide sequencing, or for con-
structing new hybrid plasmids. The degree of purity of isolated
plasmid will depends upon the methods employed for its extrac-
tion. In biochemical aspects, to purify plasmid from bacteria is to
isolate plasmid DNA from the mixture of proteins, chromosomal
DNA, plasmid DNA, and different RNAs. Of these biopolymers,
chemical properties of protein are very different from nucleic acids
(DNA and RNA) and it is rather easy to separate nucleic acids from
proteins. However, DNA and RNA are very similar molecules
exhibiting same basic biochemical properties. Uracil nucleoside
and ribose sugar in RNA at the place of thymine and deoxyribose
sugar in DNA, respectively, are the only noticeable structure differ-
ence but may have quite different conformational forms [8]. Fur-
thermore, plasmids and chromosomal DNA are both deoxyribose
nucleic acid and share similar biochemical properties. Also, in most
of the bacteria, the chromosomal DNA is closed circular form and
so is the form of plasmids, and the distinguishable difference
between chromosomal DNA and plasmid, is the reasonably smaller
size of plasmids compared to large chromosomal DNA. The tridi-
mensional structural differences along with the molecular size dif-
ference form the basis for the isolation of plasmid from other
nucleic acids. However, any nucleic acid extraction method can be
broadly divided into four basic steps, viz. lysis or disruption of cell,
removal of membrane lipids, proteins, and other nucleic acids,
purification and concentration of nucleic acid [9]. Following this
basic principle, plasmids can be isolated by a variety of methods,
many of which rely on the differential denaturation and reannealing
of plasmid DNA compared to chromosomal DNA. Moreover, with
the advancement in science and technology, various modifications
were introduced to make the process more efficient and many rapid
extraction kits using minispin column were also developed for an
early laboratory diagnosis. However, the need of the hour is the
diagnosis at the site of patient and to achieve this in molecular
Protocols for Isolation of Plasmid DNA 93
diagnosis we need to take the nucleic acid extraction protocols from

the laboratory settings to the field/onsite in an easy and rapid
format for providing point-of-care diagnosis. However, this chapter
is mostly focused on the isolation of bacterial plasmid only and
describes the classical as well as new rapid methods of plasmid
purification.
2 Materials
2.1 Media (# Note 1) 1. Luria Bertani Broth: Tryptone 10 g/L, NaCl 5 g/L, Yeast
Extract 5 g/L.
2. Brain-Heart Infusion Broth: Calf brain, infusion from 200 g/
L, Beef heart, infusion from 250 g/L, Proteose peptone 10 g/
L, Dextrose 2 g/L, Sodium chloride 5 g/L, Disodium phos-
phate 2.5 g/L.
3. Luria Bertani Agar: Tryptone 10 g/L, NaCl 5 g/L, Yeast
Extract 5 g/L, Agar 15 g/L.
4. Brain-Heart Infusion Agar: Calf brain, infusion from 200 g/
L, Beef heart, infusion from 250 g/L, Proteose peptone 10 g/
L, Dextrose 2 g/L, Sodium chloride 5 g/L, Disodium phos-
phate 2.5 g/L, Agar 15 g/L.
Adjust the pH to 7.4 0.2 using 10 N NaOH and ster-
ilized by autoclaving.
2.2 Chemicals 1. Antibiotics

and Other 2. Agarose
3. Lysozyme: Dry powder. Store at 20 C.
4. Ficoll 400,000
5. Ethidium bromide (EtBr) solution
6. Cesium chloride (CsCl)
7. Ethanol (70%)
8. Potassium acetate, pH 4.8 (3 M Potassium, 5 M Acetate)
9. Phenol, chloroform, and isoamyl alcohol (25:24:1 v/v)
10. Isopropyl alcohol
11. Isoamyl alcohol
12. Nuclease free water (NFW)
2.3 Major Equipment 1. Refrigerated microcentrifuge

2. pH meter
3. Ultracentriguge
4. UV-transilluminator or Gel documentation system
5. Water bath
6. Power supply and accessories for electrophoresis
2.4 Solutions and 1. TE buffer: 10 mM Tris, pH 8.0, 1 mM EDTA

Buffers (# Note 2) 2. 1% Sodium dodecyl sulfate (SDS) in 0.2 N NaOH
2.4.1 For Extraction and 3. Tris-borate buffer: pH 8.2; 89 mM Tris base, 12.5 mM Disodium
Purification of Plasmid DNA EDTA, and 8.9 mM Boric acid
(# Notes 3 and 4) 4. Lysozyme Mixture
(a) For Gram-negative bacteria: Lysozyme, 7500 U/mL;
Ribonuclease I, 0.3 U/mL; 0.05% Bromphenol blue in
Tris-borate buffer; 20% Ficoll 400,000 (# Note 5)
(b) For Gram-positive bacteria: Lysozyme 75,000 U/mL,
50 mM Disodium EDTA (pH 8.0); 0.1 M Sodium
chloride
5. SDS mixture.
(a) For Gram-negative bacteria: 0.2% SDS in Tris-borate
buffer in 10% Ficoll 400,000
(b) For Gram-positive bacteria: 2.0% SDS in Tris-borate
buffer in 10% Ficoll 400,000
6. Overlay mixture 0.2% SDS in Tris-borate buffer in 5% Ficoll
400,000
7. STET: 8% (w/v) Sucrose, 5% (v/v) Triton X-I 00, 50 mM
EDTA, pH 8.0, 50 mM Tris–HCl, pH 8.0
8. Glucose buffer: 50 mM Glucose, 25 mM Tris–HCl, pH 8.0, and
10 mM EDTA
9. Resuspension solution: 6.5% Sucrose, 50 mM Tris–HCl, and
1 mM EDTA; pH 8.0
10. Lysis solution: 3% SDS, 50 mM Tris–HCl, and 5 mM EDTA.
Adjust the pH to 12.2–12.4 with 5 N Sodium hydroxide just
prior to use.
11. Neutralizing solution (Potassium acetate: 3 M Potassium/5 M
Acetate): Dissolve 29.4 g of Potassium acetate in 88.5 mL
distilled water, and 11.5 mL of Glacial acetic acid. Store at
room temperature.
2.4.2 For Analysis of 1. Electrophoresis buffer (# Note 6)

Plasmid DNA (a) Tris-borate-EDTA (TBE) Buffer, 1: 89 mM Tris base
(pH 7.6), 89 mM Boric acid and 2 mM Disodium EDTA
(b) Tris-acetate-EDTA (TAE) Buffer, 1: 40 mM Tris
(pH 7.6), 20 mM Acetic acid and 1 mM EDTA
2. EtBr solution (10 mg/mL): 10 mg/mL EtBr in double distilled
water. Store in light proof vials at room temperature.
3. Sample loading Buffer (6): 0.25% (w/v) Bromophenol blue,

0.25% (w/v) Xylene cyanol FF, 30% (v/v) Glycerol in double
distilled water. Make aliquot and store at 20 C.
3 Methods of Plasmid Isolation
Plasmid isolation is a crucial step in most routine laboratory experi-

ments in biochemistry and molecular and cell biology. Over the
century the procedure has also been used as an indivisible step in
recombinant DNA technology. There are several methods pub-
lished for isolation of plasmid, majority of which makes use of the
difference in size of the chromosomal and plasmid DNAs to give
preferential release of plasmid [10–23]. The alkaline-sodium dode-
cyl sulfate (SDS) method is the classical and most popular method
for purification of plasmid [13]. With time and necessities several
modifications were introduced and new methods are still develop-
ing for purity and/or quantity and time and cost saving.
3.1 Classical The separation of plasmid DNA from chromosomal DNA is the
Methods of Plasmid major problem to overcome during purification of plasmid DNA.
Purification The physical characteristics that permit the separation of plasmids
are its relatively smaller size, covalently closed circular structure,
and the fact that they are not bound to other cellular components in
the lysate. There has been several methods developed and are being
used for the purification of plasmid DNA. Among the several
methods, the choice of the method for plasmid extraction depends
on the purpose and the required purity of extracted plasmid DNA
with each method having its own advantages and limitations. The
chromosomal DNA presence in the plasmid preparation, if any, may
interfere in downstream processes such as hybridization, RE analy-
sis, transformation and also with agarose gel electrophoresis. Also,
the other impurities such as nucleases could degrade the plasmid;
high molecular weight RNA will inhibit restriction endonucleases
while detergents and salts left over from the purification medium
may interfere with the subsequent processing. Therefore, one
should always go for some cleaning-up of the plasmid preparation
after its isolation from the bacterium. The classical methods com-
monly used for demonstrating the plasmids include agarose elec-
trophoresis method, rapid boiling method, dye-CsCl gradient
method, column chromatography and the most popular is the
alkaline lysis method.
3.1.1 Agarose Among the classical methods of plasmid isolation, agarose electro-
Electrophoresis Method phoresis method described by Eckhardt [24] is the simplest in
principle. It involves the lysis of the bacterial cells in the well of
agarose gel using lysozyme and sodium dodecyl sulfate (SDS)
followed by electrophoresis of the gel. Upon electrophoresis, the
chromosomal/genomic DNA hardly drifts into the gel matrix due

to its larger size while the much smaller plasmid DNA can penetrate
and are displayed in the gel. This method is quite simple and
convenient to carry out involving minimal manipulation with the
disadvantage of poor reproducibility of results. This method can be
useful in circumstances where primary screening of strains is the
purpose and just displays of the plasmids contained in a strain are
sufficient for identification and differentiation. However, in cir-
cumstances like DNA sequencing where highly purified plasmids
are required, methods such as dye-CsC1 gradient or column chro-
matography can be used while for recombinant DNA studies less
purified plasmid can be often satisfactory. Although, the CsC1
gradient and column chromatography methods give satisfactory
results, they are cumbersome, time taking, expensive, and not
suitable for the preparation of templates from multiple samples.
3.1.2 Rapid Boiling Rapid boiling method was developed by Holmes and Quigley
Method [19]. In this method, bacterial cells are partially lysed so that the
large chromosomal DNA remains trapped in the cell debris while
the smaller plasmid DNA can escape. Thereafter, chromosomal
DNA is denatured using high temperature, after which reannealing
allows the plasmids to reassociate. The lysate is then centrifuge to
remove the chromosomal DNA along with the cell debris while the
plasmid DNA remains in the suspension. The supernatant is col-
lected in fresh tube to which isopropanol was added to precipitate
the plasmid DNA.
3.1.3 Dye-CsC1 Gradient The base compositions of chromosomal and plasmid DNAs are
Method usually so alike that their separation on the basis of difference in
their density is quite unworkable. However, the buoyant density
can be manipulated to produce the difference using saturating
concentrations of DNA binding fluorescent dyes. Ethidium bro-
mide (EtBr) is the most commonly used fluorescent dye which
binds the DNA by intercalation between base pairs. The intercala-
tion of EtBr dye occurs only when the double helix DNA unwinds
slightly. The unwinding is unimpeded in linear or open-circular
DNA but creates strain in covalently closed circular (CCC) mole-
cules leading to binding of less dye per unit length in CCC plas-
mids. Consequently, the buoyant density will be less in case of linear
chromosomal DNA than the CCC plasmids of similar base compo-
sition and thus can be separated using equilibrium ultracentrifuga-
tion [25]. The DNA migrates to the point at which it has density
similar to that of CsCl, i.e. 1.7 g/cm3 in the gradient and the
protein molecules having lower buoyant densities remain at the
top while the RNA gets pelleted at the bottom of the tube. Cesium
chloride (CsCl)/EtBr ultracentrifugation is one of the traditional
methods being used since 1950s for purification of nucleic acids
[26–28]. After about 72–96 h of ultracentrifugation in swinging

bucket rotors using CsCl density gradient with saturated concen-
tration of EtBr, the separated CCC plasmid DNA can be visualized
under UV rays in the form of band below the linear chromosomal
DNA band [29]. Like EtBr, propidium iodide can also be used for
separation but it is quite expensive. Furthermore, in cases where the
difference of buoyant density between plasmid and chromosomal
DNA is very small, Hoechst 33258 dye can be used to augment the
buoyant density difference for a possible separation. Although, the
conventional equilibrium Dye-CsC1 ultracentrifugation leads to
isolation of ultrapure grade of plasmid DNA suitable for almost
all downstream processing [30], but requires extended hours for
ultracentrifugation and additional time and labor for the removal of
dye and CsCl salt from the preparation. However, with the
advances in rotor design and an increased understanding of centrif-
ugal force theory, researchers have come up with better controlled
protocols with significantly reduced starting material volumes and
ultracentrifugation spin time employing vertical and fixed angle
rotors [31]. Two-step CsCl-EtBr discontinuous gradient method
described by Garger et al. [32] is one such modified rapid method
capable of isolating ultrapure DNA plasmids in about 5 h with
lesser RNA contamination compared to the conventional equilib-
rium ultracentrifugation method. The detailed procedure of rapid
two-step CsCl-EtBr discontinuous gradient single ultracentrifuga-
tion method is also described in the protocol section.
Note: This method is very sensitive to CsCl concentration and a
slight change of CsCl amount may lead to a negative result; plasmid
DNA is not separated as a single band in the tube.
3.1.4 Column Although, the extended time required for separation of plasmid
Chromatography DNA by density gradient ultracentrifugation can be minimized by
using a vertical rotor, but still the removal of the EtBr and CsCl
from the recovered plasmid preparation upholds the cumbersome-
ness of the method. However, the purity and yield of plasmid DNA
comparable to density gradient ultracentrifugation method can also
be achieved with adsorption chromatography [33] and fast protein
liquid chromatography [34, 35]. Tiselius et al. [36] first developed
hydroxyapatite columns for protein chromatography which was
later extended to nucleic acids chromatography by Bernardi
[37]. Hydroxyapatite binds with proteins and nucleic acids at low
phosphate concentration while the progressive ascend in the con-
centration of phosphate leads to the earliest elution of proteins and
RNA, trailed by smaller DNA molecules, viz. plasmid DNA and
finally the high molecular weight chromosomal DNA will elute at
the highest phosphate concentrations. Furthermore, under chro-
matography methods only the protocol for most commonly used
hydroxyapatite columns chromatography will be described in
detail.
3.1.5 Alkaline Lysis Alkaline lysis method developed by Birnboim and Doly [13] is the
Method most commonly used technique applicable to a wide range of
bacterial species for isolation of plasmids. It fundamentally relies
on differential denaturation and reannealing of plasmid DNA
compared to high molecular weight chromosomal DNA and
proteins. The process involves the lysis of bacterial cells using
SDS and exposing the cell extract to high alkaline pH
(12.0–12.6) conditions [13, 38], followed by mixing the cell
extract with high concentration of low-pH potassium acetate for
neutralization to selective precipitate the chromosomal DNA, or
by direct extraction with unneutralized phenol [38, 39], which
results in the chromosomal DNA banding at the interface. This
selective precipitation occurs due to inter strand re-associations at
multiple sites owing to the very high molecular weight of the
chromosomal DNA leading to the formation of an insoluble
DNA network. The bulk of cellular RNA and protein are also
precipitated under these conditions if protein is first complexes
with SDS (anionic detergent). However, the plasmid DNA
remains in the soluble fraction due to its covalently closed circular
(CCC) nature and much smaller size and is then precipitated
using isopropanol.
Combining the different reagents appropriately, the precipita-
tion of most of the chromosomal DNA, RNA, and protein can be
accomplished in a single step and many such alterations have been
applied to the original procedure, and a large number of modified
and alternative rapid methods have been developed. The simplest
alternative to alkaline lysis is the rapid boiling method developed by
Holmes and Quigley [19]. Here, the cells are lysed partially allow-
ing plasmids to escape, whereas the bacterial chromosomal DNA
remains trapped in the cell debris. Then, the chromosomal DNA is
denatured using high temperature, after which reannealing allows
the plasmids to reassociate. Centrifugation removes the chromo-
somal DNA along with the cell debris, leaving the plasmid in
suspension, from where it is recovered by isopropanol precipita-
tion. After the initial characterization, it is possible to purify
further some or all of the plasmid DNAs by RNase digestion
and extraction with organic solvents. On the other hand, a kind
of salts such as lithium and calcium functions to make RNA as a
selective precipitate from DNA-RNA mixture [40]. This purified
plasmid DNA is suitable to be used for techniques such as
sub-cloning, sequencing, and construction of gene probes. How-
ever, in practice, simple rapid methods for plasmid preparation are
usually more dependable and preferred for plasmid DNA isolation
in most of the laboratories.
3.2 Rapid Solid In due course of time, numerous kits and systems were developed
Phase Extraction for separation of plasmid DNA from chromosomal DNA which
Methods of Plasmid does not demand CsCl/EtBr gradients [41]. These rapid methods
Purification used minispin column systems composed of silica matrices, glass
particles or powder, magnetic beads, and diatomaceous earth or ion
exchange carriers [42–45]. These methods are mainly based on
partial or complete lysis of cell followed by the removal of chromo-
somal DNA by centrifugation and selective precipitation optimized
with specific buffer and extremely precise pH and salt concentra-
tions [46]. It should be noted that the swiftness and ease of
performing plasmid isolation from multiple samples requires a
high speed microcentrifuge. Based on these properties, many isola-
tion kits were made commercially available for diagnosis and
research purposes, which could isolate the plasmid within 30 min.
Among many, the one using an anion-exchange resin and other
employing silica membrane with chaotropic solutions for plasmid
preparation are the two most commonly used kits. These kits most
oftenly used diethyl aminoethyl (DEAE) resin and guanidine
hydrochloride or guanidine thiocyanate as chaotropic agent for
adsorption of DNA. Also, both these kits utilized the alkaline-
SDS lysis principle for the separation of plasmid DNA from chro-
mosomal DNA and require addition of RNase to digest the RNA
contamination. These commercially available kits became popular
because of the limited labor requirement, ease of use, and rapid and
consistent preparation of high-quality plasmid DNA that can be
used in PCR, sequencing, restriction enzyme digestion, and trans-
formation. Also, it is worthy to note that these kits are rather
expensive.
3.3 Protocols Agarose electrophoresis method for the detection and preliminary
characterization of plasmid DNA in clinical isolates (Time: 3–4 h)
3.3.1 Protocol 1
1. Prepare 0.75–1.2% agarose gel in Electrophoresis Buffer (Note
7). Use appropriate comb to form the required number of well
for loading the samples.
2. Add 15 μL of Lysozyme Mixture in wells of mounted gel.
3. Pick 1 or 2 single colony (106 to 108 cells) of bacteria grown
overnight on LB/BHI agar with the help of flat end of tooth-
pick or micro tip and resuspend in Lysozyme Mixture poured
in wells of mounted gel. In case of liquid culture growth, take
0.1 to 0.5 mL of overnight grown culture corresponding to
106 to 108 cells. Centrifuge at 5000 g for 10 min and
resuspend the cell pellet in 10 μL of Electrophoresis Buffer
with 20% Ficoll 400,000 to prepare cell suspension. Add the
cell suspension in Lysozyme Mixture in well of mounted gel.
4. Leave the mixture at room temperature for 2–5 min in case of

Gram-negative bacteria and 30–45 min in case of Gram-
positive bacteria.
5. Add carefully 30 μL of the SDS Mixture on the top of the
bacteria-lysozyme mixture in agarose well. Gently mix the two
layers by side to side movement of a toothpick/microtip so that
the two layers should still be distinguishable. Avoid complete
mixing of the two layers.
6. Now add 100 μL of Overlay Mixture on the top without
disturbing the now viscous DNA lysate.
7. Seal the mouth of the wells containing lysate using agarose gel
(~50 C) and then fill the tanks of electrophoresis apparatus
with Electrophoresis Buffer.
8. Perform electrophoresis for 60 min at 2 mA and then for
60–150 min at 40 mA depending on the size and the desired
resolution of the plasmid(s).
9. After the completion of the electrophoresis, stain the gel for
15 min with Ethidium Bromide (0.4 pg/mL) in Electrophore-
sis Buffer (Note 8) and then observe/analyze the gel under
UV rays using transilluminator or gel documentation system
(Note 9).
3.3.2 Protocol 2 Boiling method for rapid extraction of plasmid DNA (Time:
30–45 min).
1. Culture the test bacteria in 2–3 mL BHI or LB broth with
appropriate antibiotic for overnight at 37 C in shaker incuba-
tor (120–150 rpm) (Note 10).
2. Harvest the bacterial cells from 1.5 mL of overnight grown
culture in a microfuge tube by centrifugation at 12,000 g for
1 min.
3. Discard the supernatant carefully and resuspend the obtained
cells pellet in 20 μL STET by gentle pipeting or vortex.
4. Immediately place the tubes in boiling water using a floater or
open-bottom stand for exactly 45 s.
5. Centrifuge the tubes for 10 min at 12,000 g to obtain a loose
and sticky pellet.
6. Carefully collect the pellet with the help of sterile wooden
toothpick in a fresh sterile microfuge tube and add 200 μL
isopropanol followed by centrifugation at 12,000 g for
5 min.
7. Carefully discard the supernatant and add 500 μL of 70%
ethanol for washing the pellet by centrifugation at
12,000 g for 1 min.
8. Aspirate the 70% ethanol and air dry the pellets for 10 min
(Note 11).
9. Resuspend the pellet by adding 100 μL nuclease free water
(NFW) or TE buffer for analysis/use.
3.3.3 Protocol 3 CsCl-EtBr gradient method of plasmid DNA purification.

Preparation of DNA extract
1. Grow test bacteria colony harboring plasmid in 1 L of BHI/LB
broth with appropriate antibiotic(s) in shaker incubator for
overnight (Note 10).
2. Harvest the cultured bacterial cells by centrifuging at 5000 g
for 10 min at 4 C and resuspended the pellet in 24 mL of
Glucose buffer.
3. Add freshly prepared 4 mL Glucose Buffer containing 20 mg/
mL lysozyme and keep suspension at room temperature for
10 min incubation.
4. Add 55.2 mL of 1% SDS in 0.2 N NaOH and mix by gently
swirling.
5. Place the mixture immediately in ice cold water for 5 min and
then add 28 mL of potassium acetate, pH 4.8 (3 M Potassium,
5 M Acetate).
6. Allow the mixture to remain in ice cold water for additional
15 min to precipitate the proteins, chromosomal DNA, and
high molecular weight RNA.
7. Centrifuge at 15,000 g for 10 min to remove the insoluble
contaminants and collect the supernatant in fresh tube.
8. Extract the supernatant adding an equal volume of phenol,
chloroform, and isoamyl alcohol (25:24:1) by centrifugation
12,000 g for 15 min and collect the aqueous phase contain-
ing nucleic acids carefully in fresh tube.
9. Add 0.6 volume of isopropyl alcohol and incubate for 10 min at
room temperature for precipitation.
10. Centrifuge at 15,000 g for 15 min at 4 C to collect the
plasmid DNA pellet.
11. Resuspend the pellet TE Buffer and store at 20 C for
further use.
Conventional CsCl-EtBr Equilibrium Gradient Ultracentrifugation
Method of Plasmid DNA Purification (Time: 24–48 h)
1. Make the final volume of DNA sample (500 μL) to 4.0 mL by
adding TE buffer.
2. Add 400 μL EtBr (10 mg/mL) and 4.4 g of solid CsCl. After
mixing the content the refractive index is n ¼ 1.3865 (1.35 g/
mL).
3. Load the sample solution into ultracentrifuge tube, overlay

with mineral oil and seal strictly maintain the balance of the
tubes.
4. Spin at 192,553 g at 20 C in ultraspeed centrifuge for
overnight.
5. Visualize DNA bands under long wave UV-light.
Rapid Two-Step CsCl-EtBr Gradient Ultracentrifugation Method
of Plasmid DNA Purification (Time: 4–5 h)
1. Prepare CsCl solution (density ¼ 1.470 g/mL, n ¼ 1.3780) in
TE buffer.
2. Place 8 mL of above prepared CsCl solution in large ultracen-
trifuge tubes or 4.0 mL in small ultracentrifuge.
3. Adjust the density of aliquot containing 14 mg of the buffered
total nucleic acids to 1.80 g/mL (n ¼ 1.4080) by adding solid
CsCl and ethidium bromide solution. For large tube, dissolve
4.2 g of CsCl in 2.4 mL of buffered total nucleic acid extract to
obtain a weight of 6.6 g followed by the addition of 0.4 mL of
EtBr solution (10 mg/mL) to arrive at a final weight of 7.0 g.
For small tube, dissolve 2.1 g of CsCl in 1.2 mL of buffered
total nucleic acid extract to obtain a weight of 3.3 g followed by
addition of 0.2 mL EtBr solution to arrive at a final weight of
3.5 g.
4. Layered the dense nucleic acid-containing CsCl solution
(4.0 mL final volume for the large tubes and 2.0 mL final
volume for the small tubes) beneath the less dense CsCl solu-
tion using a glass Pasteur pipette or syringe with a long cannula
without disturbing the solution interface.
5. Then fill the tube to its capacity with the less dense CsCl
solution and seal it properly.
6. Run the spin in fixed titanium rotor (Type 80 Ti or Type 75 Ti)
at 324,000 g for 4 or 5 h at 20 C.
7. Visualize DNA bands under long wave UV-light after comple-
tion of the run.
Recovery of Plasmid DNA from CsCl Gradients
1. Collect the plasmid DNA from the gradients by puncturing the
side of the tubes with an 18-gauge needle.
2. Wash repeatedly with H2O-saturated isoamyl alcohol to
remove the EtBr.
3. Dilute the plasmid DNA with 3 volumes of TE buffer and
precipitate with 2 volumes of cold ethanol.
3.3.4 Protocol 4: Small 1. Culture the test bacteria in 2–3 mL BHI or LB broth with
Scale Extraction of Plasmid appropriate antibiotic for overnight at 37 C in shaker
DNA by Alkaline Lysis incubator.
Method 2. Harvest the bacterial cells from 1.5 mL of overnight grown
culture in a microfuge tube by centrifugation at 12,000 g for
1 min.
3. Decant the supernatant carefully so as to avoid any leftover
media and add 100 μL resuspension solution to dissolve the
pellet by gentle pipeting or vortex.
4. Add 200 μL of lysis solution and mix by inverting the tube
intermittently for at least 2–3 min to allow the lysis to take
place.
5. Add 150 μL of neutralizing solution and mix by inverting the
tubes gently for several times.
6. Centrifuge the tubes at 12,000 g for 5 min in a microfuge for
phase separation.
7. Carefully remove the tubes from the microfuge without dis-
turbing the precipitate.
8. Transfer the liquid phase into fresh labeled tubes containing
250 μL isopropanol.
9. Mix by inverting tubes or gentle brief vortex and centrifuge at
12,000 g for 30 s to precipitate the plasmid DNA as a white
pellet.
10. Discard the supernatant with care not to lose the pellet. Add
750 mL 70% ethanol, vortex briefly and centrifuge at high
speed for 30 s for washing of the pellets.
11. Aspirate the 70% ethanol and air dry the pellets for 5–10 min.
12. Resuspend the pellet by adding 50 μL nuclease free water
(NFW) or TE buffer for analysis/use.
3.3.5 Protocol 5: Solid There are several commercially available kits using solid phase mini
Phase Extraction Using column for rapid extraction of plasmid DNA provided with well
Mini Column Kits for standardized procedure steps to follow for using these kits. Here,
Plasmid Purification () we describe the protocol for mdi pDNA Miniprep Kit (mdi mem-
brane technologies, Ambala, India) as per the manufacturer’s pro-
tocol with certain modifications as used in our laboratory.
1. A colony of plasmid-containing bacterial culture was inocu-
lated in 5 mL LB broth containing appropriate antibiotic in a
glass tube and incubated at 37 C overnight with 180 rpm in a
shaker incubator.
2. Centrifuge the overnight grown culture at 6000 g for 5 min
to obtain the bacterial cells pellet.
3. Decant the supernatant carefully and keep the tubes in inverted

position on a paper towel for 1 min to drain the last traces of
the media.
4. Resuspend the pellet in 250 μL of buffer AL1. Then, add
250 μL of AL2 buffer for lysis of cells and immediately mixed
by inverting the tube 4–6 times or until the solution became
viscous and slightly clear.
Note: Mixing by vortex is not advised as it may lead to
shearing of genomic DNA.
5. Without allowing the lysis reaction to proceed for more than
2 min, add 350 μL of buffer AL3 for neutralization and mix
immediately but gently by inverting the tube 4–6 times.
6. Centrifuged the solution at 15,000 g for 10 min and collect
the supernatant quickly and carefully to transfer into the spin
column with collection tube.
7. Spun the column at 13,000 g for 1.5 min and discard the
flow through.
8. Add 750 μL buffer W and centrifuged at 13,000 g for
1.5 min. Discard the flow through.
9. Run an empty spun at 13,000 g for 1 min to remove the
residual of buffer W.
10. Place the spin column in fresh sterile 1.5 mL microcentrifuge
tube and add 50 μL prewarmed buffer E directly to the center
of spin column to eluted the plasmid DNA.
11. Incubate for 5 min at 37 C, then centrifuged at 13,000 g for
2 min.
12. Store the eluted plasmid DNA at 20 C till further use.
3.4 Analysis of The plasmid preparations obtained from different methods/kits

Plasmids can easily be checked by electrophoresis in agarose gel with the
help of EtBr dye. In addition to the number and form of plasmid,
agarose gel electrophoresis also reveals the size of plasmid(s) when
converted into linear form by restriction endonucleases. In electro-
phoresis, plasmids due to their compact and small structure easily
get separated from the contaminating large fragments of chromo-
somal DNA and/or RNA, if present and indicate about the purity
of preparation. However, the separation of plasmid from smaller
fragments of chromosomal DNA is not possible in routine agarose
gel electrophoresis and requires other techniques such as pulse field
gel electrophoresis. Generally, 0.7–1.0% agarose gel is used to
observe the plasmids depending on the size. But for demonstration
of larger plasmids as low as 0.3% agarose gel can be used in hori-
zontal gel electrophoresis.
Note: The larger the plasmid, the lower the percentage of
agarose gel is required to resolve them. But at lower percentage
required for demonstration of larger plasmids, the gel became

sloppy and difficult to achieve satisfactory resolution. This became
even more difficult with some natural isolates having both small and
larger plasmid and one wants to resolve them on the same gel for
plasmid profile. In such case other techniques like pulsed-field
electrophoresis can be employed to obtain satisfactory resolution.
4 Notes
1. Suitable antibiotic(s) should be supplemented in media to

ensure retention of required plasmid.
2. Use sterilized distilled water for preparation of solutions and
buffer in DEPC treated glass bottles to minimize the risk of
DNAses contamination and do not solutions stored for more
than a month.
3. Always wear gloves while performing nucleic acid extraction to
minimize the chances of nucleases contamination from hands
and also to avoid accidental exposure to harmful solutions such
as phenol, sodium hydroxide, chloroform, and ethidium
bromide.
4. Prefer using freshly prepared solutions especially lysozyme and
sodium hydroxide for optimizing the procedure.
5. The ribonuclease is first dissolved in 0.4 M sodium acetate
buffer, pH 4.0, at 10 mg/mL and heated for 2 min at 98 C
before diluting it into the rest of the lysozyme mixture. Both
lysozyme mixtures are stable for several months at room
temperature.
6. TBE or TAE buffer can be used as per the resources and choice
of the researcher based on the purpose as both buffers have
their own advantages and limitations. TBE buffer has better
buffering capacity, whereas TAE buffer has better conductivity.
Agarose cross-linkage is better in case of TBE buffer thus gives
better resolution of large DNA fragments while smaller DNA
fragments get better resolved in TAE buffer. Also, TAE buffer
are cheaper to make than TBE buffer.
7. EtBr is carcinogenic. Use it very carefully and always wear
gloves while staining and handling the stained gel.
8. Prefer using gel documentation system for visualizing/analyz-
ing the separated plasmid(s). If using UV rays transilluminator
visualizing/analyzing, avoid observing for long time and never
forgot to wear UV-protected goggles or face shield.
9. Wherever feasible use appropriate antibiotic(s) to achieve selec-
tive growth of plasmid-containing bacterial cells.
10. Do not decant to avoid the loss of the pellet.
11. Always wear gloves while handling solutions such as sodium

hydroxide, TE-buffer-saturated phenol, chloroform: isoamyl
alcohol, and ethidium bromide.
References
1. Thomas JO, Kornberg RD (1975) Octamer of 14. Altschuler M, Heddens DK, Diveley RR Jr,
histones in chromatin and free in solution. Proc Kresheck GC (1994) Plasmid DNA isolation
Natl Acad Sci 72:2626–2630 utilizing a novel nonionic detergent. BioTech-
2. Alberts B, Johnson A, Lewis J et al (2002) niques 17:434–436
Molecular biology of the cell, 4th edn. Garland 15. Currier TC, Nester EW (1976) Isolation of
Science, New York covalently closed circular DNA of high molec-
3. Lodish H, Berk A, Zipursky SL et al (2000) ular weight from bacteria. Anal Biochem 76:
Molecular cell biology, 4th edn. W. H. Free- 431–441
man, New York 16. Ferrus MA, Alonso JL, Amoros I,
4. Broach JR (1981) The yeast plasmid 2 micron Hernandez M, Hernandez J (1999) A rapid
circle. In: Strathern JN, Jones EW, Broach JR procedure for the isolation of plasmid DNA
(eds) The molecular biology of the yeast sac- from environmental bacteria. Int Microbiol 2:
charomyces: life cycle and inheritance. Cold 115–117
Spring Harbor Laboratory, Cold Spring Har- 17. Guerry P, LeBlanc DJ, Falkow S (1973) Gen-
bor, NY, pp 445–470 eral method for the isolation of plasmid deox-
5. Hayes W (1968) The genetics of bacteria and yribonucleic acid. J Bacteriol 116:1064–1066
their viruses, 2nd edn. Blackwell, Oxford 18. He M, Wilde A, Kaderbhai MA (1990) A sim-
6. Watanabe T (1963) Infective heredity of mul- ple single-step procedure for small-scale prepa-
tiple drug resistance in bacteria. Bacteriol Rev ration of Escherichia coli plasmids. Nucleic
27(1):87–115 Acids Res 18:1660
7. Wink M (2006) An introduction to molecular 19. Holmes DS, Quigley M (1981) A rapid boiling
biotechnology: molecular fundamentals, meth- method for the preparation of bacterial plas-
ods and application in modern biotechnology. mids. Anal Biochem 114(1):193–197.
Wiley-VCH, Weinheim https://doi.org/10.1016/0003-2697(81)
8. Lesk AM (1969) Why does DNA contain thy- 90473-5
mine and RNA uracil? J Theor Biol 22(3): 20. Klein RD, Selsing E, Wells RD (1980) A rapid
537–540. https://doi.org/10.1016/0022- microscale technique for isolation of recombi-
5193(69)90021-6 nant plasmid DNA suitable for restriction
9. Ali N, Rampazzo RCP, Costa ADT, Krieger enzyme analysis. Plasmid 3:88–91
MA (2017) Current nucleic acid extraction 21. Paul B, Cloninger C, Felton M,
methods and their implications to point-of- Khachatoorian R, Metzenberg S (2008) A
care diagnostics. Biomed Res Int 2017: non-alkaline method for isolating sequencing-
9306564. https://doi.org/10.1155/2017/ ready plasmids. Anal Biochem 377:218–222
9306564 22. Serghini MA, Ritzenthaler C, Pinck L (1989) A
10. Cheng L, Li TY, Zhang Y (2004) Rapid prepa- rapid and efficient ‘miniprep’ for isolation of
ration of total nucleic acids from E. coli for plasmid DNA. Nucleic Acids Res 17:3604
multi-purpose applications. J Biochem Mol 23. Song J, Geltinger C, Sun K, Kanazawa I,
Biol 37:351–355 Yokoyama KK (1999) Direct lysis method for
11. Chowdhury EH, Akaike T (2005) Rapid isola- the rapid preparation of plasmid DNA. Anal
tion of high quality, multimeric plasmid DNA Biochem 271:89–91
using zwitterionic detergent. J Biotechnol 119: 24. Eckhardt T (1978) A rapid method for the
343–347 identification of plasmid deoxyribonucleic acid
12. Chowdhury K (1991) One step ‘miniprep’ in bacteria. Plasmid 1:584–588
method for the isolation of plasmid DNA. 25. Brown TA (2010) Gene cloning and DNA
Nucleic Acids Res 19:2792 analysis: an introduction, 6th edn. Blackwell
13. Birnboim HC, Doly J (1979) A rapid alkaline Publishing, Hoboken, NJ
extraction procedure for screening recombi- 26. Maniatis T, Fritsch EF, Sambrook J (1982)
nant plasmid DNA. Nucleic Acids Res 7: Molecular cloning, a laboratory manual. Cold
1513–1523
Spring Harbor Laboratory Press, Cold Spring 37. Bernardi G (1969) Chromatography of nucleic
Harbor, NY acids on hydroxyapatite I. Chromatography of
27. Meselson M, Stahl FW, Vinograd J (1957) native DNA. Biochim Biophys Acta 174:423–
Equilibrium sedimentation of macromolecules 434
in density gradients. Proc Natl Acad Sci U S A 38. Kado CI, Liu ST (1981) Rapid procedure for
43(7):581–588. https://doi.org/10.1073/ detection and isolation of large and small plas-
pnas.43.7.581 mids. J Bacteriol 145:1365–1373
28. Radloff R, Bauer W, Vinograd J (1967) A dye- 39. Kieser T (1984) Factors affecting the isolation
buoyant-density method for the detection and of CCC DNA from Streptomyces lividans and
isolation of closed circular duplex DNA: the Escherichia coli. Plasmid 12(1):19–36. https://
closed circular DNA in HeLa cells. Proc Natl doi.org/10.1016/0147-619x(84)90063-5
Acad Sci U S A 57(5):1514–1521. https://doi. 40. Eon-Duval A, Gumbs K, Ellett C (2003) Pre-
org/10.1073/pnas.57.5.1514 cipitation of RNA impurities with high salt in a
29. Anet R, Strayer DR (1969) Density gradient plasmid DNA purification process: use of
relaxation: a method for preparative buoyant experimental design to determine reaction con-
density separations of DNA. Biochem Biophys ditions. Biotechnol Bioeng 83:544–553.
Res Commun 34(3):328–334 https://doi.org/10.1002/bit.10704
30. Green MR, Sambrook J (2018) Preparation of 41. Tan SC, Yiap BC (2009) DNA, RNA, and
plasmid DNA by alkaline lysis with sodium protein extraction: the past and the present. J
dodecyl sulfate. Maxipreps. Cold Spring Harb Biomed Biotechnol 2009:574398. https://
Protocols 2018(1). https://doi.org/10.1101/ doi.org/10.1155/2009/574398
pdb.prot093351 42. Azimi SM, Nixon G, Ahern J, Balachandran W
31. Flamm NF, Bond HE, Burr HE (1966) (2011) A magnetic bead-based DNA extrac-
Density-gradient centrifugation of DNA in a tion and purification microfluidic device.
fixed-angle rotor: a higher order of resolution. Microfluid Nanofluid 11(2):157–165.
Biochim Biophys Acta 129:310–317 https://doi.org/10.1007/s10404-011-
32. Garger SJ, Griffith OM, Grill LK (1983) Rapid 0782-9
purification of plasmid DNA by a single centri- 43. Esser KH, Marx WH, Lisowsky T (2006)
fugation in a two-step cesium chloride- maxXbond: first regeneration system for DNA
ethidium bromide gradient. Biochem Biophys binding silica matrices. Nat Methods 3(1):
Res Commun 117(3):835–842 2005–2006. https://doi.org/10.1038/
33. Colman A, Byers MJ, Primrose SB, Lyons A nmeth845
(1978) Rapid purification of plasmid DNAs by 44. Koo K, Foegeding PM, Swaisgood HE (1998)
hydroxyapatite chromatography. Eur J Bio- Isolation of RNA and DNA fragments using
chem 91:303–310 diatomaceous earth. Biotechnol Tech 12(7):
34. Chandra G, Patel P, Kost TA, Gray JG (1992) 5 4 9 – 5 5 2 . h t t p s : // d o i . o r g / 1 0 . 1 0 2 3 /
Large-scale purification of plasmid DNA by fast A:1008859632378
protein liquid chromatography using a hi-load 45. Padhye V, York C, Adam B (1997) United
Q Sepharose column. Anal Biochem 203(1): States patent US 5658548. Promega Corpora-
169–172 tion; Nucleic acid purification on silica gel and
35. McClung JK, Gonzales RA (1989) Purification glass mixture
of plasmid DNA by fast protein liquid chroma- 46. Chacon-Cortes D, Griffiths L (2014) Methods
tography on superose 6 preparative grade. Anal for extracting genomic DNA from whole blood
Biochem 177(2):378–382. https://doi.org/ samples: current perspectives. J Biorepository
10.1016/0003-2697(89)90069-9 Sci Appl Med 2:1–9. https://doi.org/10.
36. Tiselius A, Hjerten S, Levin O (1956) Protein 2147/BSAM.S46573
chromatography on calcium phosphate col-
umns. Arch Biochem Biophys 65:132–155
Chapter 7
Recombinant Antigen-Based Diagnostic Assays of Pig Viral

Diseases
Rajib Deb, Ajay Kumar Yadav, Gyanendra Singh Sengar,
Seema Rani Pegu, Souvik Paul, Swaraj Rajkhowa,
and Vivek Kumar Gupta
Abstract
Recombinant DNA technology or genetic engineering is one of the most explored technologies in the
current era as it is one of the convincing technologies for the detection of both antigen and antibody.
Recombinant DNA-based technology involves cutting of desired genes with suitable restriction enzymes,
cloning, and expression of the immunodominant antigenic epitopes in the bacterial, yeast, baculovirus, or
cell culture system. These expressed proteins are mostly purified from the host system in the pure form
conferring its conformational structure that does not reduce its reactivity. The technology involves skill to
design primer that to in the frame of the expression vector, standardization of expression and purification,
and lesser cost utilization. Recombinant DNA technology is utilised to address a variety of diseases in
human, but it is also widely employed in the area of animal disease diagnosis, leading to the creation of
diagnostic assays like as ELISA, ELISPOT, and Lateral Flow Devices. This method is also used to create
GMOs (genetically modified organisms) (GMOs). Recombinant proteins are used in a number of diagnos-
tic tests for the detection of pig viral disease.
Key words Genetic engineering, Recombinant DNA technology, Protein, ELISA
1 Introduction
“Genetic engineering” is an important field of biotechnology hav-

ing a significant impact on agricultural sciences and its application
can be expected in the management of crops improvement, feed
industry, improvement of livestock health and genetic manipula-
tions, development of novel diagnostics and therapeutics or vac-
cines. Industrial capacity could be developed in geographic regions
which do not have any vaccine production capability. In addition,
the use of deoxyribonucleic acid (DNA) or proteins/enzymes in
health diagnostics is impending exponentially. Uses of enzymes in
food science, medicinal chemistry, leather, detergent, textile,
109
110 Rajib Deb et al.
sectors are also growing rapidly. The intensifying need of therapeu-

tic agents and other applications of enzymes or proteins could only
be met by synthesis of heterologous recombinant proteins.
Discovery of the structure of the genetic material so-called as
deoxyribonucleic acid (DNA), by Watson and Crick (1953) insti-
gated an era of biochemical exploration of cellular physiology that
has congregated momentum in the succeeding years, with more
recent advances in molecular genetics as well as biochemistry lead-
ing to a rebellion in biological research. The so-called new biology
allows investigators to polish the experimental methodologies
beyond what was believed possible only a few years ago. Applica-
tions of this research and related biotechnological applications to
various problems in agricultural sciences are now perceived as being
possible. In the year 1973, Stanley Cohen, Herbert Boyer, and their
co-workers invented a methodology for transmitting genetic infor-
mation from one organism to another and this became known as
“recombinant DNA technology” or “Genetic Engineering,” which
permitted researchers to isolate specific target genes and perpetuate
them in different host organisms.
The founding stones of “Genetic Engineering” have been laid
by strenuous efforts by various researchers across the globe and
many of which have been awarded with the prestigious Nobel
prizes. Here, we catalogued some of the milestones which molded
the destiny of genetic engineering.
1663 Cells are first described by Robert Hooke

1675 Leeuwenhoek discovers protozoa and bacteria
1855 Bacterium Escherichia coli discovered
1869 Miescher discovers the DNA in the sperm of trout
1902 Walter Sutton coins the term gene
1919 Karl Ereky coins the term biotechnology
1928 Federick Griffith’s discovers the transforming principle
1938 Edward Tatum coins the term molecular biology
1941 A. Jost coins the term genetic engineering
1943 Oswald Avery proves DNA is heritable material
1953 James Watson and Francis Crick elucidate the double helical
structure of DNA
1958 Kornberg discovers the DNA polymerase.
1964 DNA ligase was isolated
1971 Arber, Smith, and Nathans discover and characterize restriction
enzyme
1972–77 Cohen and Boyer successfully splice a gene
1973–78 Paulberg creates recombinant DNA
(continued)
Recombinant Antigen-Based Diagnostic Assays of Pig Viral Diseases 111
1975 Sanger, Maxam, and Gilbert discover nucleotide sequencing

1976–80 Formation of Genentech Inc.
1977 Genome of bacteriophage φX 174, first to be sequenced
1979 Genome of first plasmid pBR 322 was sequenced
1983 Karry Mullis discovers polymerase chain reaction
1991 Human genome project initiated
1992 Genome of first chromosome, yeast chromosome III was
sequenced
1995 Genome of first cellular organism, Haemophilus influenza was
sequenced
1996 Genome of first eukaryotic organism, Saccharomyces cerevisiae
was sequenced
1998 Human embryonic stem cells isolated by Thomson, Jones, and
co-workers
1998 Nematode C. elegans sequenced
1999 Human genome project launched
2000 Genome of first mammalian organism, Homo sapiens was
sequenced
Genome of first plant genome, Arabidopsis thaliana was
sequenced
2002 Malaria parasite, Plasmodium falciparum and mosquito,
Anopheles sequenced
Synthetic poliovirus created from the published sequence of the
virus
2003 Human genome map project completed
2004 Laboratory rat genome sequenced
2005 Draft of Chimpanzee genome released
Rice genome mapped
2006 Regulatory approval for plant-based vaccines against Newcastle
disease of poultry given to Dow Agrosciences
2008 Synthesis of artificial genome of bacteria, Mycoplasma
laboratorium derived from the genome of Mycoplasma
genitalium termed minimal genome project
2009 Draft sequence of Maize revealed
Cows and goats have been engineered to express drugs and other
proteins in their milk
FDA approved a drug produced in goat milk
2010 First synthetic genome of a bacterium, Mycoplasma mycoides
JCVI-syn 1.0 capable of self-replicating revealed by Craig
Venter’s institute
2012 Develop the CRISPR/Cas9 system
(continued)
Alipogene tiparvovec became the first gene therapy treatment to

be approved for clinical use
2013 Entire genome of a Neanderthal, an extinct species of humans

revealed
2015 CRISPR was used to edit the DNA of non-viable human
embryos
The word “cloning” refers to the fact that the method involves
the replication of a single DNA molecule starting from a single
living cell to generate a large population of cells containing identical
DNA molecules. Molecular cloning generally uses DNA sequences
from two different organisms: the species that is the source of the
DNA to be cloned and the species that will serve as the living host
for replication of the recombinant DNA. Molecular cloning meth-
ods are central to many contemporary areas of modern biology and
medicine.
DNA to be cloned is obtained from an organism of interest, the
DNA is then treated with restriction enzymes to generate smaller
DNA fragments. Afterward, these fragments are ligated with vector
DNA which is also digested by same restriction enzyme to generate
recombinant DNA molecules. The recombinant DNA is then
introduced into a competent host organism (typically an easy-to-
grow, benign, laboratory strain of E. coli bacteria). This will gener-
ate a population of organisms in which recombinant DNA mole-
cules are replicated along with the host DNA. Because they contain
foreign DNA fragments, these are transgenic or genetically mod-
ified microorganisms (GMO). This process takes the advantage of
the fact that a single bacterial cell can be induced to take up and
replicate a single recombinant DNA molecule. This single cell can
then be expanded exponentially to generate a large amount of
bacteria, each of which contain copies of the original recombinant
molecule. Thus, both the resulting bacterial population and the
recombinant DNA molecule are commonly referred to as “clones.”
Strictly speaking, recombinant DNA refers to DNA molecules,
while molecular cloning refers to the experimental methods used
to assemble them.
Competence is the ability of a cell to take up extracellular naked
DNA from its environment. Competence may be differentiated
between natural competence, a genetically specified ability of bac-
teria which is thought to occur under natural conditions as well as
in the laboratory, and induced or artificial competence, which arises
when cells in laboratory cultures are treated to make them tran-
siently permeable to DNA.
1.1 Principle
Logarithmically growing E. coli strains (e.g. DH5α cells) when

treated with chilled CaCl2 become able to take up exogenous
DNA during transformation.
Transformation is a genetic alteration of a cell resulting from
the direct uptake, incorporation and expression of exogenous DNA
from its surrounding and taken up through the cell membrane.
When a brief heat shock is given to E. coli strains (e.g. DH5α) in
a media containing exogenous DNA (plasmids), the E. coli strains
take up the DNA. During the brief period of heat shock the pores in
the bacterial membrane widens facilitating intake of the DNA.
Bacteria, such as Escheria coli, has its own chromosomal DNA,
however, also carries an extrachromosomal DNA, known as “plas-
mid” which can replicate by its own capabilities. Plasmid can carry
the foreign DNA fragment into the bacteria, ultimately made it
possible to make multiple copies of it, and thus act as “cloning
vector.” With the advancement of genetic manipulation tools, now
a days it is possible to insert foreign genes in such a way that E. coli’s
cellular machinery will be used not only for making multiple copies
of the plasmid, but also to generate mRNAs from the cloned genes
to translate into functional proteins and the so-called as expression
vector. Proteins obtained by expressing of cloned genes in bacteria
can be used in a variety of ways, such as, for investigating the
structural and biochemical functions of the protein, raising anti-
bodies, and so on. Preferably, an expression vector developed in
such a fashion that it should contain multiple cloning sites, ribo-
somal binding site, transcriptional termination site, and most
importantly a strong inducible promoter under which the foreign
gene can be expressed. With these suitable conditions, the foreign
gene thus can be transcribed, and translated, in the bacterial host
system to produce the functional proteins encoded by the target
genes. Commonly expression vector used for production of recom-
binanat protein is a pET system, which is under the controlling
power of T7 RNA polymerase promoter. pET vector is transformed
into an E. coli host strain (DE3), which contain a copy of the gene
for T7 RNA polymerase (T7 gene 1) under the control of the lac
promoter. Furthermore, the promoter for both target gene and T7
gene 1 also contain the lacO operator sequence and are, therefore,
inhibited by the lac repressor (lacI). IPTG (Isopropyl-beta-D thio-
galactoside) induction allows the transcription of the T7 RNA
polymerase gene whose protein product subsequently activates
the expression of target gene.
Plasmids expressed foreign proteins make it possible to obtain
relatively large amounts of proteins that might otherwise be very
difficult to purify by traditional methods such as gel filtration/ion-
exchange/hydrophobic interaction. These methods for purification
of heterologous recombinant proteins from E. coli are difficult as
well as time consuming. Therefore, what is required is the ability to
impart the target protein with a unique property that can be used to
separate it from all other host proteins. To facilitate the purification

of the expressed protein, “affinity tags” are mostly using. These are
a short DNA sequences that are fused to the coding sequence of the
gene, either at the 50 end or the 30 end. These sequences are
transcribed and translated together with the gene, resulting in the
formation of what is known as a “fusion protein.” The purification
of tagged proteins from host cell consists of four steps (1) Cell lysis;
(2) Binding of the tagged protein to an affinity column; (3) Washing
the column to remove the untagged ones; and (4) elution of the
tagged protein itself. Most commonly used tag is his-tag. When
histidine are used as affinity tag, the fusion protein (target gene plus
6 his residues) expressed in bacteria, bind non-covalently to a
nickel-NTA column. In a technique called immobilized metal ion
affinity chromatography (IMAC), metal nickel are bound to a resin
matrix (nitriloacetic acid or NTA) and used to capture his-tagged
proteins. NTA has four coordinated sites that bind a single nickel
ion very tightly. The charging of NTA with Ni+2 leaves two of six
possible coordination sites of the ion free, which can strongly bind
to imidazole rings of consecutive histidine residues on a polypep-
tide chain. At least six histidine residues are required to provide the
necessary binding affinity to firmly adhere the tagged proteins to
the column. The majority of the host protein will not be able to
bind to the column and thus the contaminating proteins will
washed off and fusion his-tag protein will elute using imidazole.
1.2 Lowry’s Method Lowry protein assay is a biochemical assay for determining the total
level of protein in a solution which invented by the biochemist
Oliver H. Lowry in the 1940s. The total protein concentration is
exhibited by a color change of the sample solution in proportion to
protein concentration, which can then be measured using colori-
metric techniques. The principle behind the Lowry method of
determining protein concentrations lies in the reactivity of the
peptide nitrogen with the copper [II] ions under alkaline condi-
tions and the subsequent reduction of the Folin-
Ciocalteayphosphomolybdicphosphotungstic acid to heteropoly-
molybdenumblue by the copper-catalyzed oxidation of aromatic
acids. The Lowry method is sensitive to pH changes and therefore
the pH of assay solution should be maintained at 10–10.5. The
Lowry method is sensitive to low concentrations of protein ranging
from 0.10 to 2 mg of protein per mL. The major disadvantage of
the Lowry method is the narrow pH range within which it is
accurate. However, we will be using very small volumes of sample,
which will have little or no effect on pH of the reaction mixture. A
variety of compounds will interfere with the Lowry procedure.
These include some amino acid derivatives, certain buffers, drugs,
lipids, sugars, salts, nucleic acids, and sulfhydryl reagents. It should
be noted that ammonium ions, zwitter ionic buffers, nonionic
buffers, and thiolcompounds may also interfere with the Lowry
reaction. These substances should be removed or diluted before

running Lowry assays.
Various methods have been implemented for introducing
cloned eukaryotic DNAs into cultured mammalian cells such as
calcium phosphate, DEAE-dextran mediated, protoplast fusion,
polybrene coating, electroporation, microinjection in nuclei, and
liposome driven delivery system. However, liposome mediated
transfection are most commonly used technique for delivery of
plasmids in mammalian cells. “Liposome” is an artificial membrane
vesicles described by Mannino and Gould-Fogerite (1988), which
involve encapsulation of DNA/RNA within liposome followed by
fusion of the liposome with the cell membrane and inside cell
DNA/RNA released. Liposomes are cationic charges and thus
bind with DNA. After entering inside mammalian cells, the foreign
DNA will replicate, transcribed, and followed by translated using
cellular machinery. Copy number of RNA and concentration of
protein formed inside the cell depend on cell density as well
as live: dead cell ratio. Another issue is how to extract the protein
from mammalian cells. For extraction of recombinant proteins
from mammalian cells, the crucial step is lysis. Cells need to be
lysed such a way that proteins remain in its immunoreactive state
should be undegradable and remain biologically active. In view of a
wide range of physical and biological properties of mammalian
proteins, it is not surprising that no single method of lysis is
sufficient for every purpose. The efficiency of solubilization and
subsequent immunoprecipitation depends on ionic strength and
pH of lysis buffer, concentration and or type of detergents, pres-
ence of divalent cations/cofactors/stabilizing ligands. Although
there are exception, many soluble nuclear and cytoplasmic proteins
can be solubilized by lysis buffer that contains the nonionic deter-
gents such as Nonidet P-40 (NP-40) and either no salt at all or
relatively high concentration of salts (for example, 0.5 M Nacl).
Extraction of membrane bound/hydrophobic proteins are less
effected by the ionic strength of the lysis buffer but often requires
a mixture of ionic and nonionic detergent. When attempting to
solubilize a protein for the first time there are two different strate-
gies can be employed, at one extreme, harsh conditions can be
maintained to ensure that protein is released quantitatively from
cells and on the other hand, gentle conditions can be used to help
preserve the proteins in a native confirmatory state. Denatured
proteins are much more likely to be degraded than native ones;
therefore, it is advisable to take steps to minimize proteolytic activ-
ity in cell extracts, when harsh conditions are applied. It is necessary
to keep the extracts in cold conditions and inhibitors of protease
need to be supplemented in lysis buffer.
2 Materials
1. Gene of interest (GOI).

2. Primers with restriction enzyme sites.
3. Taq DNA polymerase.
4. Restriction enzymes.
5. PCR Machine.
6. Gel Documentation system.
7. Horizontal gel electrophoresis.
8. Vertical gel electrophoresis.
9. SDS PAGE buffers (Tris Buffers, TEMED, Tris-Glycine buffer,
ammonium per sulfate (APS).
10. Acrylamide/Bis-acrylamide.
11. Western blot machine.
12. Ni-NTA agarose.
13. His-tag antibody.
14. Protein purification buffers.
15. Urea.
16. Imidazole.
17. Western blot buffers.
18. Nitro-cellulose membrane or PVDF membrane.
19. Horseradish Peroxidase (HRPO).
20. Anti-species antibody with HRPO conjugate.
21. Chemidoc system.
3 Methods
3.1 Cloning It is carried out to remove non-specific amplified products, con-

taminants, un-used primers, reagents, etc., and separate only the
3.1.1 Purification
target specific PCR product for further processing and cloning.
of Amplified PCR Product
There are several commercial kits available.
By Using Gel Extraction Kit Gel purification of amplified PCR product for cloning may be done
using any gel extraction kit. For example, QIAquick® Gel Extrac-
tion Kit (Qiagen, USA).
1. The DNA fragment was excised from the agarose gel using a
clean sharp scalpel and kept in a colorless tube.
2. The gel slice was weighed and 3 volume of buffer QG was
added to one volume of gel.
3. Incubated at 50 C for 10 min until the gel slice was completely

dissolved.
4. One gel volume of isopropanol was added to the sample and
mixed.
5. QIAquick spin column was placed in the two mL
collection tube.
6. The sample was applied to the QIAquick column and centri-
fuged for 1 min at 16,128 g.
7. The flow through was discarded and the column was placed in
the same collection tube.
8. 0.5 mL of buffer QG was added to the column and centrifuged
for 1 min at 17,900 g.
9. 0.75 mL of buffer PE was added to the column and centrifuged
for 1 min.
10. The flow through was discarded and centrifuged at 16,128
g for additional 1 min.
11. The QIAquick spin column was placed in a clean 1.5 mL
12. 50 μL of buffer EB was added into the center of QIAquick
membrane and centrifuged for 1 min.
13. The DNA was stored at 20 C.
By Using PCR Cleaning Kit The PCR cleaning can be done with any PCR clean up kit such as
QIAquick PCR purification kit (Qiagen). The protocol was
designed to purify single or double stranded DNA fragments
from PCR and other enzymatic reactions.
1. Add 5 vol. of Buffer PB to 1 volume of the PCR sample
and mix.
2. Place a QIAquick spin column in a provided 2 mL
collection tube.
3. To bind DNA apply the sample to the QIAquick column and
centrifuge for 30–60 s.
4. Discard flow through and place QIAquick column back in the
same tube.
5. To wash add 0.75 mL Buffer PE to the column and centrifuge
for 30–60 s.
6. Discard flow through and place the QIAquick column back in
the same tube. Centrifuge the column for another minute at
maximum centrifugation speed.
7. Place QIAquick column in a clean 1.5 mL
8. To elute DNA add 50 μL buffer EB (10 mM Tris-Cl, pH 8.5)

or H2O to the center of the QIAquick membrane and centri-
fuge the column for 1 min.
3.1.2 Preparation Both the vector (e.g. pET32a) and the purified PCR product need
of Gene of Interest to be double digested with respective restriction endonuclease
and Plasmid and Ligation (RE) enzymes in separate reaction conditions. For example, RE
with BamHI and XhoI as in the following.
Preparation of BamHI/XhoI
Cut Insert DNA (PCR Components Volume (μL)
Product) PCR product 10
10 RE buffer 2
BamHI 5
XhoI 3
Total volume 20
Incubate the reaction mixture at 37 C for 3–4 h. After diges-

tion inactivate the enzyme at 65 C for 10 min.
Preparation of BamHI/XhoI Set up the reaction as follows:

Cut Vector DNA (PCR
1. 5 μL pET32ab DNA (2 μg) + 4 μL 10 buffer+1 μL
Product)
BamHI + 1 μL XhoI+ 27 μL MQ water.
2. Set up 10 tubes (total volume 40 μL).
3. Incubate at 37 C for 1 h.
4. Add 1 μL BamHI and 1 μL XhoI (second addition).
6. Run 3 μL sample on gel to check the extent of digestion.
7. Incubate at 67 C for 20 min.
8. Pool all tubes into 2 tubes of 200 μL each.
9. Ammonium acetate precipitation.
10. Resuspend in 30 μL of water.
11. Add 50 μL MQ water +9 μL 10 CIAP buffer +1 μL 10
diluted Alk phosphatise (diluted in 1 buffer).
13. Add 1 μL 10 freshly diluted Alkaline phosphatise.
14. Incubate for 1 h.
15. Heat inactivation is done by heating at 67 C for 20 min.
Gel Purification of Digested Following complete digestion load the RE digested products into
Insert DNA and Plasmid separate wells of 1% agarose gel. Run the gel and follow the proce-
dure of gel extraction/purification of products as described earlier.
Quantify the product by UV spectrophotometer. Store the gel
eluted products at 20 C until further use.
3.1.3 Ligation of Insert Ligation is the process of covalent linking of two ends of insert
and Vector DNA/gene molecule with ends of vector/plasmid DNA using T4
DNA ligase enzyme.
The amount of Vector and insert required for the ligation were
calculated as per the following formula:
Size of the insert ðbpsÞ
amount of vectorðngÞ 3
Size of the vector ðbpsÞ
For efficient ligation set up the ligation reaction as follows:
Component Volume
Vector pET32a 1 μL
10 ligation buffer 1 μL
PCR product 3–4 μL
T4 DNA ligase 1 μL
Water, nuclease- free To 10 μL
Total volume 10 μL
1. Vortexed briefly and centrifuged for 3–5 s.

2. Incubated at 22 C for 2.5 h followed by 4 C for overnight.
3. 5 μL of ligation reaction was used directly for transformation.
3.1.4 Preparation 1. A single bacterial colony (e.g. DH5α) was picked up and
of Competent Cells inoculated in a fresh 15 mL autoclaved falcon tube containing
3 mL LB media for incubation at 37 C and 180 rpm in a
shaker incubator for overnight.
2. 0.5 mL of overnight culture was inoculated in to 50 mL of LB
broth and incubated as above until the bacteria reach log phase
(for around 3 hrs.) or until the OD reach 0.35–0.4.
3. After that keep the flask and the Oak Ridge tube on ice for
30 min to 1 h.
4. Pour the contents of the conical flask in four Oak Ridge tube
and centrifuge at 4032 g for 10 min at 4 C.
5. The supernatant was decanted under laminar flow and 10 mL
of ice cold 100 mM CaCl2 was added to the tube and kept on
ice for 30 min.
6. Tubes were centrifuged at 4032 g for 10 min, supernatant

was decanted under laminar flow.
7. 2 mL of CaCl2 (containing 15% glycerol) in each tube.
8. Aliquot the contents in 1.5 mL tubes and kept at 20 C for
overnight then kept at 80 C until further use.
3.1.5 Transformation 1. 50 μL of competent DH5α from 80 C was thawed on ice.

and Plating 2. 5 μL of ligated PCR product was mixed with 50 μL competent
cell and kept on ice for 30 min.
3. The mixture was subjected to shock at 42 C for 1 min, fol-
lowed by 2 min on ice.
4. 1 mL of LB broth was added to the tube and mixed by inver-
sion, incubated at 37 C in a shaker incubator for 2 h.
5. Centrifuge at 2800 g for 2 min.
6. 50 μL of supernatant was taken and rest discarded and the
pellet was reconstituted in 50 μL supernatant.
7. This 50 μL was spreaded over LB agar plate with ampicillin
(50 μg/mL), X-gal (30 μg/mL), and IPTG (40 μg/mL).
8. The Petridish was incubated at 37 C for overnight.
3.1.6 Screening Screening of recombinant clones is done by the following methods.

of Recombinant Clones
1. Observing growth of bacteria containing the recombinant plas-
mid in the presence of particular antibiotic (antibiotic selec-
tion)—the vector has the property of resistance to a particular
antibiotic and it gives the antibiotic resistance to the bacteria
which is otherwise susceptible to that antibiotic. Therefore, the
bacteria containing the recombinant plasmid will grow but the
others will not.
2. Amplification of insert DNA from recombinant plasmid con-
struct/colony PCR using specific primers.
3. Restriction analysis of the plasmid/ PCR amplicon in agarose
gel for identification of the insert.
3.2 Expression 1. Transform the plasmid into competent E. coli cells and plate on
in Prokaryotic Host LB (antibiotic added) plates. Incubate overnight at 37 C and
System then re-streak a single colony.
3.2.1 Small Scale 2. Inoculate 5 mL of LB media containing antibiotics with a
Production of Recombinant
single colony. Let the tube stand overnight at 37 C.
Protein 3. Inoculate 10 mL of LB containing antibiotics with 1/20th
(500 μL) of overnight growth culture. Incubate with aeration
at 37 C until the culture reaches 0.5–0.6 OD600.
4. Remove a 1 mL sample to a 1.5 mL microcentrifuge tube and
centrifuge for 1 min. Discard the supernatant and resuspend
the pellet in 150 μL of 2 sample buffer. This is the uninduced

protein samples which may be stored at 20 C. Immediately
induced the remaining culture by adding IPTG to a final con-
centration of 1 mM and resume incubation.
5. After -13 h, remove a sample and process it as in step 4. If
performing a time course optimization, remove and process
samples at several intervals after induction.
6. Mix the protein sample with equal volume of Lamelli buffer.
Heat all samples to 95 C for 5 min and clarify by centrifuga-
tion for 1 min in a microcentrifuge. Load 10–20 μL of each
samples on an SDS-Polyacrylamide gel. Apply protein molecu-
lar weight standards in adjoining lanes. Electrophoresis until
the bromophenol blue dye migrates to the end of the gel.
7. Fix and stain the gel with Coomassie Brilliant Blue (CBB) dye
after electrophoresis. Induced proteins are identified by com-
parison with uninduced protein control lane.
3.2.2 Large Scale 1. Transform the plasmid into competent E. coli cells and plate on
Production of Recombinant LB (antibiotic added) plates. Incubate overnight at 37 C and
Protein then re-streak a single colony.
2. Inoculate 5 mL of LB media containing antibiotics with a
single colony. Let the tube stand overnight at 37 C.
3. Use the entire 5 mL overnight culture to inoculate 500 mL LB
containing antibiotics and incubate with shaking until
OD600 ¼ 0.5.
4. Remove a 1 mL of the culture (uninduced sample) to a 1.5 mL
microcentrifuge tube and centrifuge for 1 min. Discard the
supernatant and resuspend the pellet in 150 μL of 2 sample
buffer.
5. Immediately induced the remaining culture by adding IPTG to
a final concentration of 1 mM.
6. Grow for 2 h at 37 C with shaking (the length of induction
depends upon the previous optimized times).
7. Remove a 1 mL of the culture (induced sample) to a 1.5 mL
microcentrifuge tube and centrifuge for 1 min. Discard the
supernatant and resuspend the pellet in 150 μL of 2 sample
buffer.
8. Mix the protein sample with equal volume of Lamelli buffer.
Heat all samples to 95 C for 5 min and clarify by centrifuga-
tion for 1 min in a microcentrifuge. Load 10–20 μL of each
samples on an SDS-Polyacrylamide gel. Apply protein molecu-
lar weight standards in adjoining lanes. Electrophoresis until
the bromophenol blue dye migrates to the end of the gel.
9. Fix and stain the gel with Coomassie Brilliant Blue

(CBB-R250/G-250) dye after electrophoresis. Induced pro-
teins are identified by comparison with uninduced protein
control lane.
3.3 Recombinant E. coli cells containing an inducible expression vector need to grow
Protein Purification and induced to produce the tagged fusion protein.
• The cells will be lysed and insoluble debris will be removed by
centrifugation.
• The supernatant from step 2 will be applied to a Ni+2-NTA
column.
• The column will be washed with a low concentration (20 mM)
of imidazole, which will compete with low-affinity histidine-
column interactions to remove from the column any, perhaps
histidine-rich, proteins that are non-specifically bound.
• Finally, the tagged protein itself is removed from the column by
various ways.
– Increasing the concentration of the imidazole to a high level
(250 mM).
– Alternatively, elution conditions with lowering the pH from
8 to 4.5, which will alter the protonated state of histidine
residues and results in the dissociation of the protein from
metal complex.
– The tagged proteins also can be removed by adding chelat-
ing agents for instance, EDTA, to strip nickel ions from the
column and consequently removed the tagged protein.
• Proteins will be visualized by staining of SDS gel with Coomas-
sie blue dye.
Ni-NTA Column-Based Purification of Recombinant Protein
1. Take 10 mL of prewarmed media and add 1/20th (500 μL) of
overnight grown culture.
2. Keep at 37 C for 30 min at orbital shaker and check the OD600
should reach around 0.5–0.7.
3. Induced with 1 mM IPTG and grow the culture for an addi-
tional 4–5 h.
4. Harvest the cells after centrifugation at 15,000 rpm for 1 min
and discard the supernatant.
5. Resuspend the cell pellet in 400 μL of lysis buffer (pH 8.0).
6. Lyse the cell by gentle vortexing.
7. Centrifuge the lysate for 20–30 min at 15,000 rpm.
8. Collect the supernatant in fresh tube.
9. Equilibrate the Ni-NTA spin column with 600 μL lysis buffer

(pH 8.0).
10. Centrifuge for 2 min at 448 g.
11. Load the clear lysate supernatant on equilibrate spin column.
12. Centrifuge the spin column for 2 min at 448 g.
13. Collect the flow through.
14. Wash the spin column twice with 600 μL wash buffer(pH 6.3).
15. Centrifuge for 2 min at 448 g.
16. Elute the protein with 100–200 μL elution buffer(pH 4.5)
after centrifugation for 2 min at 448 g.
17. Collect the elute and check in SDS PAGE.
3.4 Western Blot 1. After electrophoresis, the gel was washed three times in transfer
Analysis Using buffer at a 5 min interval. The nitrocellulose membrane
Anti-His Conjugate (NCM) to be utilised for transfer was pre-wetted for at least
30 min in transfer buffer.
2. Three Whatman No.3 filter papers were cut to the size of the
gel and soaked in the transfer buffer and were placed on the
anode plate over which the pre-wetted membrane was kept
making an orientation marks on it. Transfer buffer was poured
over the gel and pressed carefully to exclude the excess buffer
and air bubbles.
3. Three Whatman No.3 filter papers soaked in ice-cold transfer
buffer was placed over the gel.
4. The cathode plate was replaced in position and a constant
current of 35 mA was applied for 60 min.
5. After the transfer, the gel was removed and washed twice for
10 min with TBS-T at room temperature.
6. The membrane was blocked overnight at 4oC with blocking
buffer (5% skim milk powder in TBS-T).
7. The membrane was washed twice with TBS-T for 10 min
followed by TBS wash twice for 10 min at room temperature.
8. Post-wash the membrane was incubated with Ni-NTA HRPO
conjugate (1:1000 dilution) for 1 h at 37 C.
9. After 1 h the membrane was washed with TBS-T and TBS as
mentioned in the previous step.
10. Then the membrane was developed by dissolving 0.5 mg/mL
of 3, 30 -diaminobenzidine tetra hydrochloride (DAB)(M/s
BIO BASIC INC.) and 1 μL per mL of 3% H2O2 (M/s
Sigma-Aldrich, St. Louis, USA) solution in 20 mL of TBS.
The membrane was soaked in the developer solution for
15 min at 37 C and then the color reaction was stopped by
addition of excess distilled water. After stopping the reaction,

the membrane was air dried and documented.
3.5 Protein 1. 2% Na2CO3 in 0.1 N NaOH.

Estimation 2. 1% NaK Tartrate in H2O.
3.5.1 Reagents 3. 0.5% CuSO4.5 H2O in H2O.
4. Reagent I: 48 mL of A, 1 mL of B, 1 mL C.
5. Reagent II- 1 part Folin-Phenol [2 N]: 1 part water.
6. BSA Standard—1 mg/ mL.
3.5.2 Protocol 1. 0.2 mL of BSA working standard in five test tubes and make up
to 1 mL using distilled water.
2. The test tube with 1 mL distilled water serves as blank.
3. Add 4.5 mL of Reagent I and incubate for 10 min.
4. After incubation add 0.5 mL of reagent II and incubate for
30 min.
5. Measure the absorbance at 660 nm and plot the standard
graph.
6. Estimate the amount of protein present in the given sample
from the standard graph.
3.5.3 Bradford Method The protein in solution can be measured quantitatively by different
methods. The methods described by Bradford uses a different
concept-the protein’s capacity to bind to a dye, quantitatively.
The assay is based on the ability of proteins to bind to Coomassie
brilliant blue and form a complex whose extinction coefficient is
much greater than that of free dye.
3.5.4 Reagents • Dissolve 100 mg of Coomassie Brilliant blue G250 in 50 mL of

95% Ethanol.
• Add 100 mL of 85% phosphoric acid and make up to 600 mL
with distilled water.
• Filter the solution and add 100 mL of glycerol, then make up to
1000 mL. The solution can be used after 24 h.
• Bovine serum albumin (BSA).
3.5.5 Protocol 1. Prepare various concentration of standard protein solutions

from the stock solution (say 0.2, 0.4, 0.6, 0.8, and 1.0 mL)
into series of test tubes and make up the volume to 1 mL.
2. Pipette out 0.2 mL of the sample in two other test tubes and
make up the volume to 1 mL.
3. A tube with 1 mL of water serves as blank.
4. Add 5.0 mL of Coomassie brilliant blue to each tube and mix

by vortex or inversion.
5. Wait for 10–30 min and read each of the standards and each of
the samples at 595 nm.
6. Plot the absorbance of the standards verses their concentration.
7. Plot graph of optical density versus concentration. From graph
find the amount of protein in unknown sample.
3.6 Expression 1. Take a 45 mL sterile media in a 50 mL conical flask after passing

of Recombinant through 0.22 μm filter.
Protein in Eukaryotic 2. Add 5 mL (10%) FBS in the media.
System
3. Add 1 (500 μL) of desired antibiotics tin the media.
3.6.1 4. Now remove the pre media from the parent flask after rigorous
Lipofectamine-Based shaking.
Transfection
5. Treat with 2–3 mL of Trypsin Versene Glucose (TVG) for
in Mammalian Cells
2–3 min at 37 C.
6. Now when cells become rounded in shape, add 2–3 mL of
media, shake rigorously for several times.
7. Add 15 mL of growth media and distribute in 6 well plates,
keep at 37 C in 5% CO2 incubator till the monolayer formed.
8. Next day, remove the media and supplement the cells with
2–3 mL OptiMEM (without serum and antibiotics)/well
after 2–3 time washing with the same media and keep at CO2
incubator at 37 C.
9. Prepare the DNA-lipofectamine complex as follows.
• Dilute the plasmid DNA (500–750 ng) in 500 μL Opti-
MEM solution, mix thoroughly.
• Mix lipofectamine solution gently before use.
• Add 3.0–4.5 μL of lipofectamine directly to the diluted
DNA, mix thoroughly.
• Incubate for 1 h at room temperature.
10. Add 500 μL of DNA-lipid complex dropwise to the well con-
taining cells and mix gently by rocking the plate back and forth.
11. Keep at 37 C in 5% CO2 incubator for 4–6 h and remove the
media and supplement with fresh OptiMEM.
3.6.2 Cell Lysis 1. Add the lysis buffer of choice (pre-cooled at 0 C) to wash the
and Protein Extraction cell monolayer. Incubate for 20 min on a flat aluminum tray on
a bed of crushed ice.
Volume of lysis buffer (mL) Size of wells (mm)

1.0 90
0.5 60
0.25 35
0.25 30
2. Scrap the cells to one side of the dish with a cell scrapper.
3. Centrifuge the lysate at 12,000 g for 2 min at 4 C.
4. Transfer the supernatant to a fresh microfuge tube and store it
on ice or at 70 C depending on the sensitivity of the target
antigen for freezing and thawing.
5. Now the supernatant will be mixed with PAGE loading dye and
run the sample in SDS PAGE as described earlier,
6. Furthermore, the presence of protein can be determined by
Western blot/Immune fluorescent assay/Immune
peroxidase test.
Chapter 8
RNA-PAGE-Based Diagnosis of Viral Diseases

Naveen Kumar, Geetika Kaur, Shubhankar Sircar, Zunjar Dubbal,
R. S. Sethi, and Yashpal Singh Malik
Abstract
Regardless of low resources, many of the techniques have proven indispensable in every laboratory around
the world, particularly in developing countries. Polyacrylamide gel electrophoresis (PAGE) is one of them,
and it is a useful tool for studying RNA from biological samples. PAGE can provide information on the size,
content, and quality of RNA, as well as resolving RNA-protein complexes, depending on the type of PAGE
used. In the presence of an electric current, RNAs move toward the anode because they are negatively
charged. The polyacrylamide gel works as a sieve, preventing RNA from migrating in proportion to its mass,
assuming that its mass is primarily proportionate to its charge. Furthermore, because the chain length is
almost proportional to its mass, the length of an RNA is usually governed by its migration. This chapter
outlines the detailed step-by-step procedures for setting up instruments, preparing samples, loading and
running samples on gels, staining gels, and lastly visualizing stained gels.
Key words Electrophoresis, AGE, RNA-PAGE, Genomic Segmentation, Virus Detection, Silver
staining
1 Introduction
Polyacrylamide gel electrophoresis (PAGE) is a widely accepted

important technique in Ribonucleic acid (RNA) analysis and has
been in use in several laboratories across the world for diverse
applications such as RNA detection, purification by size, assessment
of genomic quality, and nature [1]. In principle, PAGE can be
classified as denaturing or non-denaturing PAGE. In denaturing
PAGE, the sample composition as well as the structural integrity of
individual RNA species can be assessed whereas the separation of
conformers and alternatively folded RNA species can be deter-
mined using non-denaturing gel electrophoresis [2]. Denaturing
PAGE is more appropriate for various electrophoretic procedures
and can resolve RNAs from ~600 to 20 nucleotides [3]. It
distinguishes macromolecules based on linear length and mass-to-
charge ratio. In addition, by assessing changes in the
127
128 Naveen Kumar et al.
electrophoretic mobility of the RNA samples, this method can be

employed to resolve protein-RNA complexes as well as predict the
formation of RNA complexes. However, non-denaturing or native
PAGE is more useful in differentiating RNA conformations and
RNA-protein complexes. The only limitation with PAGE is that it
cannot analyze RNAs of large sizes consisting more than
600 nucleotides. In such cases of large size RNA analysis, agarose
gel is the method of choice as it is easy to perform, less laborious,
economical, and time-saving compared to the RNA-PAGE method
which is complicated, laborious and needs an expert to perform
[4]. Similar to PAGE, acrylamide gel electrophoresis (AGE) can be
used for the differentiation of the rotavirus dsRNA not only within
a single animal species but also between various species.
PAGE utilizes the key properties of movement of charged
biomolecules (nucleic acids) under the electric fields and therefore,
RNA analysis can easily be achieved due to the RNA biomolecules
migration across the polyacrylamide meshes according to their
charge and the strength of the electric field. The negatively charged
RNA molecules migrate towards the anode under the influence of
an electric field. The sieving effect of the agarose gel allows the
RNA to pass through the gel based on the mass proportion of the
RNA segment, which is directly proportional to the possessed
charge. As the mass is invariably related to the length of RNA, it
can be principally determined through migration analysis. Further-
more, morphological characteristics such as topology also influ-
ences migration behavior, causing the RNA to appear longer than
it is. Moreover, in denaturing PAGE, the separation occurs largely
according to the size of the RNA biomolecules, whereas in
non-denaturing PAGE, RNAs mobility is determined by both the
size and conformation [5]. Usually, 0.4–1.5-mm-thick gels are
used for RNA-PAGE analysis. Depending on the type of the detec-
tion reagent, RNA can be stained and visualized in gels by using a
variety of chemicals such as Silver nitrate, Toluidine blue, SYBR
green, and ethidium bromide. Autoradiography can be used to see
radioactively labeled RNA molecules, and a fluorescence scanner
can see fluorescently labeled RNA molecules. However, we have
limited our protocol to the silver staining method, which is fre-
quently used for the visualization of RNA migration patterns with
the naked eye.
2 RNA-PAGE Principle
Poly Acrylamide Gel Electrophoresis (PAGE) is one of the best

methods in protein identification, molecular weight determination,
protein–protein or protein–DNA interaction, etc. It can be also
used for the separation of nucleic acid (RNA) from various viruses.
Protein to be analyzed needs to be mixed with a reducing agent like
RNA-PAGE-Based Diagnosis of Viral Diseases 129
Dithiothreitol (DTT) and Sodium Dodecyl Sulfate (SDS). Before

loading the samples, heating of the samples is pre-requisite that
precipitates the proteins and binding of SDS to the backbone of
denatured proteins provides a negative charge to make them solu-
ble. SDS has a hydrophobic tail and a charged polar ion, thus it gets
binds to the hydrophobic backbone of the protein. On electropho-
resis, these proteins get migrated based on the molecular weight.
Polyacrylamide gels are formed by the polymerization of the
monomer acrylamide crosslinked to the co-monomer, N,N0 -
methylenebis-acrylamide (BIS). In this process, free radicals are
generated during polymerization by ammonium persulfate, and a
catalyst such as N,N,N0 ,N0 -tetraethylmethylenediamine
(TEMED). The advantage of PAGE is that the pore size of the
gel can be controlled by using different concentrations of acrylam-
ide during gel formation.
3 Sample Preparation: RNA Extraction from Clinical or Biological Samples
Phenol-chloroform method
1. Add pre-warmed solution of 1 M sodium acetate with 1% SDS
in a microcentrifuge tube containing about 100–200 μl of
sample (fecal sample) to make 10% fecal suspension and centri-
fuge at 12,000 g for 20 min to remove coarse particles and
cellular debris.
2. Vortex for 10 s and incubate for 15 min at 37 C.
3. Add an equal amount of phenol: chloroform: Isoamyl alcohol
(25:24:1).
4. Vortex again for 1 min followed by incubation at 56 C for
15 min.
5. Vortex the mixture and centrifuge it for 3 min at 16,900 g.
6. Take a fresh 1.5 mL micro centrifuge tube and transfer the
upper aqueous phase.
7. Add chloroform: Isoamyl alcohol (24:1) solution to the same
tube and repeat the steps 4–6.
8. Further, to this, add 1/10 volume of 3 M sodium acetate
(pH 5.0) and an equal volume of isopropanol.
9. Invert tube to mix gently and incubate at room temperature for
overnight.
10. Finally, centrifuge for 15 min at 12,000 rpm (4 C) for RNA
pelleting and wash it with 70% chilled ethanol followed by
centrifuging at 16,900 g for 5 min. Immediately, decant
the ethanol and air-dry the pellet.
11. Re-suspend the RNA pellet in 30 μl RNA loading buffer using
a pipette.
4 Procedure: From Setting Up PAGE Apparatus to Stained Gels Visualization
4.1 Electrophoresis Several commercial companies sell electrophoresis equipment and

in Cylindrical Gels reagents suitable for cylindrical gel electrophoresis. Davis [6] iden-
Using Vertical tified a low-cost simple setup consisting of tubes where samples
Apparatus stack up in contiguous discs in a cylindrical gel of low concentration
before entering the higher concentration gel, in which the separa-
tion actually occurs. A distinct advantage of this set up is that the
reservoir located upwards is not adjoined/locked to that of the
reservoir located downward, preventing the entire apparatus from
being dismantled if the user wants to remove a specific tube at a
different time period during the ongoing run. The apparatus with
the rectangular boxes arranged in a straight line is considered to be
more convenient because it allows for easier monitoring of the
tracking dye during the gel electrophoresis process [7].
4.2 Apparatus Setup There are several advantages of using rectangle-shaped gel slabs as
for Gel Electrophoresis electrophoresis matrices rather than cylinder-shaped gel columns.
in Rectangle-Shaped In single gel slab methodology, the user can easily separate and
Gel Slabs compare the samples by maintaining identical conditions such as
temperature, pH, voltage gradient, and current [8].
4.3 Power Supply The best and most appropriate power supply for electrophoresis has
several constraints while facilitating and providing sufficient and
desirable current and voltage. As catalysts pass ion boundaries and
final products move out of the gel, the resistance potential of the
polyacrylamide gel gradually increases during overall electrophore-
sis. There is a detectable difference in the gel when these ion
boundaries pass out of the gel at various regions. A certain and
constant amount of current supply is needed to promote a constant
voltage gradient in the region of migrating RNA molecules. This
mechanism would also ensure that the distance traveled by these
molecules changes linearly over time. There is about a quarter of
the current shift during the overall run.
5 Method
Before starting the RNA-PAGE procedure, make sure of all of the

required equipment and reagents. To reduce the background for
direct ultraviolet scanning of gels, impurities that absorb ultraviolet
radiation should be removed. Highly purified acrylamide and
bis-acrylamide are used to prepare both diluted and
concentrated gels.
5.1 Preparation 1. Assemble the glass plates for gel casting and wash the gel plates
of Gel Plates with lab grade detergent and distilled water before each use.
Glass plates must be thoroughly dried, cleaned with ethanol on
the inside, and labeled on the outside. Furthermore, any resid-
ual dried acrylamide or any other substance on glass plates
should be properly cleaned with a fresh single-edge razor
blade so that the glass plates are not scratched.
2. A siliconizing agent or ideally a commercial alternative such as
Rainex must be applied on the inner surface of notched plates.
Cover the gel plates uniformly with Rainex, taking care not to
have any Rainex on the edges or the other surface of the gel
plate. After applying Rainex, plates should be air-dried and
washed with a lint-free tissue, such as Kim Wipes®. Further-
more, plates must be rinsed with water and then dried
before use.
5.2 Preparation Mark the top level of the resolving gel on the plate with a marker
of Solutions Used pen while ensuring to leave space above the resolving gel for the
in Gel stacking gel. Make different buffers as follows:
Buffers Methods of preparation

30% acrylamide stock Dissolve 29.2 g of acrylamide and 0.8 g of N,N0
methylene bis-acrylamide in 100 mL of
distilled water. Filter before use. Place the
solution in a dark or foil-covered bottle, and
store at 4 C
Resolving gel buffer Dissolve 18.15 g of Tris base in 60 mL of
(1.5 M, pH 8.8) distilled water. Adjust the pH to 8.8 with 1 N
HCl. Make up to 100 mL with distilled water.
Filter through Whatman no. 1 filter paper and
store at 4 C
Stacking gel buffer Dissolve 6.0 g of Tris base in 60 mL of distilled
(0.5 M, pH 6.8) water. Adjust the pH to 6.8 with 1 N HCl.
Make up to 100 mL with distilled water. Filter
through Whatman no. 1 filter paper and store
at 4 C
10% (w/v) ammonium Dissolve 0.1 g of APS in 1 mL of distilled water
per sulfate (APS) just before use. Store at 4 C for a maximum
of 3 days
1 Tris-glycine running Dissolve 3.0 g of Tris base and 14.4 g of glycine
buffer in distilled water, and make up to 1000 mL
with distilled water
2 RNA-PAGE sample Dissolve 400 μL of 0.05% bromophenol blue,
loading buffer 200 μL of 10% SDS, and 3 mL of glycerolin
5 mL of stacking gel buffer. Make up to
20 mL with distilled water
5.3 Assembling 1. Place spacers on the inside edges of the PAGE plate
of the Gel (10 8 cm 1.0 mm) and cover it with the other/second
plate (treated) side down; do not move the spacers while fixing
the plates and keep the plates in place with the help of binder
clips attached on one side of the plates.
2. Fix a piece of tape over the unclipped side and the bottom part;
smoothen the tape with a single edged razor by applying the
no-sharp side over the tape. Extend the tape over gel spacer
tabs if the small plate does not have “ears.” One-third of the
way up from the bottom, clip the tape-covered side. It is
necessary to ensure that the clip end is placed over the spacer
and tape.
3. Remove the clip from the other side and tape the plates up
using the same method as before. Apply tape to the bottom of
the plate, covering a small area next to it by a few inches.
Combs should be checked regularly to ensure that they are
fixed properly.
5.4 Prepare the 10% Prepare the resolving and stacking gels as follows:
Resolving and 5%
Stacking Gels
Resolving gel (10%) Stacking gel (5%)
Stock
solution 5 mL 10 mL 15 mL 20 mL 1 mL 2 mL 3 mL 4 mL
Distilled 1.9 4 5.9 7.9 0.68 1.4 2.1 2.7
water
30% 1.7 3.3 5.0 6.7 0.17 0.33 0.5 0.67
acrylamide
1.5 M Tris 1.3 2.5 3.8 5.0 – – – –
HCL
(pH 8.8)
0.5 M Tris – – – – 0.13 0.25 0.38 0.5
HCL
(pH 6.8)
10% APS 0.05 0.1 0.15 0.2 0.01 0.02 0.03 0.04
TEMED 0.002 0.004 0.006 0.008 0.001 0.002 0.003 0.004
1. Pour 10% resolving gel solution down the side of the balanced
plates by gently moving the plate to the other corner side when
the gel hits the bottom corner. This will allow the solution to
spread evenly over the bottom surface. Maintain a steady sup-
ply of gel solution to fill the remaining space in the plates, and
gently move the plates to ensure that they are fully filled from
the bottom. Allow the separating/resolving gel to solidify.
Then apply a layer of water-saturated iso-butanol to the gel
(to ensure the formation of an even interface and for the

exclusion of oxygen).
2. After solidification of the resolving gel, gently pour the stack-
ing gel over the resolving gel, and when the gel solution
reaches the top of the plates, immediately insert the comb in
the gel and clamp the two corners of the gel plates to keep the
comb in place. Most importantly, make sure that there are no
bubbles around the wells in the gel; if there are bubbles,
eliminate them by tightening the comb again. Otherwise, a
thin film of polymerized acrylamide will form between the
plates and wells which will subsequently interfere with the
loading of RNA samples.
3. Additionally, leave a small amount of gel solution in the flask to
confirm the polymerization of gel. Polymerization would take
about 15–20 min, and the partition between the comb’s teeth
would need to be formed. Furthermore, the refractive index of
the gel changes during the polymerization, which can be visua-
lized as a “Schlieren line” near the edge of the gel plate and
comb. The difference in refractive index between the polymer-
ized and un-polymerized acrylamide causes this effect.
5.5 Set Up the Gel 1. To begin, remove all tapes and clips from the bottom side of
After Polymerization the gel, and rinse the top side of the gel near the comb with
distilled water. The comb should then be carefully removed,
and the wells could be rinsed with the distilled water using a
syringe or pipette.
2. Place the shorter side of the gel plates inwards on the gel
apparatus. Maintain equal pressure on both sides of the clamps
to secure them in place.
3. Fill the bottom and top chambers with an appropriate amount
of 1 Tris-glycine running buffer. Rinse the wells with TBE
using a syringe to prevent the formation of air bubbles in the
wells as well as in the bottom step of the gel.
4. Close the lids gently and attach the electrodes before starting
the pre-run at 45 mA for 30–45 min. A pre-run helps in
reducing excess of persulfates and eliminates hyper focusing.
For the analysis of short RNA with a size of lesser than or equal
to 50 nucleotides, longer pre-runs of nearly 45 min are usually
recommended.
5.6 Loading the Gel 1. Firstly, turn off the power supply.
2. Add 2 RNA-PAGE sample loading buffer to RNA samples
and heat them between 90 and 95 C before snap chilling them
on ice for 5 min.
3. Rinse the wells repeatedly with the help of a syringe. This

practice will make it easier for the sample to settle and disperse
upward.
4. Load the RNA samples quickly with a RNAse and DNAse free
micropipette. Continue to prevent the bubble formation,
which may cause sample disturbance. Fix the sample loading
volume in accordance with the width and thickness of the well.
5.7 Running the Gel 1. A gel running should be carried at a stable voltage (80 V) till
the dye comes out of the gel. To monitor the heat in the
apparatus, use silicone grease to attach a thermometer to the
front plate. The average temperature range must be between
57 and 58 C. An increase in voltage and current could cause
the gel to overheat or develop cracks. This activity can result in
band distortion in the gel as well as other unusual activities
within the gel matrix. To avoid the risk of overheating, high
percentage gels should be run at a constant current rate. The
milliamps or current capacity could be determined by taking
into account the gel percentage and size.
2. Run the gel at a suitable distance to allow a better band resolu-
tion. The dyes comprising xylene cyanol and bromophenol
blue in the loading buffers still migrate to similar positions,
but they migrate along with the variable sized nucleic acids
depending on the gel percentage. To achieve higher reproduc-
ibility, gels should be run under identical conditions, and it is
therefore advised to become familiar with the gel to be used for
further work to prevent any complications or work odds.
5.8 Disassembling 1. Turn off the power supply and remove/drain the buffer from
the Gel the gel apparatus. If the RNA samples were radioactively
labeled, use caution when handling the apparatus.
2. After that, detach the plates and remove the yellow tape by
pulling it off or with a razor blade. Afterward, place the plate
on a flat surface.
3. Carefully remove the gel remnants from the gel surfaces.
4. Gels may also be processed by wrapping them in plastic wrap
and drying them thoroughly with a vacuum-driven gel drier.
5.9 Silver Staining The RNA–PAGE in combination with silver staining is a sensitive
and time-saving technique for the diagnosis of various viral dis-
eases. After electrophoretic separation on polyacrylamide gels,
nucleic acids are detected using silver staining [9]. It combines
high sensitivity with easy and inexpensive equipment and chemicals.
RNA fixation, sensitization, silver impregnation, and image devel-
opment are the stages of silver staining in order. Silver staining can
be done in a variety of ways and takes anywhere from 2 h to a day
after the electrophoretic separation is completed. Since the method

of silver staining is a delicate and temperature-dependent, so some
protocols perform poorly when the temperature is either below
20 C or above 30 C [10].
After electrophoresis, the first step in gel staining is fixation
which results in RNA binding to the gel matrix and removal of
buffer components from gels. Fixation is performed in fixing solu-
tion (10% ethanol and 0.5% acetic acid) for around 30 min
[11]. After that, rinse the gels twice in distilled water for 1 min
each time. Silver Nitrate solution (0.185 g AgNO3 in 100 mL
distilled water) is prepared just before use and the gel is dipped in
it for 30 min at room temperature on the gel rocker. After staining
with silver nitrate, the gel is treated with developing solution (add
1.5 mL of 36% formaldehyde to 6.0 g of NaOH, and make up to
200 mL with distilled water, prepare fresh before use) and kept for
about 5 min at room temperature or until RNA bands are visible.
The developing step should be carried out in dark place. After that,
drain off the developing solution, and add the stopping solution
(add 10 mL of acetic acid to 190 mL of distilled water) to prevent
further color development. Furthermore, keep the gel for
5–10 min at room temperature in the stopping solution before
rinsing in distilled water. Finally, dry the gel in a standard vacuum
gel dryer; the gel may also be temporarily stored in a 20% ethanol/
1% glycerol mixture or in a 5% acetic acid solution [10].
RNA molecules inside gels can be visualized using a variety of
methods depending on the detection reagents. There are a varieties
of dyes such as SYBR green, toluidine blue, and ethidium bromide,
which can be used to stain RNAs. Autoradiography can be used to
observe radioactively labeled RNA molecules, while a fluorescence
scanner can be used to visualize fluorescently labeled RNA
molecules [12].
Many RNA viruses have genetic material in the form of seg-
mented genomes, which can be visualized using RNA-PAGE, such
as Influenza A virus (eight segments), Bluetongue virus (ten seg-
ments), Coltivirus (Twelve segments), Rotavirus (11 segments),
Picobirnavirus (two segments), Bunyavirus (tripartite genomes),
Arenavirus (two segments), Birnavirus (two segments), and Infec-
tious Pancreatic Necrosis Virus (IPNV, two segments). RNA-
PAGE is unquestionably the most effective technique for RNA
analysis, including viral RNA identification, quantification, size
purification, and even mixed infection detection in gel electropho-
resis, and it will continue to be an indispensable tool for RNA
biology research.
References
1. Duesberg PH, Cardiff RD (1968) Structural 6. Davis BJ (1964) Disc electrophoresis II:
relationships between the RNA of mammary method and application to human serum pro-
tumor virus and those of other RNA tumor teins. Ann N Y Acad Sci 121:321–349
viruses. Virology 36:696 7. Adesnik M (1971) Polyacrylamide gel electro-
2. Rio DC, Ares M Jr, Hannon GJ, Nilsen TW phoresis of viral RNA. In: Methods in virology,
(2010a) Nondenaturing agarose gel electro- vol 5. Elsevier, pp 125–177
phoresis of RNA. Cold Spring Harb Protoc 8. Adams JM, Jepessen PGN, Sanger F, Barrel BG
2010(6):pdb.prot5445. https://doi.org/10. (1969) Nature (London) 223:1009
1101/pdb.prot5445 9. Whitton JL, Hundley F, O’donnell B, Dessel-
3. Rio DC, Ares M, Hannon GJ, Nilsen TW berger U (1983) Silver staining of nucleic
(2010b) Polyacrylamide gel electrophoresis of acids. Applications in virus research and in
RNA. Cold Spring Harb Protoc 2010(6):pdb- diagnostic virology. J Virol Methods 7(4):
prot5444 185–198
4. Dubal ZB, Mawlong M, Susngi B, Sanjukta R, 10. Chevallet M, Luche S, Rabilloud T (2006)
Puro K, Ghatak S, Sen A, Shakuntala I, Bar- Silver staining of proteins in polyacrylamide
buddhe SB, Ahuja A, Bhattacharjee U (2015) gels. Nat Protoc 1(4):1852
Comparison of agarose gel electrophoresis and 11. Minakshi P, Ranjan K, Kumar P, Prasad G
RNA-PAGE for rapid detection of rotavirus (2013) A novel method of staining of RNA in
from faecal samples. J Appl Anim Res 43(1): polyacrylamide gel electrophoresis. Adv Anim
77–82 Vet Sci 1(4S):20–23
5. Stellwagen NC (2009) Electrophoresis of 12. Petrov A, Tsa A, Puglisi JD (2013) Analysis of
DNA in agarose gels, polyacrylamide gels and RNA by analytical polyacrylamide gel electro-
in free solution. Electrophoresis 30(supple- phoresis. Methods Enzymol 530:301–313
ment 1):S188–S195
Chapter 9
Peptide Nucleic Acid (PNA): A Diagnostic Molecule

for Infectious Diseases
Vinay G. Joshi, Anu Kumari, Sushila Maan, Tarun Kumar,
and Satish Kumar
Abstract
Disease diagnosis performs an important role in the epidemiology of the disease. Genetic material identifi-
cation is the definite way of identification of the pathogen. Available techniques involved in genetic material
identification are economically as well as time consuming. Both these aspects are important in disease
diagnosis, and particularly very crucial for infectious diseases. The distinctive physiochemical properties of
PNA molecules have opened the path to use them for the development of rapid diagnostic methods. Here,
we have briefly discussed about the designing and synthesis of PNA, its two types clamp PCR and PNA and
gold nanoparticles agglomeration science in disease diagnosis.
Key words PNA, PNA-clamping PCR, Gold nanoparticles, Diagnosis
1 Introduction
Emerging infectious diseases are a serious threat to both human

and animal life. An increase in the risk of infectious diseases partic-
ularly zoonotic diseases, due to the human population explosion,
congested wet markets, climate change, globalization, pollution
has brought back health care at the center of concern. Certainly,
disease diagnosis is one of the crucial steps in the process of disease
control, eradication, and treatment. Globalization has broadened
the economic opportunities but also increases the chances of infec-
tion (contagion) spread. A localized outbreak can easily be con-
verted into a pandemic like in swine flu, SARS (severe acute
respiratory syndrome), avian influenza, and COVID 19. The con-
firmatory diagnosis helps in monitoring the disease, betters its
prognosis and treatment, and ultimately helps in disease eradica-
tion. Therefore, advancement in disease diagnosis is an exigency.
Diagnostic tests can be broadly of two types depending on the site
of testing: conventional laboratory-based and point of care (POC)
137
138 Vinay G. Joshi et al.
test. POC tests are those which can be performed near, or at, the
point of patient care [1]. Disease diagnosis is a rapidly evolving
discipline with the current scenario. Standard tests such as antibody
neutralization or identification, virus/bacteria isolation, conven-
tional PCR, RT-PCR, and real-time quantitative RT—PCR
(RT—qPCR) are being commonly used. These lab-based tests
have some constraints as they require sophisticated instrumenta-
tion, skilled personnel, poor stability of reagents, high cost, and
time. The ideal diagnostic test should fulfill the proposed
“ASSURED criteria, i.e. Affordable, Sensitive, Specific, User
friendly, Rapid and robust, Equipment-free, and Deliverable to
end-users” [2].
2 Peptide Nucleic Acid (PNA)
PNA was first reported by Nielson et al. in 1991 [3]. In PNA, the
four naturally occurring nucleobases are connected by a peptide
backbone. The backbone is made up of repetitive units of N-
(2-aminoethyl) glycine Fig. 1. The purine and pyrimidine bases
are attached via a methyl carbonyl linker to the backbone of PNA
[4]. The PNA, nucleic acid mimetic is devoid of phosphate and
glycosidic linkages [5]. This modification changes the polarity of
the molecule and makes it charge neutral. The PNA-RNA/DNA
duplex demonstrates higher binding/hybridization efficiency and
thermal stability compared to native DNA/DNA, RNA/RNA, or
DNA/RNA duplexes. The PNA being achiral and charge-neutral
molecule shows strong and specific binding to the gene target
[6]. Additionally, PNA is a chimeric molecule making it resistant
to cleavage by hydrolytic enzymes such as DNase and Proteases
[3]. The PNA is proved to be a robust molecular probe that can be
used for disease diagnosis in both conventional PCR diagnostics
and point of care diagnosis (Table 1). The use of PNA for PCR
clamping based assay and colorimetric visual detection are the most
useful applications. PNA can be synthesized by automated peptide
synthesizing platform or solid-phase peptide synthesis (SPPS).
SPPS is the standard method for PNA synthesis. Both tert-butylox-
ycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc) chemis-
try can be used for PNA synthesis. They have their advantages and
disadvantages.
3 Materials and Methods
3.1 List of Materials The Fmoc PNA monomers, Rink amide-MBHA resin, 4-(20 ,40 -
Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-nor-
leucyl-MBHA resin (100–200 mesh), (1-[Bis (dimethylamino)
methylene]-1H- 1,2,3- triazolo [4,5-b] pyridinium 3-oxide
Peptide Nucleic Acid (PNA): A Diagnostic Molecule for Infectious Diseases 139
Fig. 1 Chemical structures of PNA and DNA
hexafluorophosphate) (HATU), Pyridine, m-cresol, and triiso-

propylsilane (TIS), Specially dried analytical grade, N,N-
dimethylformamide (DMF), dichloromethane (DCM), piperi-
dine, diethyl ether and N,N-diisopropylethylamine (DIEA) and
HPLC grade acetonitrile and water. All the chemicals and biolo-
gicals should be of analytical, molecular biology, and cell culture
grade.
3.2 Designing PNA For the designing of the PNA probe, there is no set frame of rules;
Probes however, a general probe composition guideline can help to design
a successful PNA probe. The length of PNA can be varying from
6 mer to 30 mer. Synthesis of longer PNA probe results in signifi-
cantly higher Tm values. The Tm for PNA is higher than its
contemporary DNA probes. Varying reports have claimed that
the PNA probe as small as 6 mer can inhibit RNA or sufficient to
work as a clamp in clamping PCR. Ideally, PNA should not contain
loner purine-rich sequences it may reduce the product yield in
solid-phase peptide synthesis. A PNA probe should have purine
contains below 50%. More than 5–6 complementing positions
should be avoided while designing the PNA probe. Due to its
hydrophobic nature, PNA probes may have poor water solubility
generally it is observed when the probe has higher G contents. For
chemical modification PNA [2-(2-(Fmoc-amino) ethoxy) ethoxy]
acetic acid (AEEA) linker can be used. Swada et al. in 2017 reported
the utility of azobenzene linker in the PNA probe for detecting
Table 1
PNA-based disease diagnosis of pathogenic organisms
Sr.
no Diagnosis for Spp/things affected Method References
1 Porcine reproductive and Porcine PNA probe-mediated one-step [29]
respiratory syndrome real-time RT-PCR
virus genotypes
2 Antigenic discrimination of Canine PNA array [30]
canine parvovirus
3 Salmonella enterica serovar Milk, eggs and PNA fluorescence in situ [31]
Enteritidis mayonnaise hybridization (PNA FISH)
samples
4 Megalocytivirus Aquatic animal PNA-based real-time PCR assay [32]
diseases
5 Multiple strains of Avian PNA biosensor [21]
influenza A virus
6 Stenotrophomonas Respiratory tract PNA FISH [33]
maltophilia infections in
humans and
animals
7 Newcastle disease virus Avian PNA and gold nanoparticles [20]
(NDV) (AuNPs)
8 Mycobacterium tuberculosis Human PNA biosensor based on reduced [34]
graphene oxide/water-soluble
quantum dots
9 Dengue virus Human PNA and gold nanoparticles [35]
(AuNPs)
10 Human papillomavirus Human PNA probes [36]
homopurine sequences in a target gene [7]. In some cases, to

improve the water solubility of the PNA molecule the probe can
be labeled with a basic amino acid such as Lys at C terminus. PNA
probes can be labeled with suitable fluorescent labels for real-time
diagnostic studies. For PNA probe designing reader can refer to an
online PNA design tool “PNA tool.” It provides a general guideline
for probe designing.
3.3 Synthesis of PNA The PNAs are synthesized by the standard method of SPPS using
Fmoc chemistry on Rink amide-MBHA resin. Briefly, swell the
Rink amide-MBHA resin in the DMF overnight. De-blocking of
the resin-bound Fmoc protecting group is done by treating with
20% (v/v) piperidine solution in DMF. Wash the beads five times
with DMF and then DCM and followed by two DMF wash. For the
first coupling, pre-activation of amino acids is done to activate their
carboxyl group so that amide linkage will establish during the
coupling reaction. Pre-activation is done by incubating the PNA

monomers in especially dried DMF and activated using HATU in
DIEA and pyridine solution at 4 C for 20 min. Add the
pre-activated PNA into the beads and incubate it for 2 h under
shaking conditions at room temperature. After the first complete
coupling reaction, all other remaining activated sites are capped by
adding a mixture of DMF, DIEA, and acetic anhydride.
De-blocking, pre-activation, coupling, and capping reactions are
repeated until the desired length of PNA is achieved. The final
product is de-protected and cleaved from the resin by treating the
resin with TFA/m-cresol/TIS/water (85/10/2.5/2.5, v/v) for
5 h at room temperature. The cleaved PNA in the TFA extracts is
then precipitated by dried and chilled diethyl ether. At each step
progress of PNA, monomer coupling can be monitored by the
Kaiser test.
3.4 Procedure The standard protocol for the Kaiser test [8].
for the Kaiser Test
3.4.1 Reagents 1. Dissolve 8.25 mg of KCN in 12.5 mL of double-distilled water.

for Kaiser Test 2. Make 1:50 dilution of the above solution in pyridine label it as
Solution A.
Solution A
Solution B 1. Dissolve 500 mg of ninhydrin in 10 mL of 1-butanol and label

it as solution B.
Solutions C 1. Dissolve 20 g of phenol in 10 mL of 1-butanol and label it as

solution C.
3.4.2 Kaiser Test 1. Take 30–40 beads and wash it three times with excessive
ethanol.
2. Remove the ethanol and two drops of Solution A, B, and C
serially.
3. Heat this solution in boiling water for 5 min.
3.4.3 Interpretations 1. The colorless or faint blue color of beads indicates the success-
ful coupling.
2. The beads are dark blue indicates that coupling incomplete and
coupling step can be repeated.
3.5 Characterization The PNA probes can be purified by RP-HPLC using the C-8
and Quantification column. For RP-HPLC preferred buffer in PNA purification can
of PNA Concentration be Buffer A-0.08% TFA in HPLC grade water, Buffer B-0.08% TFA
in acetonitrile. A linear gradient of 95% water to 30–70% of buffer A
and buffer B for 40 min with 0.8 mL/min flow rate be used. After
the fraction collection dry lyophilized PNA probe can be sent for
Fig. 2 PNA—Clamping PCR.
the molecular weight analysis using MALDI-TOF. After confirma-

tion of desired molecular weight PNA probes can be used for the
diagnostic application. Absorbance at 260 nm can be used to
determine the concentration of PNA probe.
3.6 PNA Clamping Clamping PCR is based on the principle of competitive binding
PCR between the primer and PNA. When PNA clamps to complemen-
tary target gene sequence it does not allow DNA polymerase to
extend the sequence, hence there will be no amplification in PCR
(Fig. 2). PNA mediated clamping specifically blocks amplification
of a given gene template while allowing amplification of other
templates that differ by as little as one nucleotides. PCR clamping
can be achieved by two basic methods. First competition at the
primer binding site between the primer and the PNA. Second, by
blocking the elongation, the PNA binds near the primer binding
site and arrest the elongation [9]. This phenomenon was widely
being used for diagnostic detection of SNP in genetic diseases,
oncogenic point mutations, mitochondrial DNA mutation in
degenerative diseases, microbial mixed community analysis, analysis
of mRNA editing, cloning IgG variable regions, finding of allelic
allocations using PNA clamping PCR [10–16]. A few studies have
developed variants of clamping PCR for the detection of point
mutations in the overwhelming concentration of wild-type
sequence templates. Such a situation is generally observed in cancer
Denaturation
PNA Extension
Primer
clamping Annealing
Fig. 3 Various steps of typical clamping PCR assay
genetics where early cancer detection can be possible by detecting a

low level of point mutations using PNA clamp PCR. Real-time
assay-based detection of mutation can be achieved using these
variations of clamping PCR. Oh et al. 2010 explain the use of
unlabeled PNA probe for mutation detection using LC Green dye
[6]. While Han et al. 2016 explain the use of fluorescently labelled
PNA probes for mutation detection in clamping PCR experiments
[17]. The simplest method for the use of clamping PCR is to use
the PNA probe as a clamp in classical PCR and identify the presence
or absence of target gene.
3.6.1 Methods Clamping For a general guideline, reaction condition can be as follows
PCR 5–10 pmol of forward and reverse primers. 2 PCR master mix.
Initially, the experiment should be set with variable PNA concen-
tration from say 0–2 μM PNA with a fixed amount of template to
understand the required concentrations of PNA for inhibition of
PCR amplification. For PCR clamping identifying PNA, the
annealing temperature is a crucial step, and it can be performed
with addition PNA clamping step before primer annealing steps
Fig. 3. As PNA probes have higher Tm pre-anneal clamping step
should be preferred. After standardization of protocol PCR reac-
tion can be prepared with 2 master mix, 5–10 pmol forward and
reverse primers, 1 μL PNA probe (predetermined suitable concen-
tration) and nuclease-free water to compensate the reaction
volume.
3.7 Peptide Nucleic The PNA based label-free biosensors can be an important candidate
Acid Visual by fulfilling the ASSURED criteria. Kanjanawrut and Su first put
Diagnostics forth the idea of using PNA probes to detect a specific DNA
sequence using unmodified metallic nanoparticles [18]. This
group discovered a unique PNA metallic nanoparticle behavior of
the citrate anions-protected gold nanoparticles. These nanoparti-
cles undergo immediate agglomeration in the presence of PNA.
This agglomeration of nanoparticles is retarded when a comple-
mentary nucleic acid is present to form the PNA-nucleic acid
complex. This PNA: nucleic acid complex interaction is specific
and can be used to discriminate the presence or absence of target
nucleic acid. It suggested that the induced particle aggregation
originates from the strong PNA gold interactions with the involve-
ment of both nucleobases and peptide backbone of PNA [19]. In
addition to these factors, the presence of positive charges of the

N-terminal amines of the PNA at neutral pH may contribute to the
immediate aggregation by electrostatic interaction [18]. With the
use of this agglomeration phenomenon, label-free biosensors hav-
ing the ability of viral RNA detection could be devised. Reports on
label-free detection of Newcastle disease virus (NDV) using PNA
and gold nanoparticles based agglomeration visual assay [20] and
label-free PNA biosensor for identification of multiple strains of
influenza A virus [21], showed the way forward for application of
these techniques in POC, viral disease diagnosis. Biosensors,
according to IUPAC are integrated receptor-transducer devices,
which are capable of providing selective quantitative or semi-
quantitative analytical information using a biological recognition
element [22]. Biosensors may be classified into two broad groups
depending on the methods of detection: with and without labels
(markers). The label-free biosensor utilizes biological or chemical
receptors for direct detection of an analyte in the sample, without
the use of enzyme and radioactive or fluorescent labels [23].
3.7.1 Gold Nanoparticle Gold nanoparticles (AuNPs) assembly and disassembly cause a
Synthesis visual color change, this property is due to surface plasmon reso-
nance (SPR) which is utilized by scientists to develop colorimetric
biosensor. Here, analytes such as DNA, PNA, RNA can cause
aggregation or dispersion of AuNP directly or indirectly and leads
to a huge absorption band shift (up to ~300 nm), which can be
visualized by naked eyes [24] For AuNPs synthesis, all the glassware
should be thoroughly cleaned with freshly prepared Aqua Regia (1:
3 HNO3/HCl) and washed twice with triple-distilled water and
dried overnight. Citrate-stabilized gold nanoparticles are synthe-
sized by the reduction of gold (III) chloride trihydrate
(HAuCl43H2O) by sodium citrate [25] Briefly, 50 mL of a
1 mM tetrachloroauric (III) acid (HAuCl43H2O) solution is heated
with continuous stirring on a magnetic stirrer till boiling. Then,
add a 5 mL solution of 38.8 mM trisodium citrate quickly to the
boiling solution. Let the solution boil for 10 min with constant
stirring. Stop the heat and allow the solution to stir for another
15 min until a brick-red wine color appear. The colloidal gold
preparation is cooled to room temperature and store at 4 C until
further use, and it is characterized by UV–vis spectrophotometry
for its characteristic absorbance at 520 nm.
3.7.2 Visual Viral RNA The procedure for visual detection of viral using metallic nanopar-
Detection ticles (Silver or gold nanoparticles) and PNA is simple and can be
performed on the field. For the actual diagnostic assay, various
combinations should be tested to arrive at identifying required
PNA concentration which can induce the visual color change in
gold/silver nanoparticles. The researchers can use either gold
nanoparticles or silver nanoparticles as an indicator for color change
but the use of gold nanoparticles is more common; therefore, we

will describe the gold nanoparticle-based assay. Interested readers
can refer the article by Kanjanawaru and Su in 2009 which in detail
explanation about the use of gold/silver or combination of gold
and silver nanoparticles for PNA based visual sensing [18]. The
concentration of PNA required for induction of color change can
vary with length of PNA, a smaller size PNA can induce better color
change as it can easily cap citrate core [18]. Once the actual PNA
concentration required for the visual color change is identified, the
second step involves experiments to determine the suitable temper-
ature for PNA: RNA hybridization. If there is a shortage of target
RNA or reference RNA synthetic complementary target can also be
used to standardize the PNA: target nucleic acid complementation.
Such initial experimentations will provide baseline information
required to optimize the visual detection assay. After this step
PNA: RNA complementation can be confirmed with the addition
of gold nanoparticles to check inhibition of PNA induced gold
nanoparticle agglomeration due to its complementation with
RNA Fig. 4. Usually in the absence of complementary target
color change appears rapidly within a minute but if the PNA
probe of a larger size of about 20 mer and above wait for 2–5 min
is essential. The color retention or color change in gold nanoparti-
cles can be visually monitored. The phenomenal changes in gold
nanoparticle color can also be monitored using spectrum scanning
with recording absorption spectra of solution from 400 to 750 nm.
The stable gold nanoparticle solution of 13–18 nm particle size
gives maximum absorption at 520 nm and blue colored agglomer-
ated gold nanoparticles give maximum absorption at around
600–650 nm with a significant reduction in the pick at 520 nm.
The test has specificity to identify a single base mismatch
[20]. However, to improve the visual color discrimination and to
identify single base mismatches, one needs to optimize the reaction
with higher salt concentrations. Various combinations from
50 to150 mM NaCl can be tried.
Interpretations of visual RNA detection assay using PNA and
gold nanoparticles can be summarized as follows.
1. The red color of gold nanoparticle turns in to blue after the
addition of preincubated PNA: RNA complex indicates the
absence of a PNA complementary strand.
2. Retention of gold nanoparticles red colour after the addition of
preincubated PNA: RNA complex, indicates the presence PNA
complementary strand.
3.7.3 Essentials of Visual 1. PNA: metallic nanoparticle assay could be useful for the detec-
RNA Detection Experiments tion, identification of point mutation at target sequence, and
quantification of viral RNA.
Fig. 4 Principal of colorimetric assay based on agglomerative nature of AuNPs with PNA in presence and
absence of complementary RNA sequence
2. From the description and procedure, it is amply clear that the

color change phenomenon depends on charge interactions. It
also underlines some of the specific points to consider before
establishing an actual diagnostic assay. The importance of
which includes maintaining the no-template PNA and no
PNA, RNA controls with gold nanoparticles.
3. At the time of standardization of the assay, it is advisable to do
nonspecific RNA spiking to understand the interference of the
overwhelming negatively charged RNA molecule in stabilizing
the gold nanoparticle solution. Such RNA charge induced
stabilization of nanoparticle may result in false-positive inter-
pretations of results.
3.7.4 Viral RNA Induction of change in the color of gold nanoparticles is a function
Quantification Using PNA of free PNA available in the solution. The gradual changes with
Gold Nanoparticle increasing concentration of PNA can be appreciated using differ-
Interactions ence spectra analysis. The difference spectra can be generated using
a gold nanoparticle as reference spectra. Incremental addition of
PNA in gold nanoparticles provides a correlation in induced color
change vs concentration of PNA. These concomitant changes in the
spectra of the gold nanoparticle on difference spectra analysis will
provide a common crossover point, refereed as the isosbestic point,
indicating equilibrium between the PNA and the gold nanoparti-
cles color change. Such spectra analysis will provide two absorption
maximum one at 520 nm for gold nanoparticle showing no color
change and other at 630–640 nm for PNA induced blue colored
gold nanoparticles. The ratio of OD640/OD520 can be used as a
function of available complementary template which prevents PNA
from inducing color change. After identification PNA concentra-
tion required for stable spectral change with induction of absor-
bance maxima at 640 passing through an isosbestic point, a
standard curve can be prepared with the ratio of OD640/
OD520. For standard curve two-fold serial dilutions of comple-
mentary RNA, templates can be used. With the increase in RNA
template gold nanoparticle stability will be retained and ratio
OD640/OD520 will be minimal. This will generate a standard
curve as a function of complementary RNA concentration present
in the solution that is preventing PNA from inducing color changes
in the solution. Joshi et al. have shown quantification of viral RNA
using this method. This approach can give relative estimates of the
presence of viral RNA in test samples. The method is simple and
does not require sophisticated lab equipment.
3.7.5 PNA and Other In the previous sections, we mostly discussed the colorimetric
Nanoparticles which is based on the aggregation of unmodified metallic nanopar-
for Diagnostic Applications ticles [18, 20]. The PNA can also be used for the diagnosis of viral
diseases using SPR imaging. The SPR imaging method enhanced
with ultrasensitive nanoparticles and uses PNA probes showed
greater selectivity toward the target template with single-nucleotide
mismatches detection abilities [26]. Kerman et al. 2008 demon-
strated the electrochemical biosensor for the rapid analysis of
nucleic acids and nuclease activity [27]. Wang et al., 2006 prepared
PNA-modified magnetic nanoparticles from the 4-pyridyldithiol
derivative of PNA [28]. The PNA was attached to
3-mercapropropyloxysilane coated magnetic nanoparticles. This
nanoparticle conjugated PNA was found to hybridize with the
ssDNA target efficiently. This approach also provides a simple and
cost-effective label-free assay for diagnosis.
References
1. Kosack CS, Page AL, Klatser PR (2017) A https://doi.org/10.1128/aem.66.2.549-557.
guide to aid the selection of diagnostic tests. 2000
Bull World Health Organ 95(9):639–645. 12. Nanthakumar T, Tiwari AK, Kataria RS,
https://doi.org/10.2471/BLT.16.187468 Butchaiah G, Kataria JM, Goswami PP (2000)
2. Peeling RW, Holmes KK, Mabey D, Ronald A Sequence analysis of the cleavage site-encoding
(2006) Rapid tests for sexually transmitted region of the fusion protein gene of Newcastle
infections (STIs): the way forward. Sex Transm disease viruses from India and Nepal. Avian
Infect 82(suppl 5):v1. https://doi.org/10. Pathol 29(6):603–607. https://doi.org/10.
1136/sti.2006.024265 1080/713651205
3. Nielsen PE, Egholm M, Berg RH, Buchardt O 13. Zhong S, Nguyen NY, Eggerman TL (1999)
(1991) Sequence-selective recognition of DNA Detection of apolipoprotein B mRNA editing
by strand displacement with a thymine- by peptide nucleic acid mediated PCR clamp-
substituted polyamide. Science (New York, ing. Biochem Biophys Res Commun 259(2):
NY) 254(5037):1497–1500. https://doi. 311–313. https://doi.org/10.1006/bbrc.
org/10.1126/science.1962210 1999.0687
4. Pellestor F, Paulasova P (2004) The peptide 14. Murdock DG, Wallace DC (2002)
nucleic acids (PNAs), powerful tools for PNA-mediated PCR clamping. Applications
molecular genetics and cytogenetics. Eur J and methods. Methods Mol Biol 208:145–
Hum Genet 12(9):694–700. https://doi. 164. https://doi.org/10.1385/1-59259-
org/10.1038/sj.ejhg.5201226 290-2:145
5. Orum H, Nielsen PE, Egholm M, Berg RH, 15. Thiede C, Bayerdörffer E, Blasczyk R,
Buchardt O, Stanley C (1993) Single base pair Wittig B, Neubauer A (1996) Simple and sen-
mutation analysis by PNA directed PCR clamp- sitive detection of mutations in the ras proto-
ing. Nucleic Acids Res 21(23):5332–5336. oncogenes using PNA-mediated PCR clamp-
https://doi.org/10.1093/nar/21.23.5332 ing. Nucleic Acids Res 24(5):983–984.
6. Oh JE, Lim HS, An CH, Jeong EG, Han JY, https://doi.org/10.1093/nar/24.5.983
Lee SH, Yoo NJ (2010) Detection of low-level 16. Nanthakumar T, Kataria RS, Tiwari AK,
KRAS mutations using PNA-mediated asym- Butchaiah G, Kataria JM (2000) Pathotyping
metric PCR clamping and melting curve analy- of Newcastle disease viruses by RT-PCR and
sis with unlabeled probes. J Mol Diagn 12(4): restriction enzyme analysis. Vet Res Commun
418–424. https://doi.org/10.2353/jmoldx. 24(4):275–286. https://doi.org/10.1023/
2010.090146 a:1006403017578
7. Sawada S, Takao T, Kato N, Kaihatsu K (2017) 17. Han JY, Choi JJ, Kim JY, Han YL, Lee GK
Design of Tail-Clamp Peptide Nucleic Acid (2016) PNA clamping-assisted fluorescence
Tethered with Azobenzene linker for melting curve analysis for detecting EGFR
sequence-specific detection of Homopurine and KRAS mutations in the circulating tumor
DNA. Molecules 22(11):1840. https://doi. DNA of patients with advanced non-small cell
org/10.3390/molecules22111840 lung cancer. BMC Cancer 16:627. https://doi.
8. Wellings DAA, E. (1997) Solid-phase peptide org/10.1186/s12885-016-2678-2
synthesis. In: Methods in enzymology, vol 289. 18. Kanjanawarut R, Su X (2009) Colorimetric
Academic Press, San Diego detection of DNA using unmodified metallic
9. Orum H (2000) PCR clamping. Curr Issues nanoparticles and peptide nucleic acid probes.
Mol Biol 2(1):27–30 Anal Chem 81(15):6122–6129. https://doi.
10. Mrozikiewicz PM, Landt O, Cascorbi I, Roots org/10.1021/ac900525k
I (1997) Peptide nucleic acid-mediated poly- 19. Gourishankar A, Shukla S, Ganesh KN, Sastry
merase chain reaction clamping allows allelic M (2004) Isothermal titration calorimetry
allocation of CYP1A1 mutations. Anal Bio- studies on the binding of DNA bases and
chem 250(2):256–257. https://doi.org/10. PNA base monomers to gold nanoparticles. J
1006/abio.1997.2246 Am Chem Soc 126(41):13186–13187.
11. von Wintzingerode F, Landt O, Ehrlich A, https://doi.org/10.1021/ja046785g
Göbel UB (2000) Peptide nucleic acid- 20. Joshi VG, Chindera K, Singh AK, Sahoo AP,
mediated PCR clamping as a useful supplement Dighe VD, Thakuria D, Tiwari AK, Kumar S
in the determination of microbial diversity. (2013) Rapid label-free visual assay for the
Appl Environ Microbiol 66(2):549–557. detection and quantification of viral RNA
using peptide nucleic acid (PNA) and gold
nanoparticles (AuNPs). Anal Chim Acta 795: 29. Park J-Y, Kim S-H, Lee K-K, Kim Y-H, Moon
1–7. https://doi.org/10.1016/j.aca.2013. B-Y, So B, Park C-K (2019) Differential detec-
06.037 tion of porcine reproductive and respiratory
21. Kumar N, Bhatia S, Pateriya AK, Sood R, syndrome virus genotypes by a fluorescence
Nagarajan S, Murugkar HV, Kumar S, melting curve analysis using peptide nucleic
Singh P, Singh VP (2020) Label-free peptide acid probe-mediated one-step real-time
nucleic acid biosensor for visual detection of RT-PCR. J Virol Methods 267:29–34.
multiple strains of influenza a virus suitable for https://doi.org/10.1016/j.jviromet.2019.
field applications. Anal Chim Acta 1093:123– 02.008
130. https://doi.org/10.1016/j.aca.2019. 30. An D-J, Jeong W, Jeoung H-Y, Lee M-H, Park
09.060 J-Y, Lim J-A, Park B-K (2012) Peptide nucleic
22. Thévenot DR, Toth K, Durst RA, Wilson GS acid-based (PNA) array for the antigenic dis-
(2001) Electrochemical biosensors: recom- crimination of canine parvovirus. Res Vet Sci
mended definitions and classification1Interna- 93(1):515–519. https://doi.org/10.1016/j.
tional union of pure and applied chemistry: rvsc.2011.06.003
physical chemistry division, commission I.7 31. Almeida C, Cerqueira L, Azevedo NF, Vieira
(biophysical chemistry); analytical chemistry MJ (2013) Detection of salmonella enterica
division, commission V.5 (electroanalytical serovar Enteritidis using real time PCR, immu-
chemistry).1. Biosens Bioelectron 16(1): nocapture assay, PNA FISH and standard cul-
121–131. https://doi.org/10.1016/S0956- ture methods in different types of food
5663(01)00115-4 samples. Int J Food Microbiol 161(1):16–22.
23. Andryukov BG, Besednova NN, Romashko https://doi.org/10.1016/j.ijfoodmicro.
RV, Zaporozhets TS, Efimov TA (2020) 2012.11.014
Label-free biosensors for laboratory-based 32. Lee ES, Cho M, Min EY, Jung SH, Kim KI
diagnostics of infections: current achievements (2020) Novel peptide nucleic acid-based real-
and new trends. Biosensors 10(2):11. https:// time PCR assay for detection and genotyping
doi.org/10.3390/bios10020011 of Megalocytivirus. Aquaculture 518:734818.
24. Zhao W, Brook MA, Li Y (2008) Design of https://doi.org/10.1016/j.aquaculture.
gold nanoparticle-based colorimetric biosen- 2019.734818
sing assays. Chembiochem 9(15):2363–2371. 33. Hansen N, Rasmussen AKI, Fiandaca MJ,
https://doi.org/10.1002/cbic.200800282 Kragh KN, Bjarnsholt T, Høiby N,
25. Grabar KC, Freeman RG, Hommer MB, Natan Stender H, Guardabassi L (2014) Rapid iden-
MJ (1995) Preparation and characterization of tification of Stenotrophomonas maltophilia by
au colloid monolayers. Anal Chem 67(4): peptide nucleic acid fluorescence in situ hybri-
7 3 5 – 7 4 3 . h t t p s : // d o i . o r g / 1 0 . 1 0 2 1 / dization. New Microbes New Infect 2(3):
ac00100a008 79–81. https://doi.org/10.1002/nmi2.38
26. D’Agata R, Corradini R, Ferretti C, Zanoli L, 34. Mat Zaid MH, Abdullah J, Yusof NA,
Gatti M, Marchelli R, Spoto G (2010) Ultra- Sulaiman Y, Wasoh H, Md Noh MF, Issa R
sensitive detection of non-amplified genomic (2017) PNA biosensor based on reduced gra-
DNA by nanoparticle-enhanced surface plas- phene oxide/water soluble quantum dots for
mon resonance imaging. Biosens Bioelectron the detection of mycobacterium tuberculosis.
25(9):2095–2100. https://doi.org/10.1016/ Sensors Actuators B Chem 241:1024–1034.
j.bios.2010.02.008 https://doi.org/10.1016/j.snb.2016.10.045
27. Kerman K, Saito M, Tamiya E (2008) Electro- 35. Abdul Rahman S, Saadun R, Azmi NE,
active chitosan nanoparticles for the detection Ariffin N, Abdullah J, Yusof NA, Sidek H,
of single-nucleotide polymorphisms using pep- Hajian R (2014) Label-free dengue detection
tide nucleic acids. Anal Bioanal Chem 391(8): utilizing PNA/DNA hybridization based on
2759–2767. https://doi.org/10.1007/ the aggregation process of unmodified gold
s00216-008-2204-8 nanoparticles. J Nanomater 2014:839286.
28. Wang F, Shen H, Feng J, Yang H (2006) https://doi.org/10.1155/2014/839286
PNA-modified magnetic nanoparticles and 36. Menashi A, Cohenford WW, Brian
their hybridization with single-stranded DNA Lentrichia A, (2005) Detection and typing of
target: surface enhanced Raman scatterings human papllomavirus using pna probes US
study. Microchim Acta 153(1):15–20. 6,936,443 B2 Aug. 30, 2005
https://doi.org/10.1007/s00604-005-
0460-2
Chapter 10
Nucleic Acid Sequence-Based Amplification (NASBA)

Methods and CRISPR/Cas13 System to Detect Pig Viral
Diseases
Ajay Kumar Singh, Soumen Naskar, Pramod W. Ramteke,
and Rohit Kumar
Abstract
For the diagnosis of pathogens, amplification and detection of their genetic material is an essential step, but
the traditional method of amplification and detection requires sophisticated equipment or complex experi-
mental procedures and is not portable, precluding their deployment in the field. The distinctive physio-
chemical amplification of nucleic acid sequence-based amplification (NASBA) and detection through
CRISPR/Cas13 based system has opened the path to use them for the development of rapid diagnostic
procedures in the field. Here, we have briefly discussed the principle and protocol of NASBA and CRISPR/
Cas13 based detection system in pig viral disease diagnosis.
Key words Cas13, Isothermal amplification, CRISPR associated proteins, Nucleic acid amplification,
Disease diagnosis
1 Introduction
In the present scenario, the pig industry contributes a majority

share to the ever-increasing demand of quality food fuelled by the
increasing human population, urbanization, and social mobility.
However, the intensive production systems have made them vul-
nerable to various transboundary diseases such as African swine
fever (ASF), Classical swine fever (CSF), porcine reproductive and
respiratory syndrome (PRRS), Foot-and-mouth disease (FMD),
and Porcine epidemic diarrhea (PED).
Briefly, the virus responsible for classic swine fever (CSF) or hog
cholera is classic swine fever virus (CSFV), which causes a highly
contagious disease in domestic pigs and wild boar. It is a member of
the genus Pestivirus in the family Flaviviridae [1, 2,]. Outbreaks of
CSF lead to considerable economic losses in many countries
151
152 Ajay Kumar Singh et al.
worldwide [3]. Although, in European countries, CSFV has been

successfully eradicated by a stamping-out policy, and the China-
manufactured attenuated vaccine is being safely and efficiently
employed as prophylactics worldwide [4].
African swine fever virus, a member of genus Asfivirus, family
Asfarviridae, is a causative agent of ASF. It is a large enveloped virus
with a double-stranded DNA genome [5]. The ASF is a hemor-
rhagic viral disease that affects not only domestic and wild boars but
also ticks. The current outbreak of ASF has once again drawn
attention to the potential threat of the virus and is quite worrisome.
For ASF, as of now, no vaccine is commercially available.
PRRS virus is the etiologic agent of PPRS, a disease that causes
breathing problems in pigs and kills piglets and unborn litters. It is a
small enveloped positive-strand RNA virus that belongs to the
family Arteriviridae in the genus Arterivirus [6]. At present, due
to virus variation and unresolved issues of transmission, there is no
single successful strategy available to cure the PRRS.
FMD, it is also one of the most devastating diseases of porcine
caused by infection with a picornavirus, generically referred as
FMD virus (FMDV) [7]. Vaccination of susceptible pigs against
FMD is a well-established strategy for helping to combat the
disease [8].
PED is also a highly contagious disease of porcine caused by the
PED virus, an enveloped, ss RNA virus belonging to the Alphacor-
onavirus genus of the Coronaviridae family [9]. It causes high
mortality in neonatal piglets; however, effective and safe vaccines
are still not available [10].
The above diseases are some of the biggest threats to the pig
industry and global security. In addition, the great majority of
globally circulating pathogens go undetected, hindering outbreak
preparedness and response. To enable routine surveillance and
comprehensive diagnostic applications, there is a need for detection
technologies that can scale to test many samples while simulta-
neously testing for many pathogens [11].
Nucleic acid amplification is a key process in molecular biology
and has been widely used to detect the virus at scale—that causes
viral diseases—for surveillance and diagnostic purposes. Polymerase
chain reaction (PCR) was the first DNA amplification method,
developed in the early 1980s [12] and until now has been the
method of choice. However, it has a good number of limitations,
including high cost of equipment, contamination chances, sensitiv-
ity to certain classes of contaminants and inhibitors, requirement of
thermal cycling, etc. [13]. These limitations insisted on alternative
methods such as loop-mediated isothermal amplification (LAMP),
nucleic acid sequence-based amplification (NASBA), and most
recent clustered regularly interspaced short palindromic repeats
(CRISPR) based assay. These methods offer potential advantages
Nucleic Acid Sequence-Based Amplification (NASBA) Methods and CRISPR/Cas13. . . 153
Fig. 1 Schematic representation of the position of primers
over PCR for speed, cost, scale or portability. In this chapter, we

will focus on NASBA- and CRISPR-based assay to detect pig viral
diseases.
2 Loop-Mediated Isothermal Amplification (LAMP)
It is a simple, rapid, specific, and cost-effective nucleic acid amplifi-

cation method in comparison to PCR and nucleic acid sequence-
based amplification. It was developed by Eiken Chemical Co., Ltd.
In this method specifically designed four different primers are
employed to recognize the six distinct regions on the target gene
(Fig. 1).
Amplification and detection of gene are completed in a single
step, by incubating the mixture of samples, primers, DNA polymer-
ase with strand displacement activity and substrates at a constant
temperature (about 65 C). Combination with reverse transcrip-
tion, it can amplify RNA sequences with high efficiency [14]. It is
comparable to PCR in terms of sensitivity, but is less affected by
presence of non-targeted DNA and inhibitory molecules
[15]. Therefore, because of its simplicity, rapid amplification, and
easy detection, LAMP has been used for diagnosis of several impor-
tant emerging and re-emerging diseases such as CSF, ASF, PPRS,
and FMD. However, through this method, we cannot detect the
amplified product on a sequence basis.
3 Nucleic Acid Sequence-Based Amplification (NASBA)
It is a robust technology, amplifies nucleic acid continuously in a

single mixture at one temperature. A detailed process of amplifica-
tion was described by J. Compton in 1991. Briefly, it includes three
enzymes, namely reverse transcriptase, T7 RNA polymerase,
RNase H, two specific primers and appropriate buffer components
to amplify single-stranded RNA with opposite polarity of the tar-
get. The amplified RNA product can be detected through the use of
a target-specific capture probe bound to magnetic particles in
conjunction with a ruthenium-labeled detector probe and an

instrument called NUCLISENS capable of measuring electroche-
miluminescence (ECL) [16]. Alternatively, it can also detect in real-
time through the use of molecular beacon probes included in the
amplification reaction [17]. Molecular beacon probes possess a 50
fluorescent dye and a 30 quencher molecule and are designed to
form stem-loop structures that bring into close proximity the 50 and
30 ends of the probe, resulting in minimal fluorescence. In the
presence of a complementary target sequence, the probe is hybri-
dized to the target, resulting in a measurable increase in fluores-
cence. The template is mainly RNA, can also be DNA. It can also be
applied to single nucleotide polymorphism (SNP) analysis using
genomic DNA as a template. The combination of DNA NASBA
with multiplex hybridization of specific molecular beacons makes it
possible to unambiguously discriminate the presence of the SNP of
interest. The G-quadruplex/hemin complex can catalyze the oxi-
dation of 2,20 -azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS2) by H2O2 to produce a colorimetric signal that can be
visualized by the naked eye. Recently, Lu et al. [18] developed a
novel CSFV detection approach by combining this with NASBA
technique (Fig. 2).
4 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
In 2012, Charpentier and Doudna reported “that the Cas9 endo-

nuclease can be programmed with guide RNA engineered as a
single transcript to cleave any double-stranded DNA sequence”
[19]. Their discovery has led to widespread applications of the
CRISPR-Cas9 system as a powerful and versatile tool in genome
editing. CRISPR-Cas systems provide microbes with RNA guided
adaptive immunity to foreign genetic elements by directing
nucleases to bind and cut specific nucleic acid sequences. Through
a process termed adaptation, microbes capture snippets of foreign
genetic elements and incorporate them into their genomic CRISPR
array. Transcription of CRISPR arrays creates CRISPR RNAs
(crRNAs) that bind to Cas nucleases and provide specificity by
base-pairing with target nucleic acids [20]. Presently, on the basis
of Cas protein it is majorly divided into two classes. Class
1 CRISPR–Cas systems have effector modules composed of multi-
ple Cas proteins that form a crRNA-binding complex and function
together in binding and processing of the target. Class 2 systems
have a single, multi-domain crRNA-binding protein that is func-
tionally analogous to the entire effector complex of class
1 (Fig. 3) [21].
Recently, it was found that several Cas such as Cas12a/b,
Cas13a/b, and Cas14 protein from the CRISPR-Cas system have
collateral cleavage activity that can degrade single-stranded DNA or
Fig. 2 Schematic representation of the NASBA-DNAzyme approach for detection and differentiation of CSF
viral RNAs
cas8 or Spacers
cas3 cas10 cas11 cas7 cas5 cas6 cas1 cas2 cas4
Class 1
Large Small
subunit subunit
CRISPR
cas9 or cas12 or cas13 cas1 cas2 cas4
Class 2
tracrRNA Single eﬀector protein
Fig. 3 Schematic illustration of the generic organizations of class 1 and class 2

RNA non-specifically upon binding to their target site. This ability

can be used to detect pathogens when a short reporter sequence is
also added to the cells. This discovery has led to the emergence of
many CRISPR-Cas based virus detection protocols. This technol-
ogy could be an ideal tool in diagnostics as a result of its excellent
performance in several important areas including high sensitivity,
specificity, and accuracy as well as the fact that this technology is
rapid and easy to use [22]. Recently, Ackerman et al. develop
Combinatorial Arrayed Reactions for Multiplexed Evaluation of
Nucleic acids (CARMEN), a platform for scalable, multiplexed
pathogen detection. In the CARMEN platform, nanolitre droplets
containing CRISPR-based nucleic acid detection reagents self-
organize in a microwell array to pair with droplets of amplified
samples, testing each sample against each CRISPR RNA (crRNA)
in replicate. The combination of CARMEN and Cas13 detection
(CARMEN–Cas13) enables robust testing of more than 4500
crRNA–target pairs on a single array.
As a summary, these emerging CRISPR/Cas detection tool
shows great potentials in the detection of virally derived nucleo-
tides. Therefore, CRISPR-based methods would be more suitable
for molecular diagnosis of major epidemic outbreaks than conven-
tional techniques.
References
1. Thiel H-J, Collett MS, Gould EA, Heinz FX, 6. Conzelmann KK, Vissner N, van Woensel P,
Houghton M, Meyers G, Purcell RH, Rice CM Thiel HJ (1993) Molecular characterization
(2005) Family Flaviviridae. In: Fauquet CM, of porcine reproductive and respiratory syn-
Mayo MA, Maniloff J, Desselberger U, Ball LA drome virus, a member of the Arterivirus
(eds) Virus Taxonomy. VIIIth Report of the group. Virology 193:329–339
International Committee on Taxonomy of 7. OIE Technical Disease Cards. Foot-and-
Viruses. Academic Press, San Diego, pp Mouth Disease (2017). Available from.
979–996 http://www.oie.int/fileadmin/Home/eng/
2. Edwards S, Fukusho A, Lefevre PC, Animal_Health_in_the_World/docs/pdf/Dis
Lipowski A, Pejsak Z, Roehe P, Westergaard J ease_cards/FOOT_AND_MOUTH_
(2000) Classical swine fever: the global situa- DISEASE.pdf
tion. Vet Microbiol 73:103–119. https://doi. 8. Kahn S, Geale DW, Kitching PR, Bouffard A,
org/10.1016/S0378-1135(00)00138-3 Allard DG, Duncan JR (2002) Vaccination
3. Moennig V, Floegel-Niesmann G, Greiser- against foot-and-mouth disease: the implica-
Wilke I (2003) Clinical signs and epidemiology tions for Canada. Can Vet J 43(5):349
of classical swine fever: a review of new knowl- 9. Antas M, Woźniakowski G (2019) Current sta-
edge. Vet J 165:11–20 tus of porcine epidemic diarrhoea (PED) in
4. van Oirschot JT (2003) Emergency vaccina- European pigs. J Vet Res 63(4):465–470
tion against classical swine fever. Dev Biol 10. Hou Y, Ke H, Kim J, Yoo D, Su Y, Boley P,
114:259–267 Chepngeno J, Vlasova AN, Saif LJ, Wang Q
5. Murphy FA, Gibbs EPJ, Horzinek MA, Stud- (2019) Engineering a live attenuated porcine
dert MJ (1999) Asfarviridae and epidemic diarrhea virus vaccine candidate via
Iridoviridae. In: Murphy FA, Gibbs EPJ, Hor- inactivation of the viral 2’-O-methyltransferase
zinek MA, Studdert MJ (eds) Veterinary virol- and the endocytosis signal of the spike protein.
ogy. Academic Press, San Diego, CA, pp J Virol 93(15):e00406–e00419
293–300 11. Ackerman CM, Myhrvold C, Thakku SG,
Freije CA, Metsky HC, Yang DK, Simon HY,
Boehm CK, Kosoko-Thoroddsen TSF, Kehe J, 17. Romano JW, van Gemen B, Kievits T (1996)
Nguyen TG (2020) Massively multiplexed NASBA: a novel, isothermal detection technol-
nucleic acid detection with Cas13. Nature ogy for qualitative and quantitative HIV-1
582:277–282 RNA measurements. Clin Lab Med 16:89–103
12. Mullis K, Faloona F, Scharf S, Saiki RK, Horn 18. Lu X, Shi X, Wu G et al (2017) Visual detection
GT, Erlich H (1986) Specific enzymatic ampli- and differentiation of classic swine fever virus
fication of DNA in vitro: the polymerase chain strains using nucleic acid sequence-based
reaction. In: Cold Spring Harbor symposia on amplification (NASBA) and G-quadruplex
quantitative biology, vol 51. Cold Spring Har- DNAzyme assay. Sci Rep 7:44211. https://
bor laboratory Press, Cold Spring Harbor, doi.org/10.1038/srep44211
New York, pp 263–273 19. Jinek M et al (2012) A programmable dual-
13. Fakruddin M (2011) Loop mediated isother- RNA-guided DNA endonuclease in adaptive
mal amplification-An alternative to polymerase bacterial immunity. Science 337(6096):
chain reaction (PCR). Bangladesh Res Pub J 5: 816–821
425–439 20. Knott GJ, Doudna JA (2018) CRISPR-Cas
14. Notomi T, Okayama H, Masubuchi H, guides the future of genetic engineering. Sci-
Yonekawa T, Watanabe K, Amino N, Hase T ence 361(6405):866–869
(2000) Loop-mediated isothermal amplifica- 21. Makarova KS et al (2020a) Evolutionary classi-
tion of DNA. Nucleic Acids Res 28:E63. fication of CRISPR-Cas systems: a burst of class
https://doi.org/10.1093/nar/2 2 and derived variants. Nat Rev Microbiol
15. Kaneko, H, Kawana T, Fukushima E, Suzutani 18(2):67–83
T (2007) Tolerance of loop-mediated isother- 22. Wang X, He S, Zhao N et al (2020) Develop-
mal amplification to a culture medium and ment and clinical application of a novel
biological substances. J Biochem Biophys CRISPR-Cas12a based assay for the detection
Methods 70(3):499–501 of African swine fever virus. BMC Microbiol
16. Chan AB, Fox JD (1999) NASBA and other 20:282. https://doi.org/10.1186/s12866-
transcription-based amplification methods for 020-01966-6
research and diagnostic microbiology. Rev Med
Microbiol 10:185–196
Chapter 11
Aptamers as Diagnostic Markers for Viral Infections

of Veterinary Importance
Victoria C. Khangembam and Dimpal Thakuria
Abstract
Viral infections can cause serious diseases and remain one of the biggest challenges in animal healthcare.
Early and accurate diagnosis is crucial to prevent further spread of infections and for effective treatment. It is
also essential in case of emerging diseases to adopt correct control measures such as containment, confine-
ment, antimicrobials, and vaccines. Aptamers are promising molecules for developing biosensors to detect
infectious agents. They are a special class of small molecules consisting of single-stranded nucleic acids,
peptides, and peptide nucleic acids which can bind to a broad range of target molecules with high affinity
and specificity. Owing to its equal specificity to antibody based technologies and better sensitivity with
lower manufacturing cost, many aptamer based detection systems are being developed. Another added
advantage of aptamer based technology is its suitability for point-of-care testing in remote or less equipped
areas. This chapter aims to provide an overview of aptamers, its types and summarize the aptamer based
technologies in detection of viral pathogens of veterinary importance.
Key words Aptamers, Viral infection, Diagnostic, Nucleic acid, Peptide, Peptide nucleic acid
1 Introduction
Aptamers are a special class of single-stranded nucleic acids, pep-

tides, and peptide nucleic acids which can bind to a variety of target
molecules with high affinity and specificity [1, 2]. Nucleic acid
aptamers are 10–100 nucleotides long and form secondary and
tertiary structures ensuring the specific binding to its target mole-
cules such as amino acids, nucleotides, antibiotics, proteins, viruses,
bacteria, and even cells [3–8]. Peptide aptamers consists of 5–20
amino acids usually embedded as a loop within a stable protein
scaffold [9]. They can be regarded as smaller versions of immuno-
globulin T-cell receptors which are highly soluble, stable, and
exhibit fast folding kinetics. Both peptide and nucleic acid aptamers
are highly similar in terms of conformational flexibility and varia-
bility in functional groups [10]. An additional class of aptamers is
peptide nucleic acids (PNAs) which has high similarity to nucleic
159
160 Victoria C. Khangembam and Dimpal Thakuria
acids [2]. Structurally, PNA consists of a pseudopeptide backbone

where nucleobases are attached as side chains as in case of nucleic
acid. Since the PNA backbone is not charged unlike DNA and
RNA, there is no electrostatic repulsion during hybridization with
complementary nucleic acid sequence resulting in a more stable
PNA–DNA or PNA–RNA duplexes than the DNA–DNA or DNA–
RNA duplexes [11, 12]. Because of its non-natural polyamide
backbone, PNA is resistant to nucleases and proteases digestion
thereby extending their lifetime both in vivo and in vitro [13].
The term “aptamer” is derived from two words, “aptus”
(Latin) meaning “fit” and “meros” (Greek) meaning “part.”
These molecules particularly the nucleic acid aptamers have gained
interests due to their low production cost, easy chemical modifica-
tion, high chemical stability and binding affinity, repeatability, and
reusability. They may form secondary and tertiary structures which
gives them high specificity and affinity to a range of targets of
varying size and complexities [14]. They are also considered as
strong chemical competitors of antibodies due to their characteris-
tic features such as small size, low immunogenicity, cheaper, and
easier chemical synthesis and modification [15]. In fact, aptamers
are more amendable for on-site detection methods owing to their
stability at higher temperature and ability to recover their confor-
mation and activity upon re-annealing [14, 16]. Considering its
advantageous unique characteristics, technologies based on apta-
mers for detection of infectious diseases have achieved tremendous
progress in recent years. In addition to detection, aptamers are also
applicable in therapy, purification of target molecules, biosensors,
in vivo imaging [17–20]. Owing to its wide applicability, research
on aptamers is increasing and related information of several existing
aptamers can be accessed at the special database (http://aptamer.
icmb.utexas.edu) [1].
2 Selection of Aptamers
The method of aptamer selection called SELEX (Systematic Evolu-

tion of Ligands by EXponential enrichment) is an in vitro process.
The method was invented by two groups independently in 1990 for
selection of RNA ligands against T4 DNA polymerase and various
organic dyes [5, 21]. In the same year, another group has devised
techniques for the mutation, selection, and amplification of cata-
lytic RNA, a process that can be performed rapidly in vitro
[22]. SELEX is based on repeating cycles of binding, separation,
and amplification of nucleotides. Briefly, the first step of SELEX is
to incubate a sequence pool or nucleic acid library with targets. The
nucleic acids have a random internal region flanked by constant
sequences at both ends. The random region is tested for high
specificity and affinity to the target. The second step involves
Aptamers as Diagnostic Markers for Viral Infections of Veterinary Importance 161
removal of unbound nucleic acids. In the third step, the bound

sequences are purified and amplified that forms a new sequence
pool for the next cycle. Normally, the cycle is repeated 8–15 times
to achieve the desired aptamer sequence pool [7, 23]. Another step
of negative selection can be included to obtain aptamers with
higher specificity for the target. This is done by passing the
sequence pool over analogs or a supporting matrix in the absence
of the target which will eliminate the nucleic acids that bind to
competing analogs or matrix. The oligos selected from the final
round are then amplified and sequenced to identify aptamer. Alter-
natively, sequence data from the oligos present in the pool after
selection rounds can be obtained by next generation sequencing.
This is followed by synthesis and characterization of most potential
sequences. Frequently, aptamers with nanomolar dissociation con-
stants are identified but aptamers with picomolar dissociation con-
stants have also been isolated [24].
For peptide aptamer selection, there are different in vivo and
in vitro methods are available. The yeast two-hybrid (Y2H) strategy
and phage display are examples of in vivo and in vitro methods,
respectively [25, 26]. Initially, the method of phage display was
introduced to map epitope binding sites of immunoglobulins by
panning large phage libraries of random peptides [9, 26]. In this,
external gene is inserted into the gene of appropriate coat protein of
the bacteriophage most commonly M13 phage. The resulting
phage is amplified in E. coli and during assembly; the translated
fusion protein gets displayed on the phage surface. Then the phage
population exhibiting strong binding with the target of interest are
identified. The Y2H strategy is based on a system of two hybrid
proteins containing parts of GAL4 of Saccharomyces cerevisiae. The
hybrid proteins are GAL4 DNA-binding domain and GAL4 acti-
vating region, fused with Snf1 and Snf4 yeast proteins. The pres-
ence of both the hybrids results in high transcriptional activity in a
cell [26]. Notably, in both selection processes, peptide aptamers are
isolated together with their coding sequences [27]. Other in vitro
display based selection methods of peptide aptamer includes cell
surface display, ribosome display, mRNA display, DNA display, and
in vitro compartmentalization [9].
3 Application of Aptamer in Detection of Viral Diseases
Several viral infections can cause serious disease in animals and some
may passed to human. Therefore, rapid and accurate identification
of a causative agent is critical to ensure correct, well timed treat-
ment of the patient but also to prevent the spread of the infectious
agent. Technologies based on aptamer have great potential in virus
detection and therapeutics. In diagnosis of viral diseases, virus
isolation is regarded as gold standard which is always performed
in a designated virology laboratory [28, 29]. However, it may take a

longer period depending on the types of viruses and susceptibility
of the cell lines and may not be possible to detect the non culturable
or slow growing (non-cytopathic) viruses [28]. Hence, viral nucleic
acids or proteins involved in adsorption, penetration, and replica-
tion and finally release of virus may be targeted for aptamer selec-
tion and development of biosensor. A biosensor is an analytical
device that typically consists of a bioreceptor (enzyme/antibody/
cell/nucleic acid/aptamer) which recognizes and binds the target
with high sensitivity and selectivity and a transducer component
which translates and outputs biological signals resulting from the
interaction [30, 31]. Aptamers based biosensor also called aptasen-
sors, uses aptamers as either bioreceptor or transducer
[32, 33]. Aptasensors, which are cheap, user friendly, quick, and
specific for the target, stable and most importantly applicable at
field condition may receive wider acceptance.
4 Nucleic Acid Aptamers
Nucleic acid aptamers (DNA, RNA, oligos with modified nucleo-

tides) have high affinity and specificity for a broad range of potential
targets. They are more preferred than antibodies for use in biosen-
sor because of their flexible structure, lower cost, stability, smaller
size, and non-immunogenecity [24]. DNA aptamers have been
employed in several detection methods of animal viral diseases
and many are reported for H5N1 avian influenza virus. Highly
pathogenic avian influenza virus (HPAIV) subtype H5N1 virus
can cause severe disease in poultry often leading to huge mortality
and can also infect mammalian species including humans, rats, and
mice, weasels and ferrets, pigs, cats, tigers, and dogs. In severe form
of H5N1 infection in birds, a mortality rate up to 100% within 48 h
can be observed (OIE).
Shiratori et al. [34] have used DNA aptamers that bind to HA1
(hemagglutinin 1) proteins of multiple influenza A virus subtypes
to develop novel aptamer based sandwich detection method. DNA
aptamers were selected through a modified protocol of SELEX and
amplified by PCR. The aptamer based assay successfully detected
the H5N1, H1N1, and H3N2 subtypes of influenza A virus with
almost equal sensitivities. In another study, Bai et al. [35] have used
a DNA aptamer as the specific recognition element in a portable
Surface Plasmon Resonance (SPR) biosensor for rapid detection of
highly pathogenic avian influenza subtype H5N1 in poultry swab
samples. The aptasensor could detect 0.128–1.28 hemagglutina-
tion units (HAUs) of the virus within 1.5 h. In the similar line, a
pair of aptamers was successfully applied to develop the sandwich-
type SPR-based biosensor for detection of H5N1 whole virus
which could detect a virus concentration of 200 EID50/ml (50%
embryo infective dose/ml) in fecal samples [36]. Fu et al. [37]

proposed a method for detecting H5N1 based on exploitation of
enzymatic catalysis in ultra-low ion strength media to induce ion
strength increase for developing a novel impedance biosensing
method. The method used a bionanocomposites formed with mag-
netic beads were modified with H5N1-specific aptamer to capture
the H5N1 virus, concanavalin A (ConA), glucose oxidase (GOx),
and gold nanoparticles. The biosensor showed high sensitivity with
a detection limit of 8 104 HAU in 200 μl sample. A highly
sensitive quartz crystal microbalance (QCM) aptasensor based on
ssDNA crosslinked polymeric hydrogel was also developed for
rapid, sensitive, and specific detection of avian influenza H5N1
virus which has a detection limit of 0.0128 HAU (HA unit) and
time required from sampling to detection was only 30 min
[38]. Lum et al. [39]used a biotinylated DNA aptamer to develop
impedance biosensor using microfluidics flow cell and an interdigi-
tated microelectrode which can detect 0.0128 hemagglutinin units
(HAU) of H5N1 AIV specifically in 30 min. Another impedance
aptasensor developed using specific H5N1 aptamer and a gold
interdigitated microelectrode could detect 0.25 HAU of pure
virus and 1 HAU for the H5N1 virus spiked tracheal chicken
swab samples [40].
In poultry industry, another economically important viral dis-
ease is Newcastle disease (ND) caused by a virus in the family of
paramyxoviruses. It is a highly contagious and in severe cases vari-
able mortality as high as 100% can occur (OIE). Based on the
pathogenicity of the ND virus (NDV) strains in chickens, they are
divided into three pathotypes; velogenic (very virulent), mesogenic
(moderate virulent), and lentogenic strains (mild virulent) [41]. A
sandwich enzymatic linked aptamer assay (ELAA) using ssDNA
have been developed for rapid and sensitive detection of NDV in
farm samples [42]. The aptamers showed high specificity towards
NDV with no cross-reactivity towards other avian viruses. The
aptamers had affinity within the nanomolar range and the accuracy
of the assay was comparable with standard qRT-PCR method.
Among the economically important diseases of ruminants,
bovine viral diarrhea is a significant disease of cattle. It is caused
by bovine viral diarrhea virus (BVDV), a member of the genus
Pestivirus of the family, Flaviviridae. In bull, infection leads to fall
in semen quality and the infectious virus can be isolated from the
ejaculate. In cow, infection leads to poor conception rates, abor-
tions, and congenital defects [43]. For detection of BVDV type
1, an aptamer based sandwich-typed assay had been developed. The
ultrasensitive detection method used a ssDNA aptamer conjugated
gold nanoparticle in an aptamer–aptamer sandwich-type sensing
format which had detection limit of 800 copies/ml [44].
In pigs, porcine reproductive and respiratory syndrome (PRRS)
is one of the most important swine diseases worldwide. It is caused
by a porcine reproductive and respiratory syndrome virus (PRRSV)

of the genus, Arterivirus. The symptoms include reproductive fail-
ure, pneumonia, high levels of neonatal mortality, and increased
susceptibility to secondary bacterial infection [45]. A biosensor for
detection of PRRSV using DNA aptamer had been developed. The
aptamer could bind specifically to the PRSSV when presented with
the classical swine fever virus and a pseudo rabies virus. The devel-
oped platform was more rapid, accurate as compared to ELISA,
PCR, and RT-PCR and had a detection limit of
1.87 1010 particles [46].
5 Peptide Aptamers
Peptide aptamers resemble antibodies with a variable antigen-

binding domain with more advantageous features such as smaller
size, higher stability and solubility, high yield bacterial expression,
possibility of chemical synthesis, rapid folding properties, and in
some cases absence of disulfide bonds and free cysteine residues
[27]. The concept of peptide aptamers was originally proposed as a
short amino acid sequence fixed in a small and stable protein
backbone [9, 47]. Peptide aptamers are basically a “loop on a
frame” strategy where a peptide of around 5–20 amino acids is
embedded (constrained) in a neutral scaffold. Scaffold is rigid,
compact, stable protein core preferably monomeric, and capable
of displaying variable sites of interaction with target molecule as in
case of complementarity determining region (CDR) of immuno-
globulin molecule [9]. The embedded peptide imparts variability to
select high affinity binders to a target molecule. The binding affinity
of this constrained aptamers is much higher than the free peptide
which can be up to 1000 times [9, 48, 49].
As peptide aptamer based platforms are flexible providing bind-
ing surfaces capable of accommodating large flat protein–protein
interfaces as well as small molecules in the clefts and pockets, its
applications in biomedical and bioanalytical field are on the rise
[50]. Peptide aptamers having potentials to bind to Newcastle
disease virus (NDV) have been identified by biopanning method
using phage display technology [51]. The peptides were synthe-
sized and their binding specificities were also confirmed by com-
petitive phagemid ELISA using chicken anti-NDV antiserum as
competitor.
6 Peptide Nucleic Acid Aptamers
Peptide nucleic acids (PNAs) are chemically stable and resistant to

hydrolytic (enzymatic) cleavage and capable of sequence specific
binding to complementary DNA and RNA through Watson–Crick
hydrogen bonding with high affinity due to their uncharged and

flexible polyamide backbone [12, 52]. PNA is considered as a DNA
analogue but its chemical structure is more like a peptide or protein
with N-terminal at left and C-terminal at right side. PNAs can be
synthesized by the standard method of solid-phase peptide synthe-
sis using Fmoc-chemistry. Owing to their unique chemical, physi-
cal, and biological properties, PNA have been used to develop
powerful biomolecular tools, molecular probes, and
biosensors [53].
Using PNA and gold nanoparticles (AuNPs), a rapid label-free
visual assay have been developed for the detection, genotyping/
pathotyping, and quantification of Newcastle disease (ND) viral
RNA [54]. The developed visual assay exploited the ability of free
PNA to induce agglomeration in AuNPs leading change in color
from red to blue and prevention of aggregation by hybridization of
PNA to specific DNA [55]. In the assay, PNA probes complemen-
tary to the cleavage site of the ND virus F gene were used to detect
viral RNA. The probes could detect 5–10 ng of viral RNA in 100 μl
of biological samples, such as allantoic fluids, cell culture fluids, and
vaccines. The end result of the assay could be observed with naked
eye as plasmon color changes in the AuNPs solution and confirmed
spectrophotometrically. The assay could also detect difference in
single nucleotide thus enabling a visual viral genotyping/pathotyp-
ing. Another label-free visual assay using PNA was also developed
for multiple strains of Influenza A Virus. The method provides
accurate quantification of viral RNA on a spectrophotometer with
visual limit of detection of 2.3 ng of Influenza A viral RNA [56].
7 Advantages of Aptamers
Aptamers are considered as a promising substitute for antibodies

since their unique features can overcome some of the scientific
challenges associated with antibodies. Though antibodies have
wider range of applications, aptamers have a number of advantages
as compared to antibodies. Nucleic acid aptamers are small, 20–60
nucleotides long, single-stranded RNA or DNA that can be
arranged with a higher density on the biosensor surface. Nucleic
acid aptamers are more thermally stable and maintain their struc-
tures over repeated cycles of denaturation/renaturation. It can
recover their native conformation and can bind to targets upon
re-annealing [10]. High resistance of aptamers to degradation by
blood nucleases may be due to the formation of specific three-
dimensional structures that protect the 30 - and 50 -termini of apta-
mers against exonucleases [1, 57]. Peptide nucleic acids, due to its
non-natural polyamide backbone, are extremely stable in acidic
environment, resistant to degradation by nucleases and proteases
[13, 58]. Aptamer production is significantly easier and cheaper
than the production of antibodies [59, 60]. Furthermore, modifi-

cations of aptamers can be done during or after synthesis to increase
their stability and nuclease resistance or to introduce signal moi-
eties, such as fluorophores and quenchers to facilitate the develop-
ment of biosensors [16, 61, 62]. The most common and effective
type of modifications to increase resistance to nucleases without
affecting their binding to target molecules is the modification of 30 -
and 50 -nucleotides [63]. Another modification to make aptamer
highly resistant to degradation by nucleases is ligation of the 30 - and
50 -termini of the same [64, 65]. Unlike antibodies which are signif-
icantly immunogenic, nucleic acid aptamers are low-immunogenic
and low-toxic molecules. Aptamers has high affinity and specificity
for their targets and even for those ligands which cannot be recog-
nized by antibodies, such as ions or small molecules [16, 62].
8 Future Prospects
A rapid, user friendly, and accurate identification of causative agent

of economically important animal diseases is highly required for
mass screening of a population. Aptamer based biosensors have
enabled to detect biomarkers at a very low concentration which
can be applied for detection of infection at early stage. This will
facilitate to segregate the infected animal in time to prevent the
spread of the disease in the flock. The advantages of aptamers such
as lower production cost, easy modification to improve stability,
high sensitivity, reproducibility, specificity, and less detection time
may be exploited to develop diagnostic methods for use at the
point-of-care and simple laboratories. In addition, the ability of
aptamers to detect even those small ligands which cannot be recog-
nized by antibodies makes it a strong and versatile alternative to
antibodies. Aptamer based techniques such as electrochemical, col-
orimetric, optical using aptamers are being studied worldwide. In
therapeutic application, pegaptanib (brand name Macugen), an
anti-vascular endothelial growth factor (VEGF) aptamer was
approved by the U.S. Food and Drug Administration (FDA) in
December 2004 for the treatment of neovascular age related mac-
ular degeneration. This has created confidence among aptamer
developers and may lead to expansion of markets for aptamer
based technologies. Furthermore, low immunogenicity,
low-toxicity, and small size of these molecules make them more
versatile for different biomedical applications and may receive a
wider acceptance in the future.
References
1. Lakhin AV, Tarantul VZ, Gening LV (2013) 15. Keefe AD, Pai S, Ellington A (2010) Aptamers
Aptamers: problems, solutions and prospects. as therapeutics. Nat Rev Drug Discov 9:537–
Actanaturae 5(4):34–43 550
2. Lee EJ, Lim HK, Cho YS, Hah SS (2013) 16. Song KM, Lee S, Ban C (2012) Aptamers and
Peptide nucleic acids are an additional class of their biological applications. Sensors 12(1):
aptamers. RSC Adv 3:5828 612–631
3. Zou X, Wu J, Gu J, Shen L, Mao L (2019) 17. Yang L, Zhang X, Ye M, Jiang J, Yang R, Fu T,
Application of aptamers in virus detection and Chen Y, Wang K, Liu C, Tan W (2011)
antiviral therapy. Front Microbiol 10:1462 Aptamer-conjugated nanomaterials and their
4. Szpechciński A, Grzanka A (2006) Aptamery applications. Adv Drug Deliv Rev 63(14–15):
w diagnostyceklinicznej [aptamers in clinical 1361–1370
diagnostics]. Postepy Biochem 52(3):260–270 18. Cibiel A, Pestourie C, Ducongé F (2012) In
5. Ellington AD, Szostak JW (1990) In vitro vivo uses of aptamers selected against cell sur-
selection of RNA molecules that bind specific face biomarkers for therapy and molecular
ligands. Nature 346(6287):818–822 imaging. Biochimie 94(7):1595–1606
6. Schneider D, Tuerk C, Gold L (1992) Selec- 19. Wang K, Fan D, Liu Y, Wang E (2015) Highly
tion of high affinity RNA ligands to the bacte- sensitive and specific colorimetric detection of
riophage R17 coat protein. J Mol Biol 228: cancer cells viadual-aptamer target binding
862–869 strategy. Biosens Bioelectron 73:1–6
7. Torres-Chavolla E, Alocilja EC (2009) Apta- 20. Zhang Y, Lai, B.S. andJuhas, M. (2019) Recent
sensors for detection of microbial and viral advances in aptamer discovery and applications.
pathogens. Biosens Bioelectron 24:3175– Molecules 24(5):941
3182 21. Tuerk C, Gold L (1990) Systematic evolution
8. Ku TH, Zhang T, Luo H, Yen TM, Chen PW, of ligands by exponential enrichment: RNA
Han Y, Lo YH (2015) Nucleic acid aptamers: ligands to bacteriophage T4 DNA polymerase.
an emerging tool for biotechnology and bio- Science 249(4968):505–510
medical sensing. Sensors 15:16281–16313 22. Robertson DL, Joyce GF (1990) Selection
9. Reverdatto S, Burz DS, Shekhtman A (2015) in vitro of an RNA enzyme that specifically
Peptide aptamers: development and applica- cleaves single-stranded DNA. Nature
tions. Curr Top Med Chem 15(12): 344(6265):467–468
1082–1101 23. Davydova A, Vorobjeva M, Pyshnyi D,
10. Mascini M, Palchetti I, Tombelli S (2012) Altman S, Vlassov V, Venyaminova A (2016)
Nucleic acid and peptide aptamers: fundamen- Aptamers against pathogenic microorganisms.
tals and bioanalytical aspects. Angew Chem Int Crit Rev Microbiol 42:847–865
Ed Engl 51:1316–1332 24. Ilgu M, Fazlioglu R, Ozturk M, Ozsurekci Y,
11. Jensen KK, Orum H, Nielsen PE, Nordén B Nilsen-Hamilton M (2019) Aptamers for diag-
(1997) Kinetics for hybridization of peptide nostics with applications for infectious diseases.
nucleic acids (PNA) with DNA and RNA stud- In: Muharrem Ince, Olcay Kaplan Ince (Eds.),
ied with the BIAcore technique. Biochemistry Recent advances in analytical chemistry, Intech
36(16):5072–5077 Open. https://doi.org/10.5772/intechopen.
12. Paulasova P, Pellestor F (2004) The peptide 84867
nucleic acids (PNAs): a new generation of 25. Fields S, Song O (1989) A novel genetic system
probes for genetic and cytogenetic analyses. to detect protein-protein interactions. Nature
Ann Genet 47(4):349–358 340:245–246
13. Demidov VV, Potaman VN, Frank- 26. Smith GP (1985) Filamentous fusion phage:
Kamenetskil MD, Egholm M, Buchard O, novel expression vectors that display cloned
Sönnichsen SH, NielsenP E (1994) Stability antigens on the virion surface. Science 228:
of peptide nucleic acids in human serum and 1315–1317
cellular extracts. Biochem Pharmacol 48(6): 27. Colombo M, Mizzotti C, Masiero S, Kater
1310–1313 MM, Pesaresi P (2015) Peptide aptamers: The
14. Li HY, Jia WN, Li XY, Zhang L, Liu C, Wu J versatile role of specific protein function inhi-
(2020) Advances in detection of infectious bitors in plant biotechnology. J Integr Plant
agents by aptamer-based technologies. Emerg Biol 57:892–901
Microbes Infect 9(1):1671–1681
28. Leland DS, Ginocchio CC (2007) Role of cell as diagnostic tool for Newcastle avian virus.
culture for virus detection in the age of tech- PLoS One 15(8):e0237253
nology. Clin Microbiol Rev 20(1):49–78 43. Fray MD, Paton DJ, Alenius S (2000) The
29. Storch GA (2000) Diagnostic virology. Clin effects of bovine viral diarrhoea virus on cattle
Infect Dis 31(3):739–751 reproduction in relation to disease control.
30. Hong P, Li W, Li J (2012) Applications of Anim Reprod Sci 60–61:615–627
aptasensors in clinical diagnostics. Sensors 12: 44. Park JW, Jin Lee S, Choi EJ, Kim J, Song JY,
1181–1193 Bock Gu M (2014) An ultra-sensitive detection
31. Han K, Liang Z, Zhou N (2010) Design stra- of a whole virus using dual aptamers developed
tegies for aptamer-based biosensors. Sensors by immobilization-free screening. Biosens
10(5):4541–4557 Bioelectron 51:324–329
32. Cheng AK, Sen D, Yu HZ (2009) Design and 45. Meulenberg JJ (2000) PRRSV, the virus. Vet
testing of aptamer-based electrochemical bio- Res 31(1):11–21
sensors for proteins and small molecules. Bioe- 46. Kuitio C, Rasri N, Kiriwan D, Unajak S, Choo-
lectrochemistry 77:1–12 wongkomon K (2020) Development of a bio-
33. Hianik T, Porfireva A, Grman I, Evtugyn G sensor from aptamers for detection of the
(2009) EQCM biosensors based on DNA apta- porcine reproductive and respiratory syndrome
mers and antibodies for rapid detection of virus. J Vet Sci 21(5):e79
prions. Protein Pept Lett 16:363–367 47. Colas P, Cohen B, Jessen T, Grishina I,
34. Shiratori I, Akitomi J, Boltz DA, Horii K, McCoy J, Brent R (1996) Genetic selection of
Furuichi M, Waga I (2014) Selection of DNA peptide aptamers that recognize and inhibit
aptamers that bind to influenza A viruses with cyclin-dependent kinase 2. Nature 380:548–
high affinity and broad subtype specificity. Bio- 550
chem Biophys Res Commun 443:37–41 48. Ladner RC (1995) Constrained peptides as
35. Bai H, Wang R, Hargis B, Lu H, Li Y (2012) A binding entities. Trends Biotechnol 13:426–
SPR aptasensor for detection of avian influenza 430
virus H5N1. Sensors 12(9):12506–12518 49. Cohen BA, Colas P, Brent R (1998) An artifi-
36. Nguyen VT, Seo HB, Kim BC, Kim SK, Song cial cell-cycle inhibitor isolated from a combi-
CS, Gu MB (2016) Highly sensitive sandwich- natorial library. Proc Natl Acad Sci U S A 95:
type SPR based detection of whole H5Nx 14272–14277
viruses using a pair of aptamers. Biosens Bioe- 50. Vanhee P, van der Sloot AM, Verschueren E,
lectron 86:293–300 Serrano L, Rousseau F, Schymkowitz J (2011)
37. Fu Y, Callaway Z, Lum J, Wang R, Lin J, Li Y Computational design of peptide ligands.
(2014) Exploiting enzyme catalysis in ultra-low Trends Biotechnol 29:231–239
ion strength media for impedance biosensing 51. Ozawa M, Ohashi K, Onuma M (2005) Iden-
of avian influenza virus using a bare interdigi- tification and characterization of peptides bind-
tated electrode. Anal Chem 86:1965–1971 ing to Newcastle disease virus by phage display.
38. Wang R, Li Y (2013) Hydrogel based QCM J Vet Med Sci 67(12):1237–1241
aptasensor for detection of avian influenza 52. Ray A, Norden B (2000) Peptide nucleic acid
virus. Biosens Bioelectron 42:148–155 (PNA): its medical and biotechnical applica-
39. Lum J, Wang R, Hargis B, Tung S, Bottje W, tions and promise for the future. FASEB J 14:
Lu H, Li Y (2015) An impedance aptasensor 1041–1060
with microfluidic chips for specific detection of 53. Nielsen PE, Egholm M (1999) An introduc-
H5N1 avian influenza virus. Sensors 15: tion to peptide nucleic acid. Curr Issues Mol
18565–18578 Biol 1(1–2):89–104
40. Karash S, Wang R, Kelso L, Lu H, Huang TJ, 54. Joshi VG, Chindera K, Singh AK, Sahoo AP,
Li Y (2016) Rapid detection of avian influenza Dighe VD, Thakuria D, Tiwari AK, Kumar S
virus H5N1 in chicken tracheal samples using (2013) Rapid label-free visual assay for the
an impedance aptasensor with gold nanoparti- detection and quantification of viral RNA
cles for signal amplification. J Virol Methods using peptide nucleic acid (PNA) and gold
236:147–156 nanoparticles (AuNPs). Anal Chim Acta 795:
41. Brown C, King DJ, Seal BS (1999) Pathogen- 1–7
esis of Newcastle disease in chickens experi- 55. Su X, Kanjanawarut R (2009) Control of metal
mentally infected with viruses of different nanoparticles aggregation and dispersion by
virulence. Vet Pathol 36(2):125–132 PNA and PNADNA complexes, and its appli-
42. Marnissi B, Kamali-Moghaddam M, Ghram A, cation for colorimetric DNA detection. ACS
Hmila I (2020) Generation of ssDNAaptamers Nano 3(9):2751–2759
56. Kumar N, Bhatia S, Pateriya AK, Sood R, 60. Kulbachinskiy AV (2007) Methods for selec-
Nagarajan S, Murugkar HV, Kumar S, tion of aptamers to protein targets. Biochemis-
Singh P, Singh VP (2020) Label-free peptide try (Mosc) 72(13):1505–1518
nucleic acid biosensor for visual detection of 61. Ferreira CS, Missailidis S (2007) Aptamer-
multiple strains of influenza A virus suitable for based therapeutics and their potential in radio-
field applications. Anal Chim Acta 1093:123– pharmaceutical design. Braz Arch Biol Technol
130 50:63–76
57. Nitsche A, Kurth A, Dunkhorst A, Pänke O, 62. Jayasena SD (1999) Aptamers: an emerging
Sielaff H, Junge W, Muth D, Scheller F, class of molecules that rival antibodies in diag-
Stöcklein W, Dahmen C, Pauli G, Kage A nostics. Clin Chem 45(9):1628–1650
(2007) One-step selection of vaccinia virus- 63. Mayer G (2009) The chemical biology of apta-
binding DNA aptamers by MonoLEX. BMC mers. Angew Chem Int Ed Engl 48(15):
Biotechnol 7:48 2672–2689
58. Zhang N, Appella DH (2010) Advantages of 64. Mori Y, Nakamura Y, Ohuchi S (2012) Inhibi-
peptide nucleic acids as diagnostic platforms for tory RNA aptamer against SP6 RNA polymer-
detection of nucleic acids in resource-limited ase. Biochem Biophys Res Commun 420:440–
settings. J Infect Dis 201(1):S42–S45 443
59. Conrad RC, Giver L, Tian Y, Ellington AD 65. Di Giusto DA, Knox SM, Lai Y, Tyrelle GD,
(1996) In vitro selection of nucleic acid apta- Aung MT, King GC (2006) Multitasking by
mers that bind proteins. Methods Enzymol multivalent circular DNA aptamers. Chembio-
267:336–367 chem 7:535–544
Chapter 12
Antibody-Based Sensors for Pathogen Detection

Nirmita Dutta, Akhil Kumar, Anu Kumari, Sushila Maan,
Gorachand Dutta, and Vinay G. Joshi
Abstract
Antibodies are soluble biomolecules of the Immunoglobulin family found in serum, which can specifically
bind to and neutralize diverse antigens. Since their discovery, antibodies have been utilized for diagnostic,
therapeutic, and research purposes. The development of genetic engineering and recombinant technology
has made it possible to modify antibodies in structure and composition. Antibodies have found utility in the
field of diagnostics with the incorporation of native or recombinant antibodies in biosensing platforms,
capable of transducing the information of an antigen-antibody binding event into a measurable signal. This
platform is termed as an immunosensor. Several approaches are available for the immobilization of
antibodies on the surface of the transducer. These approaches include either a covalent or non-covalent
attachment of the active form of antibodies in proper orientation, while retaining the conductivity of the
transducing elements at the same time. The generated signal can be an electrical, optical, shear strain, or
temperature change. Accordingly, immunosensors can be broadly divided into electrochemical, optical,
piezoelectric or thermometric immunosensors. Each type has its own set of advantages and challenges in the
context of design and sensing efficiency.
Key words Antibody, Biosensor, Immobilization, Electrochemical, SPR, Piezoelectric
1 Introduction
Since the dawn of Immunology during the 1880s, Emil von Behr-
ing and Shibasaburo Kitasato first reported the presence of anti-
bodies (Abs) as anti-toxin factors (Fig. 1). Because of their high
affinity and specificity towards a target molecule, antibodies against
diverse antigens (Ags) have been produced by immunizing model
vertebrate animals. Antisera and purified antibodies have been tra-
ditionally used for diagnosis, prevention, treatment, and epidemi-
ology of animal and human diseases, and for research in life
sciences [1].
During 1975, the advent of in vitro hybridoma technology by
G. Kohler and C. Milstein marked the beginning of the modern era
in antibody production. Monoclonal Antibody (mAb) technology
171
172 Nirmita Dutta et al.
Fig. 1 Antibody production from activated B cells/plasma cells
or hybridoma technology has revolutionized the use of antibody

(Ab) as a tool for research for the prevention, detection, and
treatment of diseases (Fig. 2) [2, 3]. Because of hybridoma tech-
nology, a large number of immunological assays were developed
and are still being used for the diagnosis of different diseases
around the world. With the annual turnover of 115.2 billion US$
in 2018, the mAbs market is the fastest growing of all therapeutic
proteins [4].
In the 1980s, novel recombinant DNA (rDNA)-based technol-
ogies, including heterologous antibody expression systems, such as
bacteria, yeast, insects, and plants, as well as plant and mammalian
cell culture systems, were developed. New technologies have
enabled the production of bio-engineered Abs for application in
modern science and medicine such as diagnostics, theranostics,
therapeutics, proteomics, biosensors, etc. Desirable features can
be introduced in the recombinant Ab (rAb) formats, and various
rAbs against several clinically and biotechnologically important
antigens have already been produced [5]. Murine monoclonal anti-
bodies produced by hybridoma cultures were modified (chimerized
or humanized) using rDNA techniques for in vivo application in
humans (Fig. 3). The bio-engineered Abs have been projected as
the “Abs of the future,” which are going to replace antisera and
most, if not all mAbs in the coming decades. In fact, the engineered
Abs are making it possible to solve the problems that have defied
traditional approaches. Moreover, such antibody constructs do not
require the use of animals, thereby contributing to saving the lives
of thousands of animals and promoting animal welfare [6].
Ever since their recognition more than a century ago, antibo-
dies continue to be a class of fascinating bio-molecules globally for
researchers, clinicians, and industrialists. Advancement in molecular
biology and biotechnology open the path for the production of
customized antibodies with desired size and specificities. Ab-based
immunoassays are the most commonly used diagnostic assays and
remain one of the fastest growing technologies for the analysis of
bio-molecules. But traditional in vitro diagnostics have some lim-
itations. They are time consuming, require trained person, specific
equipment, sophisticated laboratories, pragmatic usage under field
Antibody-Based Sensors for Pathogen Detection 173
Fig. 2 Hybridoma technology for monoclonal antibody production (HGPRT Hypoxanthine-guanine phosphor-
ibosyltransferase enzyme, HAT medium Hypoxanthine-aminopterin-thymidine medium)
conditions etc. [7] However, recent advances in biosensor technol-

ogy have paved the way for the development of point-of-care
diagnostics which are more accurate, less time consuming, applica-
ble under field conditions, economical, etc. Antibodies, which are
among the most exquisitely designed and engineered molecules in
nature, are the ideal recognition elements for incorporation into
sensors. A huge number of Ab-based biosensors (Immunosensors)
are utilized clinically for the detection of a variety of analytes.
2 General Structure of an Antibody: An Overview
Antibodies are serum soluble, Y shaped glycoproteins produced by

activated B cells, also known as plasma cells, as a result of the
interaction between the antigen-specific surface receptor of naı̈ve
Fig. 3 Recombinant DNA technology for chimeric recombinant antibody (rAb) production
B lymphocytes and a specific antigen (Fig. 4). Antibodies have the

property of combining specifically with the antigen that induced
their formation. Like other proteins, antibody molecules may be
classified physio-chemically on the basis of their solubility in salt
solutions, electrostatic charge, molecular weight, and structure [8].
In the immune system, the best characterized molecules are
antibodies, which occur mainly in the globulin fraction of serum
and are known as immunoglobulins(Igs). The antibody molecule
plays the central role in humoral immunity by attaching to micro-
organisms and neutralizing it. This event leads to the activation of
one of the many killingmechanisms, such as opsonization, comple-
ment fixation, and the antibody-dependent cellular cytotoxicity
(ADCC), which involve the recruitment of effectors of the cellular
immune system. Effectors of the immune system are cells that are
activated and differentiate in response to an immune trigger. Effec-
tor B cells or plasma cells are responsible for producing antigen-
specific antibodies and mediate humoral immunity, while effector T
cells, namely helper T cells and cytotoxic T cells, help in the activa-
tion of immune cells and directly target infected cells, respectively,
contributing to cell-mediated immunity. Gerald Edelman (USA)
and Rodney R. Porter (UK) elucidated the structure of Igs and
were awarded the Nobel Prize in 1972. They studied the chemical
structure of γ (gamma)-globulin fraction of serum containing IgG
Fig. 4 General structure of an antibody.
of rabbit, using chemical solvents and proteolytic enzymes. Under

electron microscope, the Ig was observed to be a typical “Y” shaped
molecule [9].
Each IgG has two identical pairs of polypeptide chains: a light
chain (LC) about 25–30 kDa size and a heavy chain (HC) of
50–70 kDa size(Fig. 4). The LC and HC are joined together by
interchain disulphide (–S–S–) bonds. Variable (V) sequence and
constant (C) sequence regions of LC and HC are folded into
distinct domain structures, called “Ig domain” [10]. LC has two
“Ig domains,” i.e., one N-terminal VL (110 AAs) and one
C-terminal CL (110 AAs), whereas HC has 4 or 5 “Ig domains,”
i.e., one N-terminal VH (110 AAs) and three or four CH (CH1-
CH3 or CH4). Each Ig domain contains a loop (“Ig fold”) of about
60–70 AAs enclosed by intrachain (–S–S–) bonds. Three
“hypervariable” (HV1-HV3) regions, also called complementarity
determining regions (CDR1-CDR3), interspersed within four less-
variable “framework” regions (FR1-FR4) exist within each VL and
VH. The six CDRs, three each from VL and VH segments, fold to
make one antigen-binding site or “paratope” (Fig. 5) [11]. Thus,
each Ab molecule is at least bivalent, having two identical paratopes
to bind the two identical epitopes in a multivalent Ag. Each para-
tope has a unique shape and chemical complementarity to that of
the epitope for the best fit. In an immunoassay method, the most
important function of antibody molecules is to combine with their
specific antigens to form an antibody–antigen complex. The
antigen-binding sites are responsible for the specific binding of
Fig. 5 Representation of a light chain showing antiparallel β-sheets of VL and CL regions, connected by α-helix
linker, and complementarity determining regions (CDRs)
Ab to their targets. The intermolecular forces that contribute to the

stabilization of the antibody–antigen complex are hydrogen bond-
ing, electrostatic forces, hydrophobic interactions, van der Waals
forces, and stearic repulsive pulses [12].
3 Biosensor/Immunosensor
Our immune system recognizes all cells and molecules in the body
system and can differentiate between self and non-self-bio-mole-
cules. When our body encounters some foreign substances (anti-
gens), specialized immune cells get activated and produce
antibodies which are specific to these antigens. This antigen-
antibody interaction has been used by scientists to develop various
formats of immunoassays including the development of sensors. A
sensor that is based on the concept of immunology is known as an
immunosensor. This antigen-antibody (immune-complex) thus
formed in an immunosensor is measured by coupling this reaction
to the surface of a transducer. The transducer detects and converts
the reaction to an electrical signal where it can be processed,
recorded, and viewed [13]. Ideally, an immunosensor should be
designed with the following specifications: (1) the ability to identify
target antigens quickly; (2) the ability to generate immunocom-
plexes without the need to add supplementary reagents; (3) the
ability to give results with high reproducibility; and (4) the ability
to easily detect the target in real samples [14].
The term Biosensor was first defined by the International

Union of Pure and Applied Chemistry, 1992. The first biosensor
was developed by Clark and Lyons in 1962 to measure glucose in
biological samples. This biosensor coupled the biological specificity
of enzymes with an electrode and transducer [15]. The concept of
using immunological components (antibody or antigen) as sensing
agents was first described within an immunoassay for plasma insulin
in human subjects [16]. The journal Biosensors and Bioelectronics in
the abstracts of the Fifth World Conference on Biosensors define a
biosensor as an: “analytical device incorporating a biological mate-
rial, a biologically derived material, or a biomimic, intimately asso-
ciated with or integrated within a physico-chemical transducer or
transducing microsystem.”. The aim of a biosensor is to produce an
electronic signal proportional to the specific interaction of analytes
with the sensing element [17]. Biosensors can be used for the
detection of analytes ranging from small molecules to intact patho-
genic microorganisms. In the case of immunosensors, antibodies/
antibody fragments or antigens are used for detection. Immuno-
sensors employ the high Ab/Ag specificity to detect the presence of
its analyte [18]. The generated signal is proportional to the amount
of target analyte in a specific reaction. A biosensor consists of the
following constituents:
Analyte: A component or substance of interest to be detected.
Bioreceptor: A molecule that specifically recognizes the analyte.
Bioreceptors can be in the form of enzymes, cells, and antibodies.
Once bioreceptors interact with the analyte, a signal will be pro-
duced, such as heat, charge, mass change or pH, etc., and this is
called bio-recognition (or recognition receptor).
Transducer: The function of a transducer is to use the informa-
tion from the bio-recognition episode between the bioreceptor and
analyte, and convert it into a measurable signal.
Biosensors are useful and have a number of advantages over
current analytical instruments based on these two features: (1) the
proximity of the recognition receptors, e.g. antibodies, DNA, apta-
mers with the transducer; and (2) its practical size, suitable for
fieldwork. Only a minute amount of sample is required for detec-
tion, as the sensitive part of a biosensor is normally small [14].
Basically, a biosensor has two components: a selective receptor
(enzyme, Abs, etc.) and a detector (a transducer to sense the
chemical or physical change upon interaction of the analyte with
the receptor, and converts the signal into electrical/electronic).
Depending on the method of signal transduction detection, bio-
sensors can be classified as optical, electrochemical, thermometric,
piezoelectric or magnetic. Surface plasmon resistance (SPR)-based
optical biosensors are the most common. The optical biosensors
allow real time, cost-effective, sensitive, and selective detection of a
wide range of analytes including viruses, toxins, drugs, antibodies,
tumor biomarkers, and tumor cells. Biosensors using various
transducer platforms have been developed to detect various bacte-

rial pathogens and toxins of veterinary importance, such as E. coli,
Serratia marcescens, Pseudomonas aeruginosa, Acinetobacter bau-
mannii strains, Mycoplasma biomarkers, Salmonella, epsilon
toxin of Clostridium perfringens, pathogenic S. aureus and toxins
in clinical, environmental, and food samples. The interaction of
immobilized Abs with their corresponding Ags makes possible the
development of Ab-based immunosensors, which have Ab immo-
bilized onto a biosensor chip to recognize Ag specifically in a
complex medium [19].
4 Antibody Immobilization
For developing an immunosensor, the immobilization of antibo-

dies on the surface of a transducer element is of paramount impor-
tance. The understanding of the surface chemistry is required for
proper binding, optimal orientation, and free movement of anti-
body molecules to obtain a maximal functional sensor surface
[20]. Sensor surfaces consist of an inorganic material (glass, gold,
iron oxide, platinum, etc.). The performance of a bio/immunosen-
sor depends on some factors like (1) the ability to immobilize
antibodies in their active form (2) its accessibility to the relevant
analyte; and (3) low non-specific background. The immobilization
step affects the detection limit, sensitivity, and overall performance
of the immunosensor [21]. There are several factors which should
be taken into account while developing an immunosensor. The
attachment of antibodies onto the surface of a transducer may
occur in different orientations. If an antibody molecule binds on
the sensor surface through the Fc region, then the antigen-binding
regions (paratope) will be fully available to the antigen of interest,
thus maximizing sensor performance. If an antibody molecule
binds on the sensor surface through the antigen-binding regions,
it may result in decreased or no sensor activity. The extent of linkage
between the Ab and the surface may also need to be carefully
controlled. The orientation of Abs on sensor surfaces can be con-
trolled by the interaction between specific reactive groups on the
surface and on the Ab. The two main approaches that can be used
for immobilization of antibodies are non-covalent and covalent
immobilization. However, affinity-based immobilization and engi-
neered antibodies for immobilization are also being used [7].
4.1 Non-covalent Direct physical adsorption of antibodies onto the sensor surface can
Immobilization be performed via simple non-covalent forces, including electro-
static or ionic bonds, hydrophobic interactions, and van der Waals
forces. There are some limitations of direct physical adsorption.
Though easy to perform, the process is uncontrolled and may
cause protein denaturation. When protein denaturation is involved,
some affinity-based assays may lead to ligand leaching from the

surface due to extensive washing, thereby decreasing surface
bio-activity [20].The orientation of the adsorbed antibody might
not be proper as well. Another non-covalent approach used was the
entrapment of antibody molecules into a conducting polymer [22],
where Abs for human serum albumin were entrapped on a galva-
nostatically polymerized pyrrole, on to a platinum wire substrate.
Some of the common conducting polymers that have been exten-
sively used for immunosensor fabrication are polyacetylene, poly-
thiophene, polyaniline, polyindole, and polypyrrole. But
entrapment of antibody molecule may lead to poor accessibility of
the antigen-binding sites, leading to poor signal [7].
4.2 Covalent Covalent immobilization of Abs facilitates long-term storage, con-

Immobilization formational stability of the ligands, no leaching out of the analyte/
ligand and reusability of immunosensors. In covalent bonding, the
surface of the sensor is modified so that the reactive groups such as
hydroxy, thiol, carboxylic or amino groups are available on the
surface for the subsequent Ab immobilization [7]. Coupling of
Abs to the sensor surface by targeting amine groups present in
the lysine amino acid side chains of Ab is an extensively used form
of covalent immobilization, due to the relative ease of access to
these groups.
Feyssa and coworkers [23] immobilized anti-C-reactive protein
(CRP) antibody in a microfluidic platform via amine covalent link-
age. This approach showed signal enhancement in comparison with
passive binding. The reproducibility of an amine-coupled biochip
was found to be comparable with a human-CRP enzyme-linked
immunosorbent assay (ELISA) detection kit. But there are some
problems associated with amine coupling chemistry. It usually
results in random orientation and less homogeneous binding due
to the presence of excessive lysine groups in the Ab. Thiol group on
the surface can be targeted for antibody immobilization as it gives
more homogeneous immobilization or may allow defined orienta-
tion of Abs in comparison with amine coupling. Ab fragments
immobilized in defined orientation have been shown to achieve a
20-fold enhanced antigen-binding ability, compared with the ran-
domly immobilized Abs using amine groups [21]. Jarocka and
coworkers [24] immobilized Fab Ab fragment on a gold electrode
surface via thiol coupling and developed animpedimetric immuno-
sensor for the detection of peptides derived from avian influenza
haemagglutinin H5.
Another covalent approach involves the generation of active
aldehyde groups (diol groups) which can be linked efficiently on
amine-functionalized surfaces, resulting in partially oriented cova-
lent Ab coupling on the sensor surface. Shriver-Lake et al. [25]
pointed out some limitations with the use of the carbohydrate-
based immobilization procedure, like time delays due to more
steps in the procedure and increased loss of Ab. They observed 50%
loss in the Ab prior to immobilization, due to the number of steps
in the procedure.
4.3 Affinity-Based In affinity-based immobilization, the surface of the sensor is layered

Immobilization with a baselayer of intermediate binding proteins such as protein
A/Gor Fc-specific antibodies, or a nickel surface that binds
histidine-tags in the Fc region, or biotin-avidin. These proteins
have a high affinity and binding specificity towards the Fc region
of a wide range of Abs, thus encouraging Ab immobilization via the
Fc region on the sensor surface. This gives the antibody an envi-
ronment that is not restricted due to proximity to the sensor
surface, but instead has more freedom to interact with the analyte.
Furthermore, antibodies are properly oriented, so that the antigen-
binding sites are freely available for analyte interaction [18]. Hydra-
zide-containing cross-linkers provide another way of immobilizing
Abs via the glycans in the Fc region, thereby exposing the antigen-
binding sites to the solution.
de Juan-Franco and coworkers immobilized antibodies using a
fusion protein, the Protein A–gold-binding domain (PAG)
[26]. The human growth hormone-specific immunosensor fabri-
cated using the PAG immobilization approach showed better sen-
sitivity when compared with conventional methods. Barton and
coworkers [27]achieved much higher sensitivities with an avidin–
biotin interaction-based immunosensor in comparison with an
entrapment-based immunosensor. Ionescu and coworkers [28]
immobilized the anti-atrazine antibody fragments via affinity bind-
ing onto a polypyrrole film N-substituted by nitrilotriacetic acid
(NTA), electrogenerated on a gold electrode. The variable domains
of llama heavy-chain antibodies (VHH) was in vivo biotinylated at
the lysine position of the Avi-tag, which improved the analyte
binding more than 200-fold [21].
4.4 Recombinant With the advent of Recombinant DNA technology, different for-
Antibodies mats of recombinant antibodies have been generated. Recombinant
for Immobilization antibodies have several desirable characteristics including low
molecular mass, increased flexibility, high physico-chemical stabil-
ity, and easy access to the antigen, which makes them a potential
substitute for naturally generated Abs. It is now possible to intro-
duce desirable modifications in the antibody molecule by using
Recombinant DNA technology [5]. The directed immobilization
approaches developed for intact antibody based on the Fc domain
or carbohydrate moiety cannot be used with recombinant antibo-
dies or antibody fragments such as scFv (single-chain variable frag-
ment) and dsFv (disulphide-stabilized variable fragment), due to
the absence of the glycosylated Fc domain. Various approaches have
been developed to engineer scFv or scAb during their generation so
as to easily immobilize them on to the solid surface without
Fig. 6 Structure of scFv and dsFv
denaturation (Fig. 6) [29, 30]. Antibody fragments can be engi-

neered to have positively charged amino acids (e.g. arginine or Arg)
in the peptide linker or a 6-histidine amino acid sequence on the
C-terminus for immobilization via electrostatic and non-covalent
interactions, respectively. A wide variety of sensing transducers have
been developed by using rAbs to substitute for intact Abs in immu-
nosensors. The high specificity of recombinant antibodies allows
the use of a single recombinant antibody to detect an antigen, thus
eliminating the need for a second antigen-specific antibody. Addi-
tionally, the small size of recombinant antibodies permits immobi-
lization on to an immunosensor surface at high density, thus
resulting in enhanced assay avidity, sensitivity, and stability [7].
5 Biosensor Types
Depending on the method of signal transduction detection, bio-

sensors have been classified as follows:
5.1 Electrochemical An electrochemical immunosensor measures an electrical signal

Immunosensor proportionate to antigen–antibody complex formation. This tech-
nique is economical, easy to operate, portable, and simple to con-
struct. Since this is a surface based method, the reaction volume is
relatively small and samples are required in minute quantities for
detection purposes. With the use of modern and evolving technol-
ogy, the electrochemical immunosensor is an attractive candidate
for wide sensing applications (Fig. 7). One of the main areas where
sensors are the most beneficial is in clinical diagnostics [13, 31]. In
general, electrochemical immunosensors are classified as ampero-
metric immunosensors, voltammetric immunosensors, potentio-
metric immunosensors, and impedimetric immunosensors. A
more recent event in the field of electrochemical immunosensing
is the development of FET-based immunosensors.
Fig. 7 Schematic of an electrochemical immunosensor (Ag Antigen, Ab Antibody, O Oxidized species,

R Reduced species, WE Working electrode, RE Reference electrode, CE Counter electrode, V Voltage, I Current)
An amperometric immunosensor measures the changes in cur-

rent at a constant potential value. The measurable current output is
proportional to the concentration of the analyte of interest. This
type of transducer is highly selective in nature as the oxidation or
reduction potential characteristic to the target is used to determine
its identity. Also, they consume a small percentage of analytes
during measurement [32]. The sensitivity of an amperometric
immunosensor can be increased using a layer-by-layer construction
approach. In one study, gold nanoparticles and methylene blue
were layered to form a stable base for hCG sensor. Layering
increased the surface area, thereby increasing the capture of ana-
lytes, and consequently increased the sensitivity [33]. Platinum
nanoparticles have also been used for the layering purpose instead
of gold nanoparticles, for the detection of H2O2 electroreduction
[34].
In voltammetric immunosensors, current and potential are
measured over a pre-set potential range. The resulting current is
converted into a peak which represents the target of interest, and
the height of the peak corresponds to the amount of analyte in a
sample. This method is highly sensitive due to its minimal
background noise [14]. In potentiometric immunosensors,

changes in ion activity are measured which specifies the mass charge
potential. The most common instrument used in potentiometry is
the pH electrode. Other ions such as (F, I, CN, Na, K, Ca, NH) or
gas (CO, NH) selective electrodes can also be used for potentio-
metric measurement. But it lacks the sensitivity required to distin-
guish between two values within a small concentration range, and
the occurrence of non-specific binding is also very high [14, 32].
An impedimetric immunosensor measures the electrical imped-
ance of an interface by applying a small sinusoidal voltage at a
specific frequency, and the resulting current is recorded. This pro-
cedure is performed a range of frequencies. The ratio of current to
voltage provides the impedance. Impedance is altered when
antigen-antibody complex gets deposited on the surface of the
electrode, thus increasing the dielectric layer thickness. The change
in capacitance is proportional to the size and concentration of
antibody [32, 35].
An FET-based immunosensor consists of a source, drain, and
gate electrodes. An external potential (VDS) is applied across the
source and drain electrodes, and current (ID) passes from source to
drain through a semiconductor path, which has antibodies immo-
bilized on the surface for antigen binding. The gate electrode is
connected to this semiconductor path capacitatively through a
dielectric layer. The voltage (VG) applied to gate creates either an
accumulation or depletion of charge carriers in the semiconductor
path, controlling ID. Capture of antigen to this surface changes the
surface potential, and influences the magnitude of ID. For example,
a positively charged antigen induces a depletion layer in a p-type
semiconductor, and a charge (e ) accumulation layer in an n-type
semiconductor. Accordingly, the conductance either decreases or
increases, and is used to estimate the antigen concentration
(Fig. 8) [36].
5.2 Optical Optical immunosensors employ light either coming from a laser,
Immunosensor diode or white-hot light bulb for the detection of analytes. As the
light passes through or refracts from the Ag-Ab complex, the
change in intensity of light can be correlated with Ag-Ab concen-
tration. There is change in phase, polarization, speed or frequency
of input light which may correspond to the antigen-antibody com-
plex [37]. The concept involved in optical sensors for the identifi-
cation of analytes is based on the higher dielectric permittivity
acquired by all proteins, cells, and DNA, compared to air and
water, causing these biomolecules to reduce the propagation
speed of the electromagnetic fields flowing through them
(Fig. 9). Because all molecules contain atomic nuclei and electrons
in varying orbital states, these molecules are able to interact with
the electromagnetic fields that pass through them. By placing these
molecules in oscillating electromagnetic fields analogous to the
Fig. 8 Schematic of an FET-based immunosensor
Fig. 9 Schematic of an optical immunosensor
propagation of light, electrons within the molecules vibrate due to

the force subjected to them. Free electrons then polarize in the
presence of light’s magnetic field, generating a polarization current
resulting from the movement of electrons, where it moves much
slower through a biomolecule than in free space.
The target or bioreceptor molecules can also be attached to
chromogenic/fluorescent labels (e.g. dyes) that will cause a change
in the fluorescence signal signifying the existence of target analytes.
The degree of fluorescence emitted correlates to the magnitude of
Fig. 10 Schematic of a fluorimetric immunosensor
interaction between the target analytes and bioreceptors (Fig. 10).

Popular examples of optical immunosensors include surface plas-
mon resonance (SPR) based sensors, fiber-optic sensors (FOS), and
various fluorescence based sensors, with surface plasmon resonance
being the most widely employed sensor [32, 38].
In a SPR immunosensor, antibodies are immobilized on a
surface of thin metal film, typically gold, where polarized light is
radiated from the back surface through a prism and a target ligand
is introduced. The metal film reflects this light and the strength of
the reflected light can then be assessed and quantified. When the
immobilized antibodies are bound to their target, a shift in the SPR
angle can be observed that depends on the concentration of the
target (Fig. 11). SPR harnesses refractive index changes caused by
the formation of the Ab/Ag complex on a metal surface being
related to the concentration of the antigen in the sample being
measured [37, 39].Currently, SPR is a leading sensor technology
for the observation of biomolecular interactions in real time, which
has been commercialized by several companies. Biacore SPR sensor
surfaces are the most widely used and are commercially available
with various functionalities. The most widely used surface chemis-
try, called CM5, consists of a SiO2-base layer, on top of which a thin
gold film is deposited. The gold film is further modified by long-
chain hydroxyalkyl thiols to introduce organic components for
chemical modification [18].
5.3 Piezoelectric Recently, piezoelectric crystals have been used for the development
Immunosensor of piezoelectric immunosensors. When an antigen interacts with
antibodies to form immunocomplexes, there is a change in mass,
which can be examined by a quartz crystal, the main constituent of
a piezoelectric sensor (Fig. 12).The piezoelectric crystal oscillates at
Fig. 11 Schematic of an SPR immunosensor
Fig. 12 Schematic of a QCM immunosensor
a specific frequency in conjunction with the use of an electrical

signal at a certain frequency. By applying electrical voltage to the
quartz crystal via two electrodes, the orientation of the crystal is
altered and this causes a distortion in the crystal lattice that causes a
mechanical oscillation at a characteristic vibrational frequency,
i.e. the crystal’s natural resonant frequency [40]. In the event of
the formation of immunocomplexes, the surface of the crystal is
loaded with an extra mass, which changes the frequency of oscilla-
tion of the crystal and the mass change can be determined electri-
cally. Nowadays, piezoelectric crystals are becoming popular as they
are simple, highly sensitive, specific, safe, label-free, stable and give
Fig. 13 Schematic of a thermometric immunosensor
fast results. Another advantage of the piezoelectric immunosensor

is its ability to detect analytes in real time. They are being used in a
number of sectors such as in clinical diagnosis and environmental
pollution supervision [41]. In piezoelectric immunosensors, the
measurement can be direct or indirect; single step or multi step.
In a single step, the ligand binds with the analyte; and in multistep,
there is sequential binding of two or more components. In direct
measurement, the analyte or antigen binds with an antibody. While
in indirect measurement, the analyte interacts with other entities in
the solution [14].
5.4 Thermometric The thermometric immunosensor utilizes absorbance or release of

Immunosensor heat in a biological reaction as mode of detection. When the
antigen binds with the antibody, there is temperature variation
which can be converted into an electrical signal for determining
the formation of immunocomplex (Fig. 13). These immunosensors
offer the advantages of low operating cost and stability over a
relatively longer period of time, and are also not affected by the
presence of ions in the solution. However, one major disadvantage
of this type of sensors is the lack of specificity. One has to be sure
that the related enthalpy changes are not due to the immobilization
of any interfering species and are not related with dilution effect
[14, 42].
6 Biosensor Fabrication
The fabrication of a biosensor involves four primary parts, each of

which has its own set of functions and criteria for design, as dis-
cussed below.
6.1 Transducer Transducers convert a biological event into a proportionate electri-

cal signal. In electrochemical biosensors, a transducer is con-
structed with one or more materials which possess certain
properties such as high electron transfer kinetics, good
conductivity, and biocompatibility. With the advanced tools of

nanotechnology available, nanoparticles become an ideal choice,
owing to additional properties such as high surface area to volume
ratio and ease of functionalization, along with the above mentioned
properties. Some commonly used nanomaterials for the prepara-
tion of transducers include metal nanoparticles such as gold
(AuNP), silver (AgNP), platinum (PtNP) nanoparticles, etc.; car-
bon based nanomaterials such as carbon nanotubes (CNT), gra-
phene sheets and derivatives, spherical fullerene, etc.; and organic
films, dendrimers, and conductive polymers such as polypyrrole,
polyaniline, polydopamine, etc. Nanoparticles are fabricated over
an electrode surface responsible for conducting the current signal.
Common choices of electrodes are indium tin oxide (ITO) electro-
des, glassy carbon electrodes (GCE), etc. For transducer fabrication
in optical sensors, properties such as absorption of electromagnetic
radiation, reflection, scattering, refractive index, fluorescence, and
chemiluminescence are targeted. SPR involves the fabrication of a
very thin metal surface that has an extended plasmon region/
excitable electron band, QCM employs an AT-cut quartz crystal
and FET-based biosensors utilize nanomaterials which behave like
semiconductors.
6.2 Bioreceptor As discussed earlier, antibodies and antibody fragments are the
most widely used bioreceptors in biosensor fabrication. Antibodies
are immobilized on the transducer surface either covalently or
non-covalently, through adsorption, electrostatic interactions,
affinity binding, and entrapment within polymers. The free sites
might cause electrode fouling, and unnecessary background signal,
and hence are insulated with common blocking agents such as
bovine serum albumin (BSA), self-assembled monolayers (SAM)
of ethanolamine, mercaptohexanol, etc.
6.3 Electroactive In electrochemical biosensors, electroactive labels are often used to

Labels and Redox generate electrical signals following the antigen-antibody binding
Species event. Some of the common bionanolabels fabricated can be
categorized as: electrocatalytic nanoparticles combined with bior-
eceptors, enzymatic labels with redox substrates, magnetic bead
based labels with bioreceptors, heavy metal based labels, etc. One
or more redox species are added to the sensing system for signal
amplification through coupled, chemical or electrochemical, redox
cycling. Common redox species used in sensors are ferrocene
(Fe2+/3+), potassium ferricyanide/ferrocyanide, hydrazine, etc.
Electrochemical detection can also be carried out label-free, as
seen in impedimetric biosensors. Electroactive labels and redox
species do not form a part of optical, piezoelectric or thermometric
biosensors.
6.4 Buffers The samples used for detection of antigenic species often need to be
diluted, lest they cause high background signals or even damage the
electrode surface integrity. Buffers are ideal diluents, which have a
relatively stable pH similar to that of serum and other biological
fluids (~7.4), and can be added without denaturing the biochemical
components of the sample. Redox species and electroactive labels
are added to this electrolytic buffer. In fluorimetric sensors, fluo-
rescent labels are added to it. Common choices of buffers are
phosphate buffered saline (PBS), HEPES buffer, Tris-HCl
buffer, etc.
7 Methods for Biosensor Fabrication and Analyte Detection
Given below are two model antibody-based biosensors along with

the stepwise preparation of sensor surface and analyte detection.
7.1 Graphene-Based Liu et al. developed an electrochemical immunosensor with multi-

Immunosensor layered reduced graphene oxide (GO) film for transduction, [Fe
for Rotavirus Detection (CN)6]3 /4 redox system for signal generation and rotavirus
surfaceprotein-specific antibodies for bio-recognition
(Fig. 14) [43].
Preparation and detection:
1. GO solution was prepared using modified Hummer’s method,
and deposited in a free-standing multilayered film by centrifu-
gal vacuum evaporation.
2. The GO film was reduced by subjecting it to thermal annealing
at 900 C in the presence of H2/Ar flow for 10 h.
Fig. 14 Schematic for the graphene film-based immunosensor for rotavirus detection
3. The reduced GO film was cut and fixed on a polydimethylsi-

loxane (PDMS) chamber, with an exposed area of 4 mm2, used
as the working electrode. The reference and counter electrodes
were Ag/AgCl and platinum wire electrodes, respectively.
4. 1-pyrenebutyric acid N-hydroxysuccinimide ester (PSE) was
used as a linker. The pyrene ring of PSE is π-stacked on the
reduced GO electrode.
5. The N-hydroxysuccinimide group catalyzes the formation of
amide bond between PSE and rotavirus-specific antibody.
6. The free sites were blocked with BSA.
7. All fabrication steps were characterized with cyclic
voltammetry (CV).
8. The prepared sensor was incubated with a PBS solution con-
taining rotavirus, and the surface was subsequently washed.
9. Specifically bound rotavirus was quantified with CV at a scan
rate of 50 mV/s in the presence of K3Fe(CN)6 in KCl solution.
10. Viral load (measured in plaque forming units or PFUs) was
correlated to anodic peak current to obtain a standard curve,
and the linear range, limit of detection and sensitivity were
determined.
7.2 An Interdigited Kaushik et al. developed an interdigited micro-electrode of gold

Gold Micro-Electrode arrays (IDE-Au) functionalized with dithiobis(succinimidyl propi-
Impedimetric onate) (DTSP) for the covalent immobilization of anti-Zika virus
Immunosensor envelope protein antibody (Zev-Abs) (Fig. 15) [44].
for Label-Free Preparation and detection:
Zika-Virus (ZIKV) 1. IDE-Au was electrochemically cleaned and incubated with
Detection DTSP solution for 2 h for surface immobilization, followed
by washing with DI water and drying at 4 C.
Fig. 15 Schematic of IDE-Au based electrochemical ZIKV immunosensor

2. The functionalized surface was incubated with monoclonal

Zev-Abs for 2 h, leading to electrostatic immobilization of
Zev-Abs.
3. The free surface was blocked with BSA, followed by washing
with PBS, and storage at 4 C.
4. All fabrication steps were characterized with electrochemical
impedance spectroscopy (EIS).
5. The sensor was then incubated with ZIKV in PBS for 30 min,
followed by washing with DI water.
6. The specifically bound ZIKV was quantified with EIS in the
presence of the redox couple Fe(II)/Fe(III) in PBS.
7. ZIKV concentration was correlated with charge transfer resis-
tance (Rct) obtained from EIS, and the plot was used to esti-
mate the linear range, limit of detection and sensitivity of the
sensor.
References
1. Trier NH, Houen G (2020) Antibodies as 9. Ma H, O’Kennedy R (2015) The structure of
diagnostic targets and as reagents for diagnos- natural and recombinant antibodies. In:
tics. Antibodies (Basel) 9(2):15. https://doi. Houen G (ed) Peptide antibodies. Methods in
org/10.3390/antib9020015 molecular biology, vol 1348. Humana Press,
2. Kohler G, Milstein C (1975) Continuous cul- New York. https://doi.org/10.1007/978-1-
tures of fused cells secreting antibody of pre- 4939-2999-3_2
defined specificity. Nature 256:495–497 10. Weiner LM, Surana R, Wang S (2010) Mono-
3. Lu R, Hwang Y, Liu I et al (2020) Develop- clonal antibodies: versatile platforms for cancer
ment of therapeutic antibodies for the treat- immunotherapy. Nat Rev Immunol 10:317–
ment of diseases. J Biomed Sci 27:1 327
4. Kyowa Hakko Kirin Co. Ltd. Consolidated 11. Chiu ML, Goulet DR, Teplyakov A et al
Financial Summary (IFRS) Fiscal 2018. 2019, (2019) Antibody structure and function: the
February 5 basis for engineering therapeutics. Antibodies
5. Basu K, Green EM, Cheng Y, Craik CS (2019) 8 ( 4 ) : 5 5 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 /
Why recombinant antibodies - benefits and antib8040055
applications. Curr Opin Biotechnol 60:153– 12. Reverberi R, Reverberi L (2007) Factors affect-
158. https://doi.org/10.1016/j.copbio. ing the antigen-antibody reaction. Blood
2019.01.012 Transfus 5(4):227–240. https://doi.org/10.
6. Gray AC, Sidhu SS, Chandrasekera PC et al 2450/2007.0047-07
(2016) Animal-friendly affinity reagents: repla- 13. Mollarasouli F, Kurbanoglu S, Ozkan SA
cing the needless in the haystack. Trends Bio- (2019) The role of electrochemical Immuno-
technol 34(12):960–969. https://doi.org/10. sensors in clinical analysis. Biosensors 9(3):86.
1016/j.tibtech.2016.05.017 https://doi.org/10.3390/bios9030086
7. Sharma S, Byrne H, O’Kennedy RJ (2016) 14. Lim SA, Ahmed MU (2019) Chapter 1: Intro-
Antibodies and antibody-derived analytical duction to Immunosensors, Royal Society of
biosensors. Essays Biochem 60(1):9–18. Chemistry. In: Ahmed MU, Zourob M, Tamiya
https://doi.org/10.1042/EBC20150002 E (eds) Immunosensors, pp 1–20. https://doi.
8. Schroeder HW Jr, Cavacini L (2010) Structure org/10.1039/9781788016162-00001
and function of immunoglobulins. J Allergy 15. Clark LC Jr, Lyons C (1962) Electrode systems
Clin Immunol 125:S41–S52. https://doi. for continuous monitoring in cardiovascular
org/10.1016/j.jaci.2009.09.046 surgery. Ann N Y Acad Sci 102:29–45.
https://doi.org/10.1111/j.1749-6632.1962.
tb13623.x
16. Yalow RS, Berson SA (1959) Assay of plasma 30. Zeng X, Shen Z, Mernaugh R (2012) Recom-
insulin in human subjects by immunological binant antibodies and their use in biosensors.
methods. Nature 184(Suppl 21):1648–1649. Anal Bioanal Chem 402(10):3027–3038.
https://doi.org/10.1038/1841648b0 https://doi.org/10.1007/s00216-011-
17. Turner APF (2000) Biosensors—sense and 5569-z
sensitivity. Science 290(5495):1315–1317 31. Aydin EB, Aydin M, Sezgintürk MK (2019)
18. Skottrup PD, Nicolaisen M, Justesen AF Advances in electrochemical immunosensors.
(2008) Towards on-site pathogen detection Adv Clin Chem 92:1–57
using antibody-based sensors. Biosens Bioelec- 32. Holford TR, Davis F, Higson SP (2012)
tron 24(3):339–348. https://doi.org/10. Recent trends in antibody based sensors. Bio-
1016/j.bios.2008.06.045 sens Bioelectron 34(1):12–24. https://doi.
19. Singh A (2017) Next generation immunoas- org/10.1016/j.bios.2011.10.023
says for infectious diseases of animals. Res Rev 33. Chai R, Yuan R, Chai Y et al (2008) Ampero-
J Immunol 7(2):22–43 metric immunosensors based on layer-by-layer
20. Cass T, Ligler FS (1998) Immobilized biomo- assembly of gold nanoparticles and methylene
lecules in analysis: A practical approach. Oxford blue on thiourea modified glassy carbon elec-
University Press, New York trode for determination of human chorionic
21. Trilling AK, Beekwilder J, Zuilhof H (2013) gonadotrophin. Talanta 74(5):1330–1336.
Antibody orientation on biosensor surfaces: a https://doi.org/10.1016/j.talanta.2007.
minireview. Analyst 138:1619–1627 08.046
22. John R, Spencer M, Wallace GG et al (1991) 34. Fu Y, Li P, Wang T et al (2010) Novel poly-
Development of a polypyrrole-based human meric bionanocomposites with catalytic Pt
serum albumin sensor. Anal Chim Acta 249: nanoparticles label immobilized for high per-
381–385 formance amperometric immunoassay. Biosens
Bioelectron 25(7):1699–1704. https://doi.
23. Feyssa B, Liedert C, Kivimaki L et al (2013) org/10.1016/j.bios.2009.12.010
Patterned immobilization of antibodies within
roll-to-roll hot embossed polymeric microflui- 35. Bataillard P, Gardies F, Jaffrezic-Renault N et al
dic channels. PLoS One 8:e68918 (1988) Direct detection of immunospecies by
capacitance measurements. Anal Chem 60(21):
24. Jarocka U, Sawicka R, Góra-Sochacka A et al 2374–2379. https://doi.org/10.1021/
(2014) An immunosensor based on antibody ac00172a011
binding fragments attached to gold nanoparti-
cles for the detection of peptides derived from 36. de Moraes A, Kubota L (2016) Recent trends
avian influenza hemagglutinin H5. Sensors 14: in field-effect transistors-based Immunosen-
15714–15728 sors. Chemosensors 4(4):20. https://doi.org/
10.3390/chemosensors4040020
25. Shriver-Lake LC, Donner B, Edelstein R et al
(1997) Antibody immobilization using hetero- 37. González-Martı́nez MA, Puchades R,
bifunctional crosslinkers. Biosens Bioelectron Maquieira A (2007) Optical immunosensors
12:1101–1106 for environmental monitoring: how far have
we come? Anal Bioanal Chem 387(1):
26. de Juan-Franco E, Caruz A, Pedrajas JR et al 205–218. https://doi.org/10.1007/s00216-
(2013) Site-directed antibody immobilization 006-0849-8
using a protein A–gold binding domain fusion
protein for enhanced SPR immunosensing. 38. Velusamy V, Arshak K, Korostynska O et al
Analyst 138:2023–2031 (2010) An overview of foodborne pathogen
detection: in the perspective of biosensors. Bio-
27. Barton AC, Collyer SD, Davis Fet al. (2009) technol Adv 28(2):232–254. https://doi.org/
Labeless AC impedimetric antibody-based sen- 10.1016/j.biotechadv.2009.12.004
sors with pg ml 1 sensitivities for point-of-care
biomedical applications. Biosens Bioelectron 39. Phillips KS, Cheng Q (2007) Recent advances
24:1090–1095 in surface plasmon resonance based techniques
for bioanalysis. Anal Bioanal Chem 387:1831–
28. Ionescu RE, Gondran C, Bouffier L et al 1840
(2010) Label-free impedimetric immunosen-
sor for sensitive detection of atrazine. Electro- 40. Bunde RL, Jarvi EJ, Rosentreter JJ (1998) Pie-
chim Acta 55:6228–6232 zoelectric quartz crystal biosensors. Talanta
46(6):1223–1236. https://doi.org/10.1016/
29. Yan H, Shen Z, Mernaugh R, Zeng X (2011) s0039-9140(97)00392-5
Single chain fragment variable recombinant
antibody as a template for fc sensors. Anal 41. Pohanka M (2018) Overview of piezoelectric
Chem 83(2):625–630. https://doi.org/10. biosensors, Immunosensors and DNA sensors
1021/ac102087w
and their applications. Materials 11(3):448. J 5(2):123–128. https://doi.org/10.1007/

https://doi.org/10.3390/ma11030448 s13206-011-5204-2
42. Ramanathan K, Danielsson B (2001) Principles 44. Kaushik A, Yndart A, Kumar S, Jayant RD,
and applications of thermal biosensors. Biosens Vashist A, Brown AN, Li CZ, Nair M (2018)
Bioelectron 16(6):417–423. https://doi.org/ A sensitive electrochemical immunosensor for
10.1016/s0956-5663(01)00124-5 label-free detection of Zika-virus protein. Sci
43. Liu F, Choi KS, Park TJ, Lee SY, Seo TS Rep 8(9700):1–5. https://doi.org/10.1038/
(2011) Graphene-based electrochemical bio- s41598-018-28035-3
sensor for pathogenic virus detection. BioChip
Chapter 13
Lateral Flow Assay for Diagnosis of Pig Viral Diseases

Aditya Prasad Sahoo and Rajib Deb
Abstract
Several infectious pig viral diseases of economic importance such as foot and mouth disease, Hog cholera,
and African swine fever virus (ASFV) pose a serious threat to the swine industry worldwide. Control of these
viral diseases is very challenging since there is no specific treatment available and this leads to huge financial
loss to pig industries. Prevention of the viral diseases mostly rely on efficient control strategy, zoo-sanitary
measures, and culling of infected and exposed animals. Successuful implementation of disease control
programme essentially require early detection viral infection. Detection of infection can be achieved either
by detection of virus which include techniques such as virus isolation, fluorescent antibody test (FAT),
detection of viral genome using PCR or RT-PCR or detection of antibody specific to the virus. The major
bottleneck of these detection tests in implementing control measure is because of inherent nature of the test
like time consuming, require well-equipped laboratories and personnel, delaying the disease diagnosis in
remote areas. The lateral flow assay offers a rapid and simple assay which allow early detection of viral
infection in resource and equipment limited small laboratories. This chapter describes the method for
development of a lateral flow assay using gold nanoparticle probe for detection of viral antigen.
Key words Viral disease, Diagnosis, ELISA, Lateral flow assay, Gold nanoparticles
1 Introduction
Infectious viral diseases of pig such as foot and mouth disease, Hog
cholera, African swine fever virus (ASFV), Porcine reproductive and
respiratory syndrome (PRRS), and swine influenza are highly con-
tagious and these diseases outbreaks incurs a huge economic loss to
pig industry. Although few viral diseases have been eradicated from
many developed countries such as USA, Australia, New Zealand,
Canada, and few European countries, these diseases are still preva-
lent in resources limited underdeveloped countries. At present,
there is no definitive antiviral treatment for viral disease is available,
hence these diseases go unabated which leads to huge financial loss
to pig industries. Prevention of the viral diseases mostly rely on
efficient control strategy, zoo-sanitary measures, culling of infected
and exposed animals. Rapid and early diagnosis of contagious viral
195
196 Aditya Prasad Sahoo and Rajib Deb
diseases of pig is crucial in timely implementation of control mea-

sures such as restriction of animal movement and quarantine mea-
sure to prevent the spread of disease. Detection of virus by virus
isolation, direct fluorescent antibody test (FAT), and panel of
monoclonal antibodies (mAbs) or viral nucleic acid by polymerase
chain reaction (PCR) and reverse-transcription polymerase chain
reaction (RT-PCR) in whole blood is the method of choice for
detecting infected herds at an early stage of disease outbreak
[1]. Serological methods such as enzyme-linked immunosorbent
assays (ELISA), fluorescent antibody test (FAT), indirect fluores-
cent antibody test (IFAT), and complement fixation test (CFT) are
valuable for monitoring sero-prevalence in herds [2]. The
OIE-recommended tests for virus detection include virus isolation,
fluorescent antibody test, and molecular biology techniques such as
PCR and RT-PCR. However, these tests cannot be used in field
condition for detection of viral diseases in pig farms as these tests
are time consuming, expensive, and require dedicated cell culture
facilities, well-equipped laboratories, and trained laboratory per-
sonnel. In addition, logistics for transportation of clinical samples
from actual site of infection to these specialized laboratories is
cumbersome and time consuming which delay the disease diagnosis
process [3]. Hence, in order to overcome these shortcomings a user
friendly, rapid, and economical diagnostic assay is needed for
resources limited laboratories for screening clinical samples for
disease. This would help immensely in improving disease control
programs by assisting field veterinarians to make swift decision in
implementing control procedures and effectively at actual site of
virus incursion in suspected disease outbreak scenario, especially in
disease endemic areas where first evidence of the disease is based
only on clinical signs and symptoms.
Lateral flow assay (LFA) is a user friendly easy-to-use rapid
diagnostic test which require no special training to perform the
test. LFA belongs to category of rapid assays and proved its worth
in detection of pathogens, hormones, heavy metals, toxicity, and
adulterant detection. Common names used for LFA in different
sectors are Lateral flow test, Lateral flow device (LFD), Lateral flow
immunoassay (LFIA), Dipstick and Pen-side test. LFA is an immu-
nochromatographic version of ELISA based on specific antigen-
antibody interaction but unlike ELISA plate a membrane strip is
used to coat reagents required in ELISA which makes this platform
simple and rapid to perform. Usually LFA is visually interpreted
hence mostly provide qualitative estimate of the analyte, i.e. merely
screen the presence or absence of the virus or virus specific antibody
in the clinical sample. The LFA can be designed for semi-
quantitative estimation of analyte by applying a number of test
lines with known concentration of capture antibody and the colour
development is measured using a reflectometer or CCD camera.
The intensity of colour development is then corelated with the
Lateral Flow Assay for Diagnosis of Pig Viral Diseases 197
concentaion of analyte. Since LFA is an immunochromatographic

form of ELISA, it can be designed in two formats similar to ELISA
namely sandwich LFA and competitive LFA.
Successful development of LFA needs better signal-
amplification strategies. Gold nanoparticles (GNP), colored latex
beads, carbon nanoparticles, selenium, quantum dots, and enzymes
are used as label to increase signal which in turn increases sensitivity
of the test. The use of gold nanoparticles as label or probe in LFA is
very successful and most widely used. GNP is widely used as a label
because of its unique properties like easy to synthesize GNP of
different size range, easy to functionalize, and strong light extinc-
tion [4]. They can be easily synthesized in sizes ranging from 1 nm
to 200 nm using simple one-step aqueous procedures developed by
Frens [5], and their surfaces can be modified with nearly any small
molecule, polymer, peptide, oligonucleotide, protein, and DNA
[6]. Their light extinctions are enormous and can be tuned across
the visible and near infrared regions of the spectrum simply by
changing particle size and shape. Conjugation of antibodies to
the surface of GNP can be achieved by their interaction which can
be noncovalent electrostatic interaction, hydrophobic interaction
or covalent binding [7].
2 LFA Design and Principle
LFA comprises of five components, namely (1) sample pad, (2) con-
jugate pad, (3) membrane/solid phase, (4) absorption pad, and
(5) test line and control line (Fig. 1).
1. Sample pad made up of cellulose or glass fiber to filter out the
unwanted interfering materials present in sample and evenly
distribution of the sample.
2. Conjugate pad made up of fiber glass or cross-linked silica to
hold bioconjugate detector, i.e. GNP-Antibody complex till
test is performed and more importantly able to release the
bioconjugate detector once the test is performed.
3. Membrane/Solid phase made up of nitrocellulose membrane
of various pore sizes ranging from 0.05 to 12 μm is used as a
solid support for applying test line and control line. Pore size is
crucial as it is correlated with speed of flow of reagents across
the membrane and sensitivity of assay. For example, membrane
with bigger pore size results in faster flow rate which leads to
poor sensitivity. Like ELISA, blocking agents such as bovine
serum albumin (BSA), skimmed milk powder, and casein are
used to prevent the nonspecific binding of reagents to test line
and control line. The membrane is pasted onto a backing
material of polypropylene, polystyrene, or polyethylene.
Fig. 1 Schematic representation of a typical LFA test strip
4. Absorbent pad made up of cellulose filter with high wetting

capacity to absorb the excess fluid, sample or reagent at the end
of LFA test strip.
5. Test line and control line contain primary antibody or capture
antibody dried on the running surface membrane. The position
of the test line and control line is very important since longer
the distance between sample pad and test line better is the
interaction between capture antibody and analyte leading to
better sensitivity of LFA. Virus in the sample is captured and
detected as it interacts with antibody immobilized at test line.
The affinity of antibody affects the sensitivity of the test, hence
proper antibody combination is crucial foe success of LFA.
Polyclonal antibody is used as control line is dried at a distance
0.5–1 cm downstream the test line on the membrane. Control
line evaluate the validity of the test since irrespective of pres-
ence or absence of virus it should produce a color line else the
test is invalid.
The sample passes through the sample pad into the conjugate
pad remobilize the already dried GNP-Antibody (secondary anti-
body) conjugates present on conjugate pad. If virus is present in the
sample, it will bind to the GNP-Antibody to form GNP-Antibody-
virus complexes which pass through the membrane due to capillary
force and reach the test line where the primary antibody against
virus is immobilized. This primary antibody binds to the GNP-
Antibody-virus complex and produces a color line on test line due
to GNP label. Anti IgG antibody immobilized on control line
which can bind to secondary antibody with or without analyte
bound to it and produce a color line. The control line should always
appear as an indicator of a valid test. Excess sample will pass
through the nitrocellulose membrane into the absorbent pad
which absorb the excess sample.
3 Materials
Materials required for preparation of colloidal gold nanoparticles

(GNP) and optimization of pH and concentration of IgG/anti-
body for bioconjugation with GNP.
1. Hydrogen tetrachloroaurate (HAuCl4).
2. Trisodium citrate (C6H5Na3O72H2O).
3. Milli-Q water.
4. Magnetic stirrer with hot plate and magnetic bar.
5. K2CO3.
6. NaCl.
7. IgG.
8. 0.45-μm filters.
9. Spectrophotometer.
Materials required for development and assembly of the lateral
flow device are as follows.
1. Sample pad.
2. Conjugate pad.
3. Membrane.
4. Backing card.
5. Reagent dispensing system to dispense the test and control line.
6. Centrifuge.
7. Spectrophotometer.
8. Capture antibodies.
4 Methods
4.1 Gold Gold Nanoparticle can be synthesized in a one-step aqueous prep-

Nanoparticle Synthesis aration in which hydrogentetrachloroaurate (HAuCl4) is brought
to boiling and reduced by rapid addition of trisodium citrate.
1. Prepare100 ml of 0.01% of HAuCl4 solution and 2 ml of 1%
trisodium citrate solution in Milli-Q water.
2. Heat the solution on a magnetic stirrer with hot plate up to
95 C (avoid boiling of the solution) with vigorous stirring
using a magnetic bar.
3. While heating quickly add 2 ml of trisodium citrate to the
stirring solution. The color should change from a pale yellow
to a wine-red color in 2–3 min.
4. Allow the solution to boil for another 10 min with vigorous

stirring.
5. Finally, cool down the solution to room temperature and filter
through 0.8 μm membrane filter.
6. Store the colloidal gold nanoparticle preparation at 4 C till
further use in a dark colored bottle.
7. Measure the size of GNP spectrophotometrically by scanning
colloidal gold solution from 200- to 550-nm wave length.
4.1.1 Conjugation of mAb Antibodies can nonspecifically adsorbed onto GNP by noncovalent
on GNP electrostatic and hydrophobic interactions of the antibody and the
gold surface. In addition, positively charged amino acids present in
antibody form ionic bond with negatively charged citrate capped
surface of the GNP [8].
4.2 Optimization 1. Take 138 mg K2CO3 and add 100 ml of Milli-Q water then
of pH of the GNP filter it with 0.45 μm filter.
and mAb 2. Take 100 μl GNP in 9 tubes as depicted in the following table.
Concentration
for GNP-mAb Tubes 1 2 3 4 5 6 7 8 9
Conjugation pH of GNP 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
4.2.1 Optimization of pH
of GNP 3. Adjust pH of GNP from 5 to 9 by K2CO3.
4. Add 10 μg IgG to 100 μl GNP and mix properly.
5. Incubate for 15 min at room temperature.
6. Add 50 μl of 10% NaCl and incubate for 15 min.
7. Observe the color change.
8. Lowest pH of GNP which maintained wine-red color is the
optimum pH of conjugation.
4.2.2 Optimization Optimization of mAb concentration for GNP-mAb conjugation is

of mAb Concentration required to determine minimum antibody concentration necessary
for GNP-mAb Conjugation to stabilized GNP to avoid agglomeration. Optimization of work-
ing concentration of antigen and GNP conjugated mAb is crucial
for successful development of LFA as excessive concentration of any
one reagent adversely affect sensitivity of the test.
1. Take 1 ml GNP in 5 tubes.
2. Prepare series of mAb dilutions in 5 mM Borax buffer and add
1 μg, 5 μg, 10 μg, 20 μg, 50 μg IgG/ml GNP.
Tube No. 1 2 3 4 5
IgG conc. 1 μg 5 μg 10 μg 20 μg 50 μg
3. Incubate for 5–15 min at room temperature.

4. Add 50 μl of 10% NaCl to each tube.
5. Incubate for 2 h at room temperature.
6. Observe the change in colour of GNP after NaCl addition. The
lowest concentration of mAb which prevent colour change is
selected as the the minimum concentration of mAb required to
protect GNP from flocculation.
7. Mix optimized concentration of mAb with GNP and stir at
room temperature for 1 h to allow complete conjugation
between mAb and surface of GNP by noncovalent and ionic
interactions.
8. Centrifuge the complex at 12,000 g for 30 min. Decant the
supernatant and resuspend the pellet in Milli-Q water.
4.3 Preparation In order to detect virus in clinical sample LFA use monoclonal
of Lateral Flow Assay antibody (MAb) specific for an immunodominant epitope of virus
like major capsid protein VP72 of ASFV and the structural protein
E2 of CSFV immobilized as test line. In addition, a second MAb
specific for another epitope of virus conjugated with GNP as the
indicator system applied in conjugate pad. The control band con-
tain recombinant protein A/G or polyclonal antibody IgG.
1. MAb specific for virus is used as the test line capture reagent.
Dilute MAb to 1 mg/ml in Tris–HCl 20 mM buffer at pH 7.5
containing 5% sucrose and 0.1% sodium azide as preservative.
2. Polyclonal antibody IgG is used as control line capture reagent.
Dilute IgG to 1 mg/ml in the same dilution as the test line
capture reagent.
3. Dispense the test and control capture reagents at 1 μl/cm in
two parallel lines on nitrocellulose membrane. Keep the dis-
tance between two lines at least 5 mm.
4. Dry the test line and control line for 5 min at 45 C and store
the membranes in a desiccator at room temperature.
5. Additionally, membranes can be blocked with PBS containing
1% BSA at room temperature for 5 min to avoid nonspecific
binding.
6. Dispense the conjugate mixture (GNP-MAb) at a concentra-
tion of 0.2%, in a 25-mM phosphate buffer with 1% BSA onto
the conjugate pad and dry for 30 min at 45 C. Store the
conjugate pad in a desiccator at room temperature under dry
condition. (The conjugate pad should facilitate the release of
label. For effective release of label from the conjugate pad the
use of detergents (tween-20) and alcohols (methanol) is
recommended in the running buffer).
7. Paste the nitrocellulose membrane, sample pad, conjugate pad,

and absorbent pad on the plastic backing with adhesive.
Arrange all the components of LFA strip in a manner that
they overlap each other for uninterrupted capillary flow.
8. Cut the master card into strips of 4–5 mm width, which are
placed individually in a plastic device. Store the LFA strips
under dry conditions at 4 C.
9. Dispense 10 μl of the sample onto the sample pad followed by
120 μl of running buffer (Tris–HCl pH 7.5, NaCl, casein, and
NaN3 as preservative), which allows the mixture to migrate
through the conjugate pad and the nitrocellulose membrane
by capillarity.
4.4 LFA Test The LFA devices should be in dry conditions before test to avoid
Procedure any negative effect on the result. Sample once applied to the sample
pad it migrates through the conjugate pad and the nitrocellulose
membrane by capillary force. Results are to be interpreted within
the specified time limit after adding the sample. In the presence of
virus, the major immunodominant protein of virus is captured by
the mAb coated GNP, forming a GNP-mAb-virus immune com-
plex. This immune complex then migrates across the membrane by
capillary action and reacts with the immobilized mAb (specific for
another epitope of virus) on the test line of membrane, making the
test line visible. Irrespective of positive or negative sample control
line must appears else the test is invalid.
5 Notes
1. Gold particle diameter can be tuned via citrate:[AuCl4]1 stoi-

chiometry, i.e. ratio of reducing agent trisodium citrate:tetra-
chloroauric acid (HAuCl4) regulates gold nanoparticle
diameter where citrate act as a reducing as well as capping
agent. Gold nanoparticles size ranging from 12 to 147 nm
can be achieved by changing the ratio of Na3Citrate:HAuCl4,
i.e. higher the ratio, smaller the size of particles [9]. While
preparing gold nanoparticle formation of a deep red color
solution indicated the formation of spherical gold nanoparti-
cles and the colors depend strongly on their size and shape
[10].
2. The maximum absorbance of surface plasmon band (SPB) of
gold nanoparticles is generally around 520 nm. The extinction
maximum of SPB shifts from 518 nm to 533 nm when the
particle means diameter changes from 10 nm to 48 nm and
20 nm diameter particles has peak absorption at 520 nm [9].
3. Larger size nanoparticle provides better sensitivity but stability

of GNP decreases with the increase in size as it tends to aggre-
gate. 40-nm GNP provide best result in terms of sensitivity,
better flow through and high signal-to-noise ratio. The
GNP-antibody bioconjugate has a high affinity leads to non-
specific binding with interfering molecules present in clinical
samples. This problem can be solved by adding blocking agents
such as BSA, skimmed milk powder directly to GNP to block
nonspecific binding of antibody.
4. Several factors influence the performance of LFA like type of
capture reagents, membrane (pore size and flow rate), and
complexity of the sample. The capture reagents used in devel-
oping LFA influence the specificity and sensitivity of the assay.
5. Sufficient GNP-Antibody-virus should bind to primary anti-
body at test line to form a colored line at test line as well as
sufficient secondary antibody at conjugate pad should be
applied to bind with the antibody present at control line.
6. Primary antibody and secondary antibody dilutions must be
optimized for successful development of LFA.
7. More complex samples such as muscle tissue and blood contain
interfering background which have adverse impact on overall
test performance. Hence, complex samples need
pre-processing before the actual test to minimize interfering
substances [11]. Migration speed of analyte and antibody-gold
conjugate on membrane the test strip has the impact on sensi-
tivity of assay. For effective immobilization of capture reagent
on the membrane incubation temperature, time, volume of
antibody immobilized on the test line must be optimized.
Membrane drying temperature after applying test line and
control line affects pore structures of membrane as high drying
temperature results in smaller pores which in turns decreases
the flow rate. Thus, while developing lateral flow assays, the
membrane, sample pad, absorbent pad, conjugate pad, and
capture antibodies should be very carefully chosen. For more
sensitive assays, membranes with slow migration rate are better,
whereas for those assays where sensitivity is not an issue but
faster result is important, membranes with fast migration rates
should be chosen.
References
1. Fernandez-Pinero J, Gallardo C, Elizalde M, 2. Gallardo C, Nieto R, Soler A, Pelayo V,

Robles A, Gomez C, Bishop R, Heath L, Fernandez-Pinero J, Markowska-Daniel I,
Couacy-Hymann E, Fasina FO, Pelayo V et al Pridotkas G, Nurmoja I, Granta R, Simon A
(2013) Molecular diagnosis of African swine et al (2015) Assessment of African swine fever
fever by a new real-time PCR using universal diagnostic techniques as a response to the epi-
probe library. Transbound Emerg Dis 60(1): demic outbreaks in eastern European union
48–58 countries: how to improve surveillance and
control programs. J Clin Microbiol 53(8): Synthesis and bioconjugation of gold nanopar-
2555–2565 ticles as potential molecular probes for light-
3. Oura CA, Edwards L, Batten CA (2013) Viro- based imaging techniques. Int J Biomed Imag-
logical diagnosis of African swine fever–coming 2007:29817. https://doi.org/10.1155/
parative study of available tests. Virus Res 2007/29817
173(1):150–158 9. Daniel MC, Astruc D (2004) Gold nanoparti-
4. Baudhuin P (1989) Colloidal gold: principles, cles: assembly, supramolecular chemistry,
methods, and applications, vol 2. Academic quantum-size-related properties, and applica-
Press, New York, pp 1–17 tions toward biology, catalysis, and nanotech-
5. Frens G (1973) Controlled nucleation for reg- nology. Chem Rev 104(1):293–346
ulation of particle-size in monodisperse gold 10. Kelly KL, Coronado E, Zhao LL, Schatz GC
suspensions. Nat Phys Sci 241(105):20–22 (2003) The optical properties of metal nano-
6. Mirkin CA (1996) A DNA-based method for particles: the influence of size, shape, and
rationally assembling nanoparticles into macro- dielectric environment. J Phys Chem 107:
scopic materials. Nature 382(6592):607–609 668–677
7. Milan RC, Marzan LML (2014) Gold nano- 11. Lee JY, Kim YA, Kim MY, Lee YT, Hammock
particle conjugates: recent advances toward BD, Lee HS (2012) Importance of membrane
clinical applications. Expert Opin Drug Deliv selection in the development of immunochro-
11(5):741–752 matographic assays for low-molecular weight
compounds. Anal Chim Acta 757:69–74
8. Rayavarapu RG, Petersen W, Ungureanu C,
Post JN, Leeuwen TG, Manohar S (2007)
Chapter 14
Droplet Digital PCR-Based Diagnosis for Porcine Viral

Diseases
Abstract
Polymerase chain reaction (PCR) is a common and indispensable technique that has been employed since its
discovery for the diagnosis of infectious diseases. A biotechnological refinement of the conventional PCR
led to the third generation of PCR, called the droplet digital polymerase chain reaction (ddPCR), that can
be used to directly quantify and amplify nucleic acids. Presently, ddPCR is widely used in low-abundance
nucleic acid detection and is useful in the diagnosis of infectious diseases. The distinctive feature of ddPCR
is the separation of the reaction mixture into partitions, followed by a real-time or end-point detection of
the amplification. As Poisson distribution describes the distribution of target sequences into partitions, it
allows accurate and absolute quantification of the target from the ratio of positive against all partitions at the
end of the reaction. ddPCR enables the absolute quantification of nucleic acids without the need to use
reference materials with known target concentrations as used commonly in qPCR. A higher resilience to
inhibitors in a number of different types of samples is an additional feature of ddPCR. ddPCR provides
more sensitive, accurate and reproducible detection of low-abundance pathogens and definitely serves as a
better choice than qPCR for clinical applications in the future.
Key words Diagnostics, Droplet digital polymerase chain reaction, Porcine, Viral pathogens
1 Introduction
Almost four decades later, polymerase chain reaction (PCR) still

continues to be used as a standard and has become a ubiquitous
laboratory tool. During the early 1990s, a concept of “limit dilu-
tion PCR” arose when researchers began exploring the possibility
of diluting the template to an extent such that, on average, any
single PCR reaction contained only a single template molecule. A
key advantage of limit dilution PCR is that each DNA molecule is
amplified separately, killing interferences between template mole-
cules during PCR and greatly reducing background noise in com-
plex samples. Additionally, when many reactions are performed at
this level of dilution, the frequency of positive and negative reac-
tions follows the Poisson distribution and therefore allows for
205
206 Yoya Vashi and Sachin Kumar
calculating the abundance of the target molecule based on the

dilution factor. The concept of digital PCR was first described in
1992 [1] using the principles of limiting dilution, PCR, and Pois-
son statistics. The term “digital PCR” was first used in the 1999
paper by Kinzler and Vogelstein [2] in which they described the
quantitation of ras mutations in a sample by partitioning the sample
in order to perform a series of PCRs in 384 well microplates. Later,
the scientific community recognized key advantages of digital PCR
over traditional end-point or real-time PCR. The key advantages
offered by digital PCR were that it does not rely on a standard
curve, had improved accuracy, provides absolute quantification and
offered improved detection of low copy-numbers nucleic acids.
Other key advantages of digital PCR became evident later, as the
method was used more broadly [3]: repeatability of assays over time
and across different labs; robustness and tolerance to PCR
inhibitors.
Researchers and equipment makers have also made more radi-
cal modifications to PCR by borrowing technologies from other
fields. One such effort yielded droplet digital PCR (ddPCR), which
combines aspects of fluorescence-activated cell sorting with con-
ventional PCR. Though the protocol for ddPCR is somewhat
complicated, it has also become highly automated. The ddPCR is
a method of dPCR in which a 20 μl sample reaction including assay
primers and either Taqman probes or an intercalating dye, is
divided into ~20,000 nanoliter-sized oil droplets through a water-
oil emulsion technique, thermocycled to end-point in a 96-well
PCR plate, and fluorescence amplitude read for all droplets in each
sample well in a droplet flow cytometer [4].
ddPCR is a recent technology that has become commercially
available since 2011 [5]. As with qPCR, ddPCR technology utilizes
Taq polymerase in a standard PCR reaction to amplify a target
DNA fragment from a complex sample using pre-validated primer
or primer/probe assays. However, there are two distinct differ-
ences: (1) the partitioning of the PCR reaction into thousands of
individual reaction vessels prior to amplification and (2) the acqui-
sition of data at the reaction end-point (Fig. 1). These factors offer
the advantage of direct and independent quantification of DNA
without standard curves giving more precise and reproducible data
versus qPCR, especially in the presence of sample contaminants that
can partially inhibit Taq polymerase and/or primer annealing
[7, 8]. In addition, end-point measurement enables nucleic acid
quantitation independently of the reaction efficiency, resulting in a
positive–negative call for every droplet and greater amenability to
multiplexed detection of target molecules [9]. Thereby, ddPCR
technology can be used for extremely low-target quantitation
from variably contaminated samples where the sample dilution
requirements to assure consistent and acceptable reaction efficiency,
Droplet Digital PCR-Based Diagnosis for Porcine Viral Diseases 207
Fig. 1 Principles of digital PCR. The sample is divided into many independent partitions such that each
contains either a few or no target sequences. The distribution of target sequences in the partitions can be
approximated with a Poisson’s distribution. Each partition acts as an individual PCR microreactor and
partitions containing amplified target sequences are detected by fluorescence. The ratio of positive partitions
(presence of fluorescence) over the total number allows determining the concentration of the target in the
sample. (Reproduced from [6])
primer annealing and Cq values for qPCR would likely lead to

undetectable target levels [7, 10].
Some of the major applications of ddPCR include absolute
quantification, detection of genomic alteration such as gene copy
number variation (CNV), detection of rare sequences, gene expres-
sion and microRNA analysis, single-cell analysis, etc.
2 ddPCR Experimental Workflow
In this section, we focus on the ddPCR workflow for viral pathogen

(DNA or RNA) quantification using the Bio-Rad QX100
(or QX200) system.
2.1 Preparation The concentrations of primers and probe for each assay with the
of Reaction Mixture corresponding 2 ddPCR master mix should be mixed in nuclease-
free tubes. Concentrations of primers and probe should be prefera-
bly optimized previously (See Note 1). The preparation of duplex
or multiplex assays is also possible. It is to be noted that manganese
acetate needs to be added in the case of the one-step master mix.
The final reaction volume should be planned to 20 μl. Before
adding the sample, the prepared mix should be distributed into
nuclease-free tubes, strips, or 96-well plates. Sample (DNA/RNA
samples and controls, see Tables 1 and 2) should be added into each
tube containing master mixes and mixed thoroughly by pipetting,
followed by brief centrifugation (See Note 2). Each tube should
contain 20 μl of the reaction mixture.
2.2 Droplet A DG8™ droplet generation cartridge is placed into the cartridge
Generation holder. To each of the 8 wells indicated as “sample” in the droplet
generation cartridge, 20 μl of each prepared reaction mixture is
Table 1
Reaction setup for DNA amplification in ddPCR
Component Volume (μl) Final concentration

2 ddPCR super mix for probes (See Note 3) 10 1
20 target primers/probe (FAM) 1 1 (i.e., 900 nM/150 nM)
20 second target primers/probe (VIC) 1 1 (i.e., 900 nM/150 nM)
Nuclease-free water Variable –
DNA sample Variable 50 fg to 100 ng
Final volume 20 –
Table 2
Reaction setup for RNA amplification in one step RT-ddPCR
Component Volume (μl) Final concentration

2 ddPCR super mix for probes 10 1
Manganese acetate 0.8 1 (i.e., 900 nM/250 nM)
20 target primers/probe (FAM) 1 1 (i.e., 900 nM/150 nM)
20 second target primers/probe (VIC) 1 1 (i.e., 900 nM/150 nM)
Nuclease-free water Variable –
RNA sample Variable 50 fg to 100 ng
Final volume 20 –
transferred (See Note 4). Precautions should be taken not to form

bubbles in the bottom of the well, as they could interfere with the
droplet formation. In the wells indicated as “oil,” 70 μl of droplet
generation oil is added. The oil bottle should not be left open for
extended periods of time to avoid evaporation and stability of
components. The gasket is then hooked over the cartridge holder
using the holes in both sides and the holder is placed with the
cartridge in the QX100 droplet generator unit, initiating the drop-
let generation (See Notes 5 and 6). Oil and sample are pushed
through microfluidic channels and mixed in the cartridge in the
process, forming droplets. Droplets are accumulated in the droplet
well. The process takes 2–3 min for each cartridge. Once droplets
are generated, the gasket is removed and 40 μl of the droplet
suspension (“droplets” lane in the cartridge) is transferred from
the cartridge to a 96-well PCR plate. The pipetting both for col-
lecting the droplet suspension and for dispensing it in the PCR
plate wells should be slow to protect the integrity of the droplets.
After all the samples have gone through droplet generation and
have been transferred to the 96-well PCR plate, the plate is heat-
sealed with a pierceable foil.
Table 3
PCR cycling conditions
DNA samples RNA samples
Step T ( C) Time Cycle T ( C) Time Cycle

RT – – – 60 30 min Hold
Enzyme activation 95 10 min Hold 95 5 min Hold
Denaturation 94 30 s 40 94 30 s 40
Annealing and extension 60 1 min 60 1 min
Heat deactivation 98 10 min Hold 98 10 min
Hold 4 1 Hold 4 1 Hold
2.3 PCR The sealed plate is transferred to the thermocycler and the PCR is
Amplification run as per the conditions shown in Table 3.
2.4 Droplet Reading Once the PCR is over, the PCR plate is transferred to the QX100
and Data Analysis droplet reader. In the QuantaSoft software (Bio-Rad), the “Setup”
button is clicked and the information for each well/sample, includ-
ing name, type of experiment, type of sample and detectors or
channels (FAM and/or VIC), is defined. The reading is started by
clicking the “Run” button. The droplet reader acts as a flow cyt-
ometer and reads each droplet to determine their signal and ampli-
tude in the selected detectors. Once the run is over, click “Analyze”
to start analyzing the results. The critical point is to set a threshold
that allows the software to differentiate between negative and
positive droplets. The software offers the possibility of defining
this automatically or manually. However, other approaches to ana-
lyses are available and may, in specific cases, be more suitable (See
Note 7). The software offers different ways of viewing the results
(1D amplitude of one channel, 2D amplitudes of both channels,
copy number in each well/channel) that are more or less informa-
tive depending on the type of experiment. It ultimately gives a table
with parameters resulting from the analysis, such as the concentra-
tion of target copies/microliter of reaction, the number of total
accepted droplets, the positive ones, and the negative ones.
3 Notes
1. The widely accepted quantitative PCR (qPCR) design guide-

lines also apply to ddPCR primer design. Some of the impor-
tant guidelines include the primers to have a GC content of
50–60%, avoiding repeats of Gs or Cs longer than 3 bases and
ensuring that no 30 complementarity exists. It is to be noted

that the QX100™ Droplet Digital PCR system (Bio-Rad) is
compatible only with TaqMan hydrolysis probes and QX100™
system is compatible with TaqMan hydrolysis probes and Eva-
Green® double-stranded DNA binding dye. The advantage of
using hydrolysis probes includes high specificity, a high signal-
to-noise ratio, and the ability to perform multiplex reactions.
Few considerations during designing probes include choosing
the probe sequence between the two primers of the amplicon,
higher Tm than that of the primers, probe length of <30
nucleotides, etc.
2. For RNA or DNA isolation, same protocols or kits as those
used for qPCR can be used for ddPCR. The higher resilience of
ddPCR to inhibitors reported in several recent studies [7, 11–
13] allow the use of a broader range of nucleic acid purification
methods, including more simple extraction protocols.
3. At present, the Bio-Rad platform requires the use of their
proprietary master mixes enabling stable droplets formation,
while platforms based on partitioning into chambers allow for
testing different commercially available PCR mixes.
4. The highest accuracy when pipetting is required. In addition,
when handling the droplets, the pipetting should be done very
carefully to preserve their integrity.
5. The QX100 and QX200 systems allow duplexing with fluores-
cence data acquisition in both the FAM and HEX-VIC spectral
regions, respectively. There is an increasing number of studies
where multiplexing with ddPCR has been reported [11, 14–
16].
6. The process of droplet generation is repeated as many times as
required by the sample number. When longer preparation
times are expected due to the high number of samples, it is
recommended to keep both samples and droplet suspensions at
4 C using refrigerated blocks. In the case that the droplet
reader is unavailable after PCR cycling, the plate can be stored
at 4 C for several hours or even overnight.
7. The variation of signals depends on many factors, e.g., assay
characteristics [17], matrix or presence of inhibitors [12], sin-
gle nucleotide polymorphisms (SNP) in the probe annealing
region and others. All these factors can affect the separation
between negative and positive droplets in different ways, result-
ing for example, in the presence of higher signal droplets in
negative samples or in the induction of the so-called droplet
rain effect. Therefore, in certain cases, automatic analysis can
sometimes be misleading, and setting the threshold manually
can be necessary. Manual definition of the threshold can also
help overcoming other droplet particularities, such as rain
effect. Both in diagnostic and quantitative applications, it is

important to evaluate the signal amplitude obtained with neg-
ative samples (NTC, isolation controls, matrix controls, closely
related pathogens). The threshold can be then manually
defined to consider such signals as negative.
4 ddPCR for Porcine Viruses
During the last decades, effective treatments, vaccination, and

improved diagnostics have resulted in a reduction in the direct
burden of infectious diseases on livestock production [18]. Never-
theless, emerging and re-emerging swine viruses are an emerging
threat already ravaging the pig producers worldwide. An early field
diagnosis is considered the first line of defense and plays an impor-
tant role in the treatment of viral infection. However, to date, it can
take up to several weeks or months from the time between initial
disease outbreak and laboratory confirmation of the viral pathogen.
To further prevent the spreading of the disease, reliable, quick, and
simple diagnostic testing is crucial so that targeted control strate-
gies can be implemented. Currently, rapid diagnostics systems rely
on nucleic acid extraction, purification, and PCR-based amplifica-
tion and detection. PCR amplification methods are very sensitive
but often produce false positives from trace contamination of the
specimen/equipment.
The ddPCR is the third generation of PCR technology, which
enables absolute quantification of nucleic acid targets without the
need to construct a calibration curve as used commonly in qPCR
[5, 19]. Therefore, this method has highly precise and sensitive,
which is rapidly replacing qPCR as an efficient method for indepen-
dent nucleic acid quantifications. ddPCR has been used to detect
low-abundance nucleic acids in many laboratories and is proving
itself to be a powerful tool in detecting the swine viral pathogens.
ddPCR has been used to detect major viral pathogens of swine like
African swine fever virus [20], porcine circovirus type 2 and 3 [21–
23], porcine reproductive and respiratory syndrome virus [24],
porcine epidemic diarrhea virus [25], Senecavirus A [26, 27], pseu-
dorabies virus [28], Japanese encephalitis virus [29], and hepatitis
E virus [30]. In a few cases, the detection limit for ddPCR was
1 copy/μl, which was about 10 times greater sensitivity than Taq-
Man real-time PCR [22].
The ddPCR has become an emerging tool for the diagnosis of
porcine viral pathogens. The method overrides the variation of
amplification with a high level of reliability and reproducibility.
Development of ddPCR for more porcine viral pathogens known
to affect the swine industry will surely help in the establishment of
reliable countermeasures to disease outbreaks and will limit severe
animal’s health and socio-economic consequences.
5 Conclusion
Nonetheless, researchers worldwide continue to push the capabil-

ities of PCR and find ways to propel it into new territories. The
advantages of ddPCR lie in many aspects. Many studies have shown
that ddPCR is technically more accurate and sensitive. Also, during
disease diagnosis, a faster readout is provided by PCR-based tech-
niques as compared to serological or pathogen culturing tests, and
ddPCR overcomes the problems in the accuracy of qPCR, which
makes it more suitable as a diagnostic tool. The ddPCR is now a
potential diagnostic method for porcine viral pathogens.
References
1. Sykes PJ, Neoh SH, Brisco MJ, Hughes E, quantification of oseltamivir-resistant subpo-
Condon J, Morley AA (1992) Quantitation of pulations. J Virol Methods 224:58–66
targets for PCR by use of limiting dilution. 9. Hughesman CB, Lu XJ, Liu KY, Zhu Y, Poh
BioTechniques 13(3):444–449 CF, Haynes C (2016) A robust protocol for
2. Vogelstein B, Kinzler KW (1999) Digital PCR. using multiplexed droplet digital PCR to quan-
Proc Natl Acad Sci U S A 96(16):9236–9241 tify somatic copy number alterations in clinical
3. Kuypers J, Jerome KR (2017) Applications of tissue specimens. PLoS One 11(8):e0161274
digital PCR for clinical microbiology. J Clin 10. Verhaegen B, De Reu K, De Zutter L,
Microbiol 55(6):1621–1628 Verstraete K, Heyndrickx M, Van Coillie E
4. Wood-Bouwens CM, Ji HP (2018) Single (2016) Comparison of droplet digital PCR
color multiplexed ddPCR copy number mea- and qPCR for the quantification of Shiga
surements and single nucleotide variant geno- toxin-producing Escherichia coli in bovine
typing. Methods Mol Biol 1768:323–333 feces. Toxins (Basel) 8(5):157
5. Hindson BJ, Ness KD, Masquelier DA, 11. Morisset D, Stebih D, Milavec M, Gruden K,
Belgrader P, Heredia NJ, Makarewicz AJ, Zel J (2013) Quantitative analysis of food and
Bright IJ, Lucero MY, Hiddessen AL, Legler feed samples with droplet digital PCR. PLoS
TC, Kitano TK, Hodel MR, Petersen JF, Wyatt One 8(5):e62583
PW, Steenblock ER, Shah PH, Bousse LJ, 12. Racki N, Morisset D, Gutierrez-Aguirre I, Rav-
Troup CB, Mellen JC, Wittmann DK, Erndt nikar M (2014b) One-step RT-droplet
NG, Cauley TH, Koehler RT, So AP, Dube S, digital PCR: a breakthrough in the quantifica-
Rose KA, Montesclaros L, Wang S, Stumbo tion of waterborne RNA viruses. Anal Bioanal
DP, Hodges SP, Romine S, Milanovich FP, Chem 406(3):661–667
White HE, Regan JF, Karlin-Neumann GA, 13. Yang R, Paparini A, Monis P, Ryan U (2014)
Hindson CM, Saxonov S, Colston BW (2011) Comparison of next-generation droplet digital
High-throughput droplet digital PCR system PCR (ddPCR) with quantitative PCR (qPCR)
for absolute quantitation of DNA copy num- for enumeration of cryptosporidium oocysts in
ber. Anal Chem 83(22):8604–8610 faecal samples. Int J Parasitol 44(14):
6. Quan PL, Sauzade M, Brouzes E 1105–1113
(2018) dPCR: a technology review. Sensors 14. Kelley K, Cosman A, Belgrader P, Chapman B,
(Basel) 18(4):1271 Sullivan DC (2013) Detection of methicillin-
7. Racki N, Dreo T, Gutierrez-Aguirre I, resistant Staphylococcus aureus by a duplex
Blejec A, Ravnikar M (2014a) Reverse tran- droplet digital PCR assay. J Clin Microbiol
scriptase droplet digital PCR shows high resil- 51(7):2033–2039
ience to PCR inhibitors from plant, soil and 15. Leibovitch EC, Brunetto GS, Caruso B,
water samples. Plant Methods 10(1):42 Fenton K, Ohayon J, Reich DS, Jacobson S
8. Taylor SC, Carbonneau J, Shelton DN, Boivin (2014) Coinfection of human herpesviruses
G (2015) Optimization of droplet digital PCR 6A (HHV-6A) and HHV-6B as demonstrated
from RNA and DNA extracts with direct com- by novel digital droplet PCR assay. PLoS One
parison to RT-qPCR: clinical implications for 9(3):e92328
16. Sedlak RH, Kuypers J, Jerome KR (2014) A 24. Yang Q, Xi J, Chen X, Hu S, Chen N, Qiao S,
multiplexed droplet digital PCR assay performs Wan S, Bao D (2017) The development of a
better than qPCR on inhibition prone samples. sensitive droplet digital PCR for quantitative
Diagn Microbiol Infect Dis 80(4):285–286 detection of porcine reproductive and respira-
17. Dreo T, Pirc M, Ramsak Z, Pavsic J, tory syndrome virus. Int J Biol Macromol 104
Milavec M, Zel J, Gruden K (2014) Optimis- (Pt a):1223–1228
ing droplet digital PCR analysis approaches for 25. Cao WW, He DS, Chen ZJ, Zuo YZ, Chen X,
detection and quantification of bacteria: a case Chang YL, Zhang ZG, Ye L, Shi L (2020)
study of fire blight and potato brown rot. Anal Development of a droplet digital PCR for
Bioanal Chem 406(26):6513–6528 detection and quantification of porcine epi-
18. Perry BD, Grace D, Sones K (2013) Current demic diarrhea virus. J Vet Diagn Investig
drivers and future directions of global livestock 32(4):572–576
disease dynamics. Proc Natl Acad Sci U S A 26. Pinheiro-de-Oliveira TF, Fonseca-Junior AA,
110(52):20871–20877 Camargos MF, Laguardia-Nascimento M,
19. Pinheiro LB, Coleman VA, Hindson CM, Giannattasio-Ferraz S, Cottorello ACP, de Oli-
Herrmann J, Hindson BJ, Bhat S, Emslie KR veira AM, Goes-Neto A, Barbosa-Stancioli EF
(2012) Evaluation of a droplet digital polymer- (2019) Reverse transcriptase droplet digital
ase chain reaction format for DNA copy num- PCR to identify the emerging vesicular virus
ber quantification. Anal Chem 84(2): Senecavirus a in biological samples. Trans-
1003–1011 bound Emerg Dis 66(3):1360–1369
20. Wu X, Xiao L, Lin H, Chen S, Yang M, An W, 27. Zhang Z, Zhang Y, Lin X, Chen Z, Wu S
Wang Y, Yang Z, Yao X, Tang Z (2018) Devel- (2019) Development of a novel reverse tran-
opment and application of a droplet digital scription droplet digital PCR assay for the sen-
polymerase chain reaction (ddPCR) for detec- sitive detection of Senecavirus a. Transbound
tion and investigation of African swine fever Emerg Dis 66(1):517–525
virus. Can J Vet Res 82(1):70–74 28. Ren M, Lin H, Chen S, Yang M, An W,
21. Liu Y, Meng H, Shi L, Li L (2019a) Develop- Wang Y, Xue C, Sun Y, Yan Y, Hu J (2018)
ment of a droplet digital polymerase chain reac- Detection of pseudorabies virus by duplex
tion for sensitive and simultaneous droplet digital PCR assay. J Vet Diagn Investig
identification of porcine circovirus type 2 and 30(1):105–112
3. J Virol Methods 270:34–37 29. Wu X, Lin H, Chen S, Xiao L, Yang M, An W,
22. Liu Y, Meng H, Shi L, Li L (2019b) Sensitive Wang Y, Yao X, Yang Z (2017) Development
detection of porcine circovirus 3 by droplet and application of a reverse transcriptase drop-
digital PCR. J Vet Diagn Investig 31(4): let digital PCR (RT-ddPCR) for sensitive and
604–607 rapid detection of Japanese encephalitis virus. J
23. Zhao S, Lin H, Chen S, Yang M, Yan Q, Virol Methods 248:166–171
Wen C, Hao Z, Yan Y, Sun Y, Hu J, Chen Z, 30. Mykytczuk O, Harlow J, Bidawid S,
Xi L (2015) Sensitive detection of porcine Corneau N, Nasheri N (2017) Prevalence and
circovirus-2 by droplet digital polymerase molecular characterization of the hepatitis E
chain reaction. J Vet Diagn Investig 27(6): virus in retail pork products marketed in
784–788 Canada. Food Environ Virol 9(2):208–218
Chapter 15
Protocols for Immunofluorescence Techniques

Deepika Bisht, Shikha Saxena, Nitish Singh Kharayat,
and Siddharth Gautam
Abstract
Since the initial description of immunofluorescence (IF) assay by Coons, Creech, and Jones in 1941, it has
evolved from being an academic technique to a routine laboratory investigative practice. Immunofluores-
cence technique is a very useful tool to detect protein expression and localize cellular as well as viral antigen
(in case of virus infected cell). Immunofluorescence technique detects the target optically using fluorophore
conjugated-antibodies. In this chapter, we have described various types of IF techniques, the advantages
and limitations associated with each variant. Here we also describe the protocol for performing immuno-
fluorescence technique along with the important points to note and alternative ways for each step involved
while performing the IF assay.
Key words Immunofluorescence, Fluorophore, Antibody, Antigen, Fixatives, Permeabilizers
1 Introduction
Protein expression in a biological sample of cellular origin can be

evaluated by various techniques such as enzyme linked immunosor-
bent assay (ELISA) and western blotting, etc. [1, 2]. However, the
inability of cellular and subcellular localization of the protein is the
most significant limitation of these techniques. Immunohistochem-
istry (IHC) assays are generally performed to localize the protein;
however, Immunofluorescence technique is preferred, when there
is a need for co-localization of proteins. Immunofluorescence
(IF) technique enables us to visualize and localize the protein in
the cells or tissues using antibodies conjugated to a fluorophore
[3]. The antigen–antibody interaction and fluorescent signal pro-
duced by the conjugated fluorophore on excitation with suitable
Ultraviolet (UV) light forms the base of immunofluorescence tech-
nique [4]. The fluorophore absorbs UV light in a defined wave-
length range and emits the light of higher wavelength. This emitted
light is detected by the detector of fluorescent microscope,
215
216 Deepika Bisht et al.
Table 1
Commonly used fluorophores: excitation and emission wavelengths
Fluorophore Excitation wavelength (nm) Emission wavelength (nm)

Fluorescein isothiocyanate 492 518
Texas Red 568 590
Rhodamine 555 620
B-Phycoerythrin 546 575
R-Phycoerythrin 490 575
equipped with specific filter for the wavelength of emitted light.

Selection of fluorophore to be used in IF imaging is critical to
obtain good quality immunofluorescence images (see Note 1).
Some of the most frequently used fluorophores are summarized
in Table 1.
Albert H. Coons and his colleagues first described IF, while
reporting that antigens in mammalian tissues were detectable opti-
cally in ultraviolet (UV) light with a fluorophore (fluorescein isocy-
anate) conjugated antibody [5, 6]. Various studies have shown that
the IF technique is a useful tool in studying bacterial and viral
proteins [7–9]. Immunofluorescence technique possesses great
value in virology for identification of viral antigens in virus-infected
cells [10]. Rapid viral diagnosis using IF staining was first described
by Liu in 1956 [11] for the detection of influenza virus. Later, this
virus detection system was pioneered by Gardner and McQuillan
for other viruses [12]. The detection of viral proteins in the cell
indicates active replication, as opposed to latent viral infections. For
detecting viral proteins, the fluorophore is conjugated either to the
primary/antiviral antibody itself (direct immunofluorescence) or to
the anti-antibody/secondary antibody (indirect immunofluores-
cence), and visualized using ultraviolet light. The type of primary
antibody (monoclonal/polyclonal) used depends upon the amount
of viral target protein expected in the sample (sensitivity) and cross-
reactivity of the antibody with non-target proteins of the host or
unrelated pathogen (specificity). The major advantage of immuno-
fluorescence is its ability to localize protein of interest. Another
advantage is that it requires lesser time and resources than isolation
of virus in cell culture.
Different variants of IF assay, namely, direct immunofluores-
cence, indirect immunofluorescence, and multicolor immunofluo-
rescence, have their own advantages and disadvantages. Before
initiating the immunofluorescence protocol, these advantages/dis-
advantages should be considered.
Protocols for Immunofluorescence Techniques 217
1.1 Direct In this protocol, the fluorophore is directly conjugated to the pri-
Immunofluorescence mary antibody for target antigen. Simple protocol and shorter
incubation time are few advantages of direct IF. For visualizing
multiple proteins, each of the antibody should be tagged with
distinct/different fluorophores. The antibodies developed in the
same species are compatible and do not pose a problem. Low
staining intensity due to the lack of signal amplification phenomena
and requirement of tagging fluorophore to each primary antibody
are its major limitations.
1.2 Indirect In this protocol, secondary antibodies are conjugated with fluor-
Immunofluorescence ophore. These secondary antibodies are specific to the unlabelled
primary antibodies. As multiple secondary antibodies bind to the
target bound primary antibody, indirect IF protocol leads to signal
amplification. This phenomenon becomes more desirable while
dealing with low abundance targets. Secondary antibodies are
able to recognize all primary antibodies derived from a target host
species. This improves the flexibility and cost-effectiveness of the
protocol. However, while visualizing multiple antigens, the primary
antibodies need to be raised in distinct species to prevent cross
reactivity. Major limitation of this protocol is high background
interfering with fluorescence imaging, when used against proteins
in abundance.
The difference of direct and indirect IF is summarized in Fig. 1.
1.3 Multicolor Different fluorophores possess specific excitation and emission

Immunofluorescence wavelength spectra, multiple antigens can be localized on the
same sample by conjugating their respective antibodies to different
fluorophores with non-overlapping excitation and emission spectra.
This form of immunofluorescence is known as Multicolor
immunofluorescence [13].
Fig. 1 Direct and Indirect Immunofluorescence. In case of direct immunofluorescence, fluorophore is

conjugated to antibody binding to target antigen. Indirect IF protocol involves detection of target antigen,
utilizing fluorophore conjugated secondary antibody bound to unlabelled primary antibody specific to target
antigen. Signal amplification is seen in case of indirect IF
2 Materials
1. Phosphate-buffered saline (1): 137 mM NaCl, 10 mM

Phosphate, 2.7 mM KCl in double distilled water (pH 7.4).
2. Fixative solutions (Paraformaldehyde solution): 4% Parafor-
maldehyde (w/v) in PBS (see Note 5).
3. Permeabilizer (0.4% Triton X-100):0.4% (v/v) Triton X-100
in PBS.
4. Blocking buffer: 5% (v/v) normal goat serum, 0.05% (v/v)
Triton X-100 in PBS.
5. Antibody dilution buffer: 1% (v/v) normal serum, 0.05%
(v/v) Triton X-100 in PBS.
6. Primary Antibody.
7. Secondary antibody (FITC conjugated).
8. Counterstaining stock solution, DAPI (40 ,6-diamidino-2-
phenylindole) (5 mg/mL stock): 14.3 mM of DAPI dihy-
drochloride in DMF. It is diluted to 300 nM in PBS before use.
9. Mounting medium: 50% (v/v) glycerol in PBS (see Note 13).
3 Method
In general, immunofluorescence technique using a single fluoro-

phore conjugated antibody involves the following steps:
1. Sample Preparation.
2. Fixation of cells.
3. Permeabilization of cell membrane.
4. Blocking.
5. Incubation with primary antibody.
6. Incubation with secondary antibody.
7. Mounting.
8. Imaging.
9. Post-imaging analysis.
3.1 Sample Immunofluorescence can be performed on adherent cell culture,

Preparation suspension cell culture as well as on a tissue sample. The main
objective of this step is to promote adherence of the specimen to
a solid substrate that is optically suitable for imaging at the end of
the IF assay.
3.1.1 In Case of Adherent 1. The cells can be cultured on coverslips, kept inside sterile multi-
Culture well tissue culture plate or petri-dish (see Note 2). The sterili-
zation of coverslip can be done by keeping it under UV light in
biosafety cabinet for 30 min (see Note 3).
2. On day 1, the cells are seeded generally at low confluency (cell
density approximately 10,000/cm2) in sufficient volume of cell
culture medium (e.g. 500 μL for a well of four well cell culture
plate) for immunofluorescence assay.
3. Next day, the cultured cells are washed thrice with 1
phosphate-buffered saline (PBS) to remove cell culture
media, unattached dead cells, and debris before fixation.
3.1.2 Suspension Cell 1. Cells grown in suspension can be coated on a slide using the
Culture cytospin technique.
2. Approximately 150–200 μL of cells suspension in (1) PBS are
allowed to attach onto the glass slide by spinning at low speed.
3. The slides are pre-treated with L-polylysine to encourage cell
attachment. The slide is then air-dried, followed by fixation (see
Note 4).
3.1.3 Tissue Samples Tissue samples from animals need fixation prior to be cut into
smaller pieces (6–8 μm), to get smooth and fine sections. This
improves the quality and visibility of immunofluorescence signifi-
cantly. The fixation is done either by flash freezing the freshly
dissected tissue or by embedding formalin fixed tissue in
paraffin block.
Flash freezing fixes the tissue by avoiding ice crystal formation.
In this method, the freshly dissected tissue is placed on a tissue
mold and is covered with a cryo-embedding medium and kept in
the dry ice. The storage of the sample is done at 80 C or in liquid
nitrogen. The blocks are cut into thin sections of approximately
4–8 μm and mounted on the glass slide.
For paraffin embedding, the tissue is fixed by suspending it
overnight in neutral buffered formalin followed by embedding with
paraffin. Paraffin embedded tissue is cut into fine sections which are
mounted onto the glass slide. Deparafinization is done by multiple
xylene washes and rehydration by graded alcohol washes.
In case of paraffin embedding, the antigens get masked, which
needs to be unmasked (retrieval of antigen) before proceeding to
immunofluorescence assay (see Note 4).
3.2 Fixation of Cells Fixation maintains the architecture of the cells as close to native
state as possible. It stops the proteolytic enzyme induced cellular
autolysis and the process of putrefaction (cellular decay) occurring
due to the loss of nutrient supply to the cells. The choice of fixative
used may need optimization for each type of antigen-antibody
combination as there may be damage of some of the antigenic
sites due to certain type of fixative (see Notes 5 and 6).
3.2.1 Cell Fixation Paraformaldehyde (polymerized formaldehyde, PFA) is the most

for Membrane-Associated commonly used fixative while staining membrane-associated pro-
Antigens teins. Being a cross linking fixative, Paraformaldehyde, causes
chemical crosslinking of free amino groups, resulting into proteins
interactions that preserve cellular architecture. PFA is fixative of
choice while dealing with 3D cultures.
For fixation, freshly prepared 4% Paraformaldehyde in warm 1X
PBS solution is used, to cover cells to a depth of 2–3 mm and
incubated for 15–20 min at room temperature. The fixative is
removed by three washes with 1 PBS for 5 min each (see Note 7).
3.2.2 Cell Fixation Methanol is an example of precipitating fixative. Such fixatives act as
for Intracellular Antigens strong dehydrating agents and precipitate the cellular proteins.
While such fixatives are efficient in maintaining the architecture of
cell, they tend to remove small soluble molecules and lipids from
the cell.
For fixation of cells with methanol, cells are treated with chilled
100% methanol and incubated at 20 C for 15–20 min. The
methanol is removed by washing three times with 1 PBS for
5 min each.
This fixative is used mostly to stain cytoskeletal proteins or
epitopes buried within the internal protein structure as it disturbs
hydrophobic bonds of proteins. Its inherent nature to reduce pro-
tein solubility limits its use for lipid-associated proteins and proteins
localized to the nucleus and mitochondria.
3.3 Permeabilization Typically, antibody molecules are very large and ionic to penetrate
of Cell Membrane the cell membrane and interact with intracellular proteins. Permea-
bilization allows the antibodies to penetrate the fixed cell by dis-
turbing the cell membrane and subsequently interact with
intracellular antigen. Therefore, while performing IF for markers
on cell surface, the permeabilization step is not recommended (see
Note 8).
The results of immunofluorescence assay vary, depending upon
the type and concentration of permeabilizer used and incubation
time given to permeabilizer to act upon the sample. For example,
most commonly used permeabilizer, Triton X-100 has been used at
1% in PBS for 1–5 min or at 0.1–0.4% in PBS for 10–20 min. This
concludes that for each IF study, the protocol needs to be opti-
mized in such a way to develop good immunofluorescence with
minimal distortion of cell morphology. Some of the permeabilizers
are enlisted with their commonly used working concentration in
Table 2.
3.4 Blocking The non-specific interaction of primary or secondary antibodies

of Non-specific Sites with the sample leads to false results in IF assays. These
for Antibody Binding non-specific interactions occur due to trapping of antibody in
hydrophobic structure in cell or by cross reactive binding of
Table 2
Commonly used permeabilizers, their working concentrations and preferred uses
Working concentration (Needs

Permeabilizer optimization for each IF assay) Use as permeabilizer
Triton X 100 0.1–0.4% in PBS for 15–20 min Non-selective detergent that can extract proteins
along with lipids to permeabilize cellular bilayers
including the nuclear membrane. Therefore,
preferred for staining intracellular and intranuclear
antigens
Tween-20 0.1–0.5% in PBS for 15–20 min Similar to Triton X 100 with milder activity.
Saponin/ 0.1–0.5% in PBS for 5–7 min Reversible nature of action as mainly solubilizes
Digitonin cholesterol from cell bilayer. Maintains the
integrity of protein surface antigens
Cannot permeabilize the nuclear membrane.
Common alternative for cells sensitive to triton
X-100
Preferred for staining target antigen located at cell
membrane
polyclonal antibodies to non-target antigenic sites. The specificity

of IF staining is enhanced by blocking these non-specific interac-
tions. Treatment with blocking agent prior to incubation with
primary antibodies prevents the non-specific bindings.
The fixed and permeabilized cells are incubated with blocking
agent such as serum, bovine serum albumin (BSA), skimmed milk,
and gelatine. Generally, 5% serum or 1–5% BSA in 1 PBS with
0.05% Tween-20 or triton X-100 is incubated with the sample for
1 h (see Note 9).
After blocking, the extra blocking buffer is removed by washing
three times with PBS with 0.05% Tween 20 or Triton-X 100 con-
taining washing buffer.
3.5 Incubation After blocking, the sample is ready for incubation with the primary
with Primary Antibody antibodies. The primary antibody is diluted as per prior optimized
dilution in antibody dilution buffer and allowed to incubate with
the sample. The dilution of primary antibody used depends on
abundance of target antigen in the sample, concentration of anti-
body stock, and affinity of antibody to target antigen (see Note 10).
The incubation of primary antibody with the sample can be
done either in two ways, i.e., an hour at room temperature or
overnight at 4 C. To remove unbound antibodies, washing is
done 3–5 times for 5 min each with PBS containing either 0.05%
Tween-20 or 0.05% Triton X-100. Care should be taken so that the
sample cells do not get dried up between different steps.
In case of direct immunofluorescence assay, incubation of pri-
mary antibody (fluorophore conjugated) with the sample is directly
followed by mounting and imaging. In case of indirect immunoflu-

orescence assay, before mounting and imaging, sample is incubated
with fluorophore conjugated secondary (anti-species) antibodies.
3.6 Incubation In indirect IF, fluorophore conjugated secondary antibody is

with Secondary diluted to a pre-optimized dilution using the antibody dilution
Antibody buffer and incubated with the sample (see Note 11). Like primary
antibody, the secondary antibody incubation is done either for 1 h
at room temperature or overnight at 4 C. To remove excess
unbound secondary antibodies, the sample is washed thrice with
PBS containing 0.05% Tween 20 or Triton X-100.
3.7 Counterstaining Counterstaining (for cell nuclei or cytoskeleton) is perforemend to

and Mounting visualize the cell with respect to its location and morphology. After
incubation with secondary antibody, the sample is equilibrated with
PBS and cell nuclei are counterstained with DAPI (40 ,6-diamidino-
2-phenylindole). The DAPI stock solution is diluted to 300 nM in
PBS. This diluted DAPI staining solution is added to the sample, so
that the cells are completely covered. Incubation is done for
1–5 min. Sample is then rinsed several times with PBS. Excess
buffer is drained from the coverslip followed by mounting (see
Note 12).
For mounting, a few small drops of mounting medium (50%
glycerol in PBS), just sufficient to make a thin film over sample, is
added prior to gently covering the sample with a coverslip (see Note
13). The sample is kept on a flat surface for drying and the edges of
the coverslips can be sealed with nail polish. After mounting, in
order to preserve the fluorescence signal, the sample needs to be
stored at 20 C until imaging.
3.8 Imaging After IF staining, the sample is visualized by a fluorescence micro-

scope or confocal microscope, depending on the requirement. If
only expression of certain protein needs to be confirmed, a regular
fluorescent microscope is generally used however for co-localizing
proteins inside the cell, a confocal microscope becomes the instru-
ment of choice (Fig. 2). Confocal microscope eliminates outoffocus
light and thus captures high optical resolution images in the confo-
cal planes. These images of different confocal planes of any sample
can be integrated together to give rise to full three-dimensional
picture of any sample [14].
3.9 Post-imaging Like other biochemical techniques, IF staining results may be

Analysis and Issues affected by many factors. The major issues which are generally
Encountered in IF encountered are listed in the following.
Staining
Fig. 2 Confocal Image showing Immunofluorescence assay: Hela cells expressing a recombinant protein were
immunostained and confocal imaging was done to localize the recombinant protein inside the cell. Cells grown
for 24 h on coverslip kept inside the well of sterile tissue culture plate. Cells fixed with 4% paraformaldehyde
for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, blocked with 5% goat serum for 1 h at room
temperature. As primary antibody, cells were incubated for 1 h with polyclonal sera raised in rabbit and finally
incubated with Goat anti-rabbit IgG conjugated with FITC. Cells were counterstained with DAPI and examined
under confocal microscope
3.9.1 Specificity In order to analyze specific signals in immunofluorescence staining,

Validation of Antibodies we need to validate specific binding of the primary and secondary
antibody. For this, it is essential to include appropriate controls in
the assay (see Note 14). In addition, issues regarding antibody
specificity may be resolved by optimizing the process of fixation
and blocking or by using different specific antibodies having high
affinity to the target antigen.
3.9.2 Photobleaching During the IF imaging, fluorophore conjugated to the bound

secondary antibody gets excited and a light of a specific wavelength
is emitted which is detected by the detector of the fluorescent
microscope. This also results in the generation of reactive oxygen
species (ROS). These ROS interact chemically with the fluorophore
and cause impediments in its optimal excitation over time. There-
fore, after light exposure, the quality of the image is deteriorated
overtime, giving a bleached appearance. This phenomenon is
known as photobleaching. Photobleaching is prevented by opti-
mizing the exposure time by the excitation light or by using mount-
ing medium containing ROS scavengers (see Note 13). Another
approach is using more robust fluorophores, which are less prone to
photo bleaching (e.g., Alexa Fluors, DyLight Fluors, and Seta
Fluors).
3.9.3 Autofluorescence Biological samples contain coenzymes that are important in regu-
lating cellular metabolic activities. Some of them may exhibit auto-
fluorescence such as reduced NADH (Absorption wavelength:
340 nm, emission: 460 nm) and flavin coenzymes like FMN (flavin
mononucleotide) and FAD (flavin adenine dinucleotide)
(absorption wavelength: 450 nm, emission: 460 nm). Therefore,

sometimes during detection of fluorophores which emit light in the
green spectrum, low signal-to-noise ratio is observed. This issue
can be resolved by using a different specific antibody having higher-
affinity to antigen.
Fixation methods using aldehydes such as glutaraldehyde may
also result in high autofluorescence. Quenching the fixation agent
to eliminate any free aldehyde groups which could non-specifically
bind antibody, reduces the autofluorescence (see Note 15).
3.9.4 High Background Due to inefficient blocking or too high concentration of primary
Fluorescence antibody used or insufficient washing out of fixative, sometimes
there occurs the problem of high background fluorescence. To
resolve this issue, we can take following measures:
1. Increase the percentage of blocking agents used in the blocking
buffer as well as in antibody dilution buffers. Increase the time
for blocking step and/or reducing the time given for antibody
incubations.
2. Increasing the dilution of primary and/or secondary
antibodies used.
3. Proper washing and quenching of free aldehyde in case of
aldehyde fixatives used (see Note 15).
3.9.5 Fluorophore This problem is encountered when multiple target antigens are
Overlap tagged using different fluorophores having emitted light in similar
spectral wavelengths (see Note 11). For example, if Alexa Fluor
430 (excitation wavelength: 434 nm, emission wavelengths:
539 nm) and Alexa Fluor 514 (excitation wavelength: 518 nm,
emission wavelength: 540 nm) are used to stain different antigen in
same sample, the detector of fluorescence microscope will not be
able to distinguish the light emitted from these two fluorophores
due to a significant overlap of their emission wavelengths. Alterna-
tively, if Alexa Fluor 430 is used along with another fluorophore
such as Alexa Fluor 594 (excitation wavelength: 590 and emission
wavelength: 617 nm), there will be no overlap of the emitted light
and the proteins stained with this combination of dyes will be
optically differentiated.
4 Notes
There are many tips to follow and alternative methods to perform

IF staining in order to achieve better results. Some of them are
listed in the following:
1. The thickness of coverslip plays a key role in the quality and

intensity of the image. Generally, coverslips having thickness of
approximately 170 μm are found compatible with most
microscopes.
2. As a crude method for sterilization, coverslip can be dipped in
70% ethanol and kept in the flame using forceps for approxi-
mately 15–20 s and directly put inside the well of sterile tissue
culture plate. Use of coverslip reduces the volume of primary as
well as secondary antibody during incubation, but the fragile
nature of coverslip requires extra care in handling during the
wash steps.
3. For cells having less adherent strength, the coverslip may be
pre-treated with 50 μg/mL L-poly-lysine to aid cell
attachment.
4. Unmasking of the antigen is done by heat treatment preferably
or by saponin/pepsin treatment. Before the advent of antigen
retrieval methods involving heating, techniques to improve
immunostaining included treating sections at room tempera-
ture with 5 M urea [15] or with detergents [16]. Some of the
substances that have been included in solutions for heat
induced antigen retrieval are 1% Zinc sulfate, 1% lead thiocya-
nate [17], 0.1 M Citrate buffer, pH 6 [18], 0.01 M EDTA,
pH 8 [19], and Tris buffer, pH 9 [20].
5. Commonly used fixatives other than paraformaldehyde and
methanol are as follows:
(a) Formalin (10%)
A 3.7% Paraformaldehyde solution is equivalent to a
10% formalin solution. Cells can also be fixed with 10%
formalin solution for 10–20 min at room temperature
followed by three washes with 1 PBS for 5 min each.
(b) Acetone
Acetone is also a precipitating fixative but it is milder
than methanol. For acetone fixation, chilled acetone is
used to cover the sample in a thin layer followed by
incubation at 20 C for 5–20 min, depending upon
the antigen of interest. Acetone maintains antigenic integ-
rity as compared to methanol. It may be used when meth-
anol fixation is ineffective. Acetone is also used for
cytoskeletal proteins. Acetone is very good at cell permea-
bilization but shares the same limitations as that of meth-
anol fixation.
In some cases where neither methanol nor acetone
alone is effective, 1:1 ratio of methanol and acetone is
used (10 min incubation at 20 C).
Precipitating fixatives generally denature overex-
pressed fluorescent proteins (e.g., Green fluorescent
Table 3
Fixatives: Major advantages and disadvantages
Fixatives Advantages Disadvantages

Formaldehyde Universal fixative Sometimes it may reduce the signals due to
Preserves cellular morphology excessive cross linking
Good for staining membrane
proteins
Methanol Choice of fixative for aldehyde Highly volatile and flammable
sensitive epitopes Not suitable for overexpressed fluorescent protein
No requirement of additional Reduces solubility of proteins
permeabilization
Acetone Choice of fixative for aldehyde Highly volatile and flammable
sensitive epitopes Not suitable for overexpressed fluorescent protein
No requirement of additional Reduces solubility of proteins
permeabilization
Milder than methanol
protein, GFP). Therefore, they are not recommended to

fix cells with such proteins.
The advantages and disadvantages of mentioned fixa-
tives are compiled in Table 3.
6. Cell fixation using Paraformaldehyde may sometimes leads to
auto-fluorescence-mediated artefacts and therefore it is cru-
cial to have a control sample that skips the step of sample
incubation with the primary antibody to differentiate any
non-specific background signal.
7. Unlike precipitating fixatives such as methanol and acetone,
aldehyde-based fixatives cannot permeabilize the cell mem-
brane effectively and the samples need to be permeabilized
before proceeding to staining of intracellular biological
molecules.
8. Different permeabilizing agents in increasing order of permea-
bilizing efficiency are saponins, Tween-20, Triton X-100, and
sodium dodecyl sulfate (SDS). Fixatives such as acetone and
methanol also perform permeabilization while other fixatives
are not very efficient permeabilizers. The permeabilization step
can be skipped if the antigen of interest is located on the cell
membrane or if acetone is used as fixative. To achieve good
signal with minimal cell distortion, it is advised to use the
mildest detergent possible that allows antibody penetration
into the cell. Milder detergents such as saponin, Tween™
20, or digitonin at 0.1–0.5% (v/v) for 5–10 min can be used
if Triton X-100 or NP-40 do not give good visualization of the
target.
9. When using serum as blocking agent, its source should be

different animal species than that of primary antibody. In case
of indirect IF, the blocking serum should be the same animal
species in which secondary antibody was raised. For example, if
secondary antibody raised in goat is being used, then blocking
is done with 5% normal goat serum. Various commercially
available proprietary protein-free compounds or highly purified
single proteins may also be used as blocking reagents in IF
assays.
10. In multicolor immunofluorescence assay, for staining multiple
target proteins, their respective primary antibodies can be
combined, provided they are derived from different animal
species. This combination is then allowed to incubate with
the sample. Alternatively, the primary antibodies can be used
sequentially. In order to avoid chances of non-specific binding,
the source of the primary antibody should preferably be a
species distinct from the species being studied.
11. In immunostaining for multiple target antigens, the secondary
antibody corresponding to each primary antibody needs to be
conjugated with distinct fluorophore having non-overlapping
emission wavelength spectrums.
12. Some mounting media also contains counterstain DAPI to
visualize nuclei. DAPI interacts with DNA, emitting light in
the blue spectrum. When using such mounting media, coun-
terstaining step is skipped and after secondary antibody incu-
bation, directly mounting step can be performed.
13. The function of 50% glycerol in PBS (mounting medium) is to
preserve the sample and to increase the refractive index for
obtaining high-quality images using an oil immersion lens.
Some commercially available mounting media, such as Fluor-
omount-G(anti-fade) from Southern Biotech media and Pro-
long Gold from Molecular Probes also help in minimizing
photobleaching caused by free reactive oxygen species
(ROS) scavengers.
14. For validating the specificity of antibodies used, appropriate
controls should be included to the IF assay.
Specificity of primary antibody can be tested by the
following:
(a) Blocking primary antibody with its complementary anti-
gen and then performing IF staining using this blocked
primary antibody.
(b) Another way is to produce a sample with targeted deletion
or RNAi mediated silencing of the antigen of interest and
performing IF staining using this sample as control.
In both cases, specificity of the primary antibody can be

validated by the lack of signal on IF staining.
Specificity of secondary antibody can be tested by
confirming the lack of signal in a sample incubated only
with the secondary antibody and not incubated with
the primary antibody.
15. For quenching/attenuation formaldehyde, washing with
0.1 M Tris or Glycine buffer and for quenching glutaraldehyde
washing with 0.1% sodium borohydride in PBS prior to anti-
body incubation is done.
5 Conclusion
In conclusion, immunofluorescence technique is very useful tool to

detect protein expression and localize cellular antigen. IF technique
detects the target optically using fluorophore-conjugated antibo-
dies. The IF assay has been very useful in rapid identification of viral
antigens in the infected cells. Various modifications in the tech-
nique have been adopted to develop multiplex immunofluores-
cence staining systems. The Tyramide signal amplification–avidin–
biotin complex (TSA-ABC) method of IF staining is one such
example [21]. Such continuous modifications indicate that the IF
technique will become more adapted and useful tool for laboratory
investigations and identification of pathogenic agents.
References
1. Burnette WN (1981) “Western blotting”: elec- 6. Coons AH, Creech HJ, Jones RN, Berliner E
trophoretic transfer of proteins from sodium (1942) The demonstration of pneumococcal
dodecyl sulfate—polyacrylamide gels to antigen in tissues by the use of fluorescent
unmodified nitrocellulose and radiographic antibody. J Immunol 45(3):159–170
detection with antibody and radio-iodinated 7. Cherrywbmoody MD (1965) Fluorescent-
protein A. Anal Biochem 112(2):195–203 antibody techniques in diagnostic bacteriology.
2. Engvall E, Perlmann P (1972) Enzyme-linked Bacteriol Rev 29:222–250
immunosorbent assay, Elisa. 3. Quantitation of 8. Hers JF (1963) Fluorescent antibody tech-
specific antibodies by enzyme-labeled anti- nique in respiratory viral diseases. Am Rev
immunoglobulin in antigen-coated tubes. J Respir Dis 88(SUPPL):316–338
Immunol 109(1):129–135 9. Kraft SC, Kirsher JB (1964) Immunofluores-
3. Liu JJ, Liu C, He W (2013) Fluorophores and cent studies of chronic nonspecific ulcerative
their applications as molecular probes in living colitis. Gastroenterology 46:329–332
cells. Curr Org Chem 17(6):564–579 10. Bourgeois M, Oaks J (2014) Laboratory diag-
4. Joshi S, Dihua Y (2017) nosis of viral infections. In: Sellon DC, Long
Immunofluorescence. In: Jalali M, MT (eds) Equine infectious diseases, 2nd edn.
Saldanha F, Jalali M (eds) Basic science meth- W.B. Saunders, Philadelphia, Pennsylvania
ods for clinical researchers, 1st edn. Academic 11. Liu C (1956) Rapid diagnosis of human influ-
Press, Cambridge, Massachusetts enza. Proc Soc Exp Biol Med 92:883–887
5. Coons AH, Creech HJ, Jones RN (1941) 12. Gardner PS, McQuillin J (1980) Rapid virus
Immunological properties of an antibody con- diagnosis. In: Application of immunofluores-
taining a fluorescent group. Proc Soc Exp Biol cence, 2nd edn. Butterworths, London
Med 47(2):200–202
13. Clark Brelje T, Wessendorf MW, Sorenson RL oven heating of tissue sections. J Histochem
(1993) Multicolor laser scanning confocal Cytochem 39:741–748
immunofluorescence microscopy. In: Matsu- 18. Shi SR, Chaiwun B, Cote RJ, Taylor CR
moto B (ed) Methods in cell biology practical (1993) Antigen retrieval technique utilizing
application and limitations, vol 38. Academic citrate buffer or urea solution for immunocy-
Press, Cambridge, pp 97–181 tochemical demonstration of androgen recep-
14. Kogata N, Howard BA (2013) A whole-mount tor in formalin-fixed paraffin sections. J
immunofluorescence protocol for three- Histochem Cytochem 41:1599–1604
dimensional imaging of the embryonic mam- 19. Gown AM, Willingham MC (2002) Improved
mary primordium. J Mammary Gland Biol detection of apoptotic cells in archival paraffin
Neoplasia 18(2):227–231 sections: immunohistochemistry using antibo-
15. Hausen P, Dreyer C (1982) Urea reactivates dies to cleaved caspase 3. J Histochem Cyto-
antigens in paraffin sections for immunofluo- chem 50:449–454
rescent staining. Stain Technol 57:321–324 20. Koopal SA, Coma MI, Tiebosch ATMG, Suur-
16. Feldmann G, Maurice M, Bernuau D, meijer AJH (1998) Low-temperature heating
Rogier E, Durand AM (1983) Penetration of overnight in tris-HCl buffer pH 9 is a good
enzyme-labelled antibodies into tissues and alternative for antigen retrieval in formalin-
cells: a review of the difficulties. In: fixed paraffin-embedded tissue. Appl Immuno-
Avrameas S, Druet P, Masseyeff R, Feldmann histochem 6:228–233
G (eds) Immunoenzymatic Techniques. Else- 21. Toda Y, Kono K, Abiru H, Kokuryo K,
vier, Amsterdam, pp 3–15 Endo M, Yaegashi H, Fukumoto M (1999)
17. Shi SR, Key ME, Kalra KL (1991) Antigen Application of tyramide signal amplification
retrieval in formalin-fixed, paraffin-embedded system to immunohistochemistry: a potent
tissue: an enhancement method for immuno- method to localize antigens that are not detect-
histochemical staining based on microwave able by ordinary method. Pathol Int 49(5):
479–483
Chapter 16
Polymerase Spiral Reaction (PSR) for the Diagnosis

of Porcine Viral Diseases
Vikas Gupta, Nihar Nalini Mohanty, and Vinod Kumar Singh
Abstract
Polymerase spiral reaction is a novel and emerging isothermal nucleic acid-based amplification assay for the
detection and diagnosis of microbial pathogens of both veterinary and medical importance. The assay is
very cost effective as it does not require any sophisticate equipment or laboratory facilities. The test can be
completed within 1 h by employing one set of specifically designed primers unique to target, MgSO4,
Betaine, dNTP mix, Bst enzyme, and isothermal buffer in a single tube by incubating in water bath or heat
block at constant temperature (isothermal). Amplification of target could be detected by agarose gel
electrophoresis, real-time measuring of increase in turbidity in turbidimeter or by naked eye visualization
of change in fluorescence of colorimetric dyes such as calcein, hydroxynaphthol blue, SYBR green-I or Eva
green. The assay is an amalgamation of isothermal assay and convention polymerase chain reaction and has
immense potential to detect the pathogens of animals including swine in a simple, rapid, sensitivity, and
specific manner.
Key words Diagnosis, Isothermal, Simple, Sophisticated equipment-free, Naked eye visualization
1 Introduction
Pig production is an important source of global economy and food

security. Pigs are susceptible to various types of infectious diseases
and among them viral diseases such as African swine fever, Porcine
reproductive and respiratory syndrome, Classical swine fever, Pseu-
dorabies, Porcine epidemic diarrhea, Porcine multisystemic wasting
syndrome impose constant threat to pig population in the form of
heavy morbidity, mortality, restriction in local and international
trade leading to massive economical loss and lack of food security.
Rapid and accurate diagnosis of infectious diseases play crucial role
in the prevention and control of disease. Conventional diagnosis of
infectious agents such as agent identification, antigen detection,
biochemical identification, and serology are reliable and confirma-
tory but these approaches are time consuming and require sophis-
ticated laboratory setups. “ASSURED” (Affordable, Sensitive,
231
232 Vikas Gupta et al.
Specific, User-friendly, Rapid and robust, Equipment-free, and

Delivered) concept of disease diagnosis is the main focus of research
in developing countries for timely diagnosis of diseases
[1]. Sequence-based amplification of specific genes has many appli-
cations in molecular biology research as well as veterinary and
medical diagnostics. At present, for amplifying target sequence,
various kinds of polymerase chain reactions (PCR) and isothermal
amplification are used. The PCR requires sophisticated instrument
for amplifying the target sequence by following chain of three cyclic
thermal conditions followed by visualization of products by agarose
gel electrophoresis while isothermal amplification assays do not
requires any specialized instruments to amplify target sequence at
specific temperature. Various types of isothermal reaction such as
self-sustained sequence replication reaction (3SR) [2], nucleic acid
sequence-based amplification (NASBA) [3], strand displacement
amplification (SDA) [4], rolling circle replication (RCR) [5],
loop-mediated isothermal amplification (LAMP) [6], helicase-
dependent amplification (HDA) [7] and single primer isothermal
amplification (SPIA) [8], cross-priming amplification (CPA) [9],
transcription-based amplification system (TAS) [10], and polymer-
ase spiral reaction (PSR) [11] have been employed for amplification
and detection of genomes of infectious micro-organism in the last
three decades. Most of these mentioned assays such as TAS, 3SR,
NASBA, SDA, HAD, and SPIA require three or more enzymes and
rigorous optimization. Only a few of these isothermal amplification
methods (e.g., RCR, LAMP, CPA, and PSR) can efficiently amplify
the target sequence by using single enzyme at a constant tempera-
ture. However, RCR method can only amplify circular DNA while
initial denaturation step is required for the satisfactory result in
LAMP assay. Polymerase spiral reaction (PSR) technique was
invented by Liu et al. [11] for the detection of antibiotic resistance
gene blaNDM-1 in E. coli pGEX-NDM-BL21 plasmid. This tech-
nique is the amalgamation of isothermal amplification techniques,
namely RCR and 3SR, and conventional PCR in which only one
pair of primers is needed. In contrast to RPA and HDA which need
multiple enzymes, the PSR assay requires DNA polymerase enzyme
with strand displacement activity (Bst DNA polymerase) showing
the similarity with LAMP technique for amplification of target
sequence at a constant incubation temperature (60–66 C). The
product can be visualized by DNA binding intercalating dye or with
the aid of gel electrophoresis showing specific ladder pattern of
amplicons. PSR, when employed for detection and identification
of pathogens of both medical and veterinary importance, the assay
accurately and efficiently detected both RNA and DNA virus such
as Porcine epidemic diarrhea virus [12], Canine provirus-2 [13],
Bovine herpes virus-1 [14], Hepatitis C virus [15], Porcine circo-
virus type-3 [16] from clinical samples. The assay has also been
evaluated for diagnosis of pathogens of zoonotic and public health
Polymerase Spiral Reaction (PSR) for the Diagnosis of Porcine Viral Diseases 233
importance, namely Brucella sp. [17], M. tuberculosis [11], E. coli

[11], Salmonella sp [18, 19], Mycoplasma synoviae [20]; Candida
albicans [21], Pseudomonas aeruginosa [22], and Vibrio parahae-
molyticus ([23]). Also, [24] developed polymerase cross link spiral
reaction (a modification of PSR) for detection of African swine fever
virus (ASFV) from blood of infected pigs and wild boars.
2 Materials Required for PSR
1. Isothermal DNA polymerase having strand displacement

activity e.g. Bst DNA polymerase large fragments (8 U/μl,
New England Biolab); Bst 2.0 WarmStar (New England Bio-
lab); GspssD DNA polymerase large fragment (8 U/μl, Opti-
gen west Sussex, UK) or Bsm DNA polymerase large fragment
(8 U/μl, Thermo Fisher Scientific) (see Note 1).
2. Isothermal reaction buffer e.g. 10 thermopol (200 mM
Tris–HCl, 100 mM KCl, 100 mM (NH4)2SO4, 20 mM
MgSO4, 1% Tween 20); 10 Bsm buffer (200 mM Tris–HCl,
100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1%
Tween 20,) or 10 Gsp buffer.
3. Magnesium sulfate (100 mM): Generally Magnesium sul-
phate is required in the range of 2 mM to 10 mM and it is
most important factor, affecting the annealing temperature of
primers and optimum activity of isothermal DNA polymerase.
4. dNTPs (10 mM): Generally required in the range of
0.4–1.6 mM.
5. Betaine (N,N,N-trimethylglycine) (5 M): It is required for
unwinding of duplex form of DNA and generally required in
the range of 0.4–1.5 M.
6. Polymerase spiral reaction primers: Specifically designed sin-
gle pair of primer generally required in the range of
20–40 pmole.
7. Water bath or heat block: All the isothermal DNA poly-
merases enzymes have their optimum activity in the range of
60–65 C.
8. Visualization device and dyes:
(a) Gel electrophoresis apparatus and accessories: Ampli-
fied product form ladder pattern in gel but generally not
recommended to visualize the amplified product due to
significant risk of carry over contamination.
(b) Real-time fluorimeter (qPCR machine) or turibidi-
meter for real-time monitoring.
(c) pH indicator dyes: Combination of 0.025 mM phenol red

and 0.08 mM cresol red dyes that become yellow from
purple-red.
(d) SYTO®-9 for real-time fluorescence.
(e) SYBR® Green 1, Calcein, hydroxynaphthol blue, mala-
chite green or Eva green for endpoint analysis.
3 Methods
3.1 Primer Designing Generally PSR technique requires one pair of special primer
(Table 1) [11, 13–17] and it could be designed by using any PCR
primer designing software such as Primer 5, Oligo v7.37, DNA-
MAN, etc. The software is used to design a pair of specific primer
for target sequence. The exogenous sequences (N and Nr) are
added to 50 end of forward and reverse primers. The exogenous
sequences must be taken from non-relative source such as from
plant to avoid any nonspecific amplification. Both the exogenous
sequences should have the same set of nucleotides but in the reverse
order and the melting temperature of exogenous sequence should
fall 5 C lower than the PCR primer sequences in order to ensure
annealing of complementary forward and reverse sequence to tar-
get region occurred earlier than the formation of spiral structure
[11]. Further, some time auxiliary/accelerated primers are also
used in PSR to enhance the reaction velocity [19, 21, 23].
Step to design PSR primer:
1. Step-1: Design a pair of common PCR primer (F and B) for
target sequence with product length of around 150–160 bp
following the guideline of PCR primer design.
2. Step-2: Design a single primer (exogenous sequence N) from
non-relative source following the guidelines of PCR primer
design but keep in mind the melting temperature of this primer
falls 5 C lower than the target primer. Nr sequence is the
reverse of exogenous sequence N.
3. Step-3: Add the N and Nr sequences to the F and B respec-
tively at 50 end to design PSR primers Ft and Bt.
4. Step-4: Check the specificity of the PSR primers (Ft and Bt) by
primer-blast analysis of NCBI sequence database to avoid the
nonspecific reaction.
3.2 Procedure 1. Setting up of reaction mixture for PSR amplification: Pre-

pare an initial reaction mixture composition for setting up the
amplification run of PSR is as follows (Table 2) (see Notes 2,
3, 4 and 5).
Table 1
Example of primers set for polymerase spiral reaction
Forward primer for target(F) 5-GAACTAGTGGCACACCAACA-3

Reverse primer for target(B) 5-TGGTAAGCCCAATGCTCTATTT-3
Exogenous sequence (N) 50 -acgattcgtacatagaagtatag-30
Exogenous sequence (Nr) 50 -gatatgaagatacatgcttagca
(reverse of N)
PSR-forward primer (Ft) 50 -acgattcgtacatagaagtatagGAACTAGTGGCACACCAACA-30
PSR-reverse primer (Bt) 50 -gatatgaagatacatgcttagcaTGGTAAGCCCAATGCTCTATTT-30
Table 2
Initial reaction set up for polymerase spiral reaction
Sl. No. Reagents Volume (25 μl)

1 10 isothermal reaction buffer 2.5 μl (1)
2 100 mM MgSO4 1.5 μl (6 mM)
3 10 mM dNTPmix 3.5 μl (1.4 mM)
4 5 M Betain 4 μl (8 M)
5 40 pmole forward PSR primer 1 μl
6 40 pmole reverse PSR primer 1 μl
7 Bst 2.0 large fragment (8000 U/ml) 1 μl (8 U/μl)
7 DNA template 1 μl
8 Nuclease free water 9.5
2. Optimization of reaction composition and conditions: Var-

ious concentration combinations of MgSO4, Betaine, Bst
enzyme, and different incubation temperature and time could
be used to get optimum reaction mixture composition and
temperature and time for the rapid and specific amplification
(see Note 6).
3. Visualization of isothermal amplified product
(a) Visualization of isothermal amplified product (LAMP,
PSR, and CPA) can be done by agarose gel electrophoresis
or by using colorimetric dyes (see Note 7).
(b) In agarose gel electrophoresis (2.5 to 3%), amplified
product after staining with ethidium bromide or other
commercial dyes shows specific ladder pattern on excita-
tion with UV light on UV transilluminator at 302 nm.
(c) Monitoring of turbidity: Pyrophosphate released during
amplification process form a complex with divalent ion
Table 3
Colorimetric dyes used for detection amplification on visual basis
Type of dye Concentration Change in fluorescence

Hydroxynaphthol 1 μl of 0.2% solution Dye changes its color from violet to sky blue
blue
Calcein/MnCl At a final concentration of 0.5 mM Orange color of calcein changes to yellow
MnCl and 0.05 mM calcein green on excitation either with visible or UV
light
pH indicator dye 0.025 mM phenol red and Become yellow from purple-red
0.08 mM cresol red dyes at the
rate of 1 μl
SYBR green, At a final concentration of 1 Fluoresce strongly on binding with double
Evagreen stranded DNA
SYBR green dye is added after end point of
reaction
Evagreen can be used while setting reaction
mix
such calcium, magnesium, and manganese leading to

increase in turbidity of the reaction mixture. Increase in
turbidity could be recorded in real time in the form of OD
at 400 nm at every 6 s by using turbidimeter. Turbidity
can also be visualized by naked eyes as pellet on
centrifugation.
4. Visual fluorescence: Colorimetric dyes such as hydroxy-
naphthol blue (HNB), calcein, SYBR green, Evagreen, and
combination of 0.025 mM phenol red and 0.08 mM cresol
red dyes can be employed for visualization of amplified product
via naked eyes or with the help of UV light. Various colorimet-
ric dyes, their concentration and change in fluorescence in
presence of amplified product are described in Table 3.
3.3 Determination 1. Specificity of PSR could be determined by employing naturally

of Specificity of PSR occurring restriction site within the original target sequence
and digestion of the amplified sequence with restriction
enzyme [11, 13].
2. Excised band from the gel after purification could also be
sequenced for the determining authenticity of the amplified
product.
3. Restriction enzyme site may be artificially incorporated in vari-
able exogenous sequences (N & Nr) to design PSR primers
having restriction cutting site [11, 12]. After the completion of
amplification reaction, the product can be digested with
corresponding restriction enzyme and digested product can
also be sequenced after purification.
4 Notes
1. dUTP and Thermolabile Uracil DNA Glycosylase for carryover

contamination prevention (Bst 2.0 is strongly recommended
for use with dUTP/UDG systems).
2. Before starting to set the reaction mixture for PSR, keep all the
reagents on ice.
3. Do not set the reaction in room used for visualization of PSR
product by agarose gel electrophoresis or any other proce-
dure that needs to open the reaction vessel, to avoid possible
carry over contamination.
4. For organism having RNA as genomic material, reverse tran-
scriptase enzyme for synthesis of cDNA and then use it as a
template for setting the reaction.
5. A layer of mineral oil (~25 μl) could be used to prevent the
volatilization of PSR products and subsequently to avoid the
possibility of aerosol formation.
6. It is strongly recommended to run a non-template control to
ensure amplification specificity and to avoid the possibility of
carry over contamination.
7. Gel electrophoresis and other post-amplification detection sys-
tem which requires opening of reaction tubes are not recom-
mended due to significant risk of carry over contamination
leading to false positive results in subsequent reaction.
References
1. Mabey D, Peeling RW et al (2004) Diagnostics 7. Vincent M et al (2004) Helicase-dependent

for the developing world. Nat Rev Microbiol 2: isothermal DNA amplification. EMBO Rep 5:
231–240 795–800
2. Fahy E, Kwoh DY et al (1991) Self-sustained 8. Kurn N et al (2005) Novel isothermal, linear
sequence replication (3SR): an isothermal nucleic acid amplification systems for highly
transcription-based amplification system alter- multiplexed applications. Clin Chem 51:
native to PCR. PCR Methods Appl 1:25–33 1973–1981
3. Compton J (1991) Nucleic acid sequence- 9. Fang R et al (2009) Cross-priming amplifica-
based amplification. Nature 350:91–92 tion for rapid detection of mycobacterium
4. Walker GT et al (1992) Strand displacement tuberculosis in sputum specimens. J Clin Micro-
amplification—an isothermal, in vitro DNA biol 47:845–847
amplification technique. Nucleic Acids Res 10. Kwoh DY et al (1989) Transcription-based
20:1691–1696 amplification system and detection of amplified
5. Banér J, Nilsson M, Mendel-Hartvig M et al human immunodeficiency virus type 1 with a
(1998) Signal amplification of padlock probes bead-based sandwich hybridization format.
by rolling circle replication. Nucleic Acids Res Proc Natl Acad Sci U S A 86:1173–1177
26:5073–5078 11. Liu W, Dong D, Yang Z et al (2015) Polymer-
6. Notomi T et al (2000) Loop-mediated isother- ase spiral reaction (PSR): a novel isothermal
mal amplification of DNA. Nucleic Acids Res nucleic acid amplification method. Sci Rep 5:
28:E63 12723. https://doi.org/10.1038/srep12723
12. Wang Y, Jiao W, Wang Y et al (2019) Simulta- 18. Momin KM, Milton AAP, Ghatak S et al
neous nucleic acids detection and elimination (2019) Development of a novel and rapid poly-
of carryover contamination with nanoparticles- merase spiral reaction (PSR) assay to detect
based biosensor- and Antarctic thermal sensi- salmonella in pork and pork products. Mol
tive UracilDNA-glycosylase-supplemented Cell Probes 50:101510. https://doi.org/10.
polymerase spiral reaction. Front Bioeng Bio- 1016/j.mcp.2020.101510
technol 7:401. https://doi.org/10.3389/ 19. Xu W, Gao J, Zheng H et al (2019) Establish-
fbioe.2019.0040 ment and application of polymerase spiral reac-
13. Gupta V, Chakravarti S, Chander V et al (2017) tion amplification for salmonella detection in
Polymerase spiral reaction (PSR): a novel, food. J Microbiol Biotechnol 29:1543–1552
visual isothermal amplification method for 20. Wu Q, Xu X, Chen Q et al (2019) Rapid and
detection of canine parvovirus 2 genomic visible detection of mycoplasma synoviae using
DNA. Arch Virol 162:1995–2001 a novel polymerase spiral reaction assay. Poult
14. Malla JA, Chakravarti S, Gupta V et al (2018) Sci 98:5355–5360
Novel polymerase spiral reaction (PSR) for 21. Jiang X, Dong D, Bian L, Zou D, He X et al
rapid visual detection of bovine herpesvirus (2016) Rapid detection of Candida albicans by
1 genomic DNA from aborted bovine fetus polymerase spiral reaction assay in clinical
and semen. Gene 644:107–112 blood samples. Front Microbiol 7:916.
15. Sun W, Du Y, Li X, Du B (2020) Rapid and https://doi.org/10.3389/fmicb.2016.00916
Sensitive Detection of Hepatitis C Virus in 22. Dong D, Zou D, Liu H, Yang Z, Huang S et al
Clinical Blood Samples Using Reverse Tran- (2015) Rapid detection of Pseudomonas aeru-
scriptase Polymerase Spiral Reaction. J Micro- ginosa targeting the toxA gene in intensive care
biol 30(3):459–468. https://doi.org/10. unit patients from Beijing, China. Front Micro-
4014/jmb.1910.10041. PMID: 31893596 biol 6:1100
16. Ji J, Xu X, Wang X et al (2019) Novel polymer- 23. He S, Jang H, Zhao C, Xu K, Wang J et al
ase spiral reaction assay for the visible molecular (2019) Rapid visualized isothermal nucleic
detection of porcine circovirus type 3. BMC acid testing of Vibrio parahaemolyticus by
Vet Res 15:322. https://doi.org/10.1186/ polymerase spiral reaction. Anal Bioanal Chem
s12917-019-2072-9 412(1):93–101. https://doi.org/10.1007/
17. Das A, Kumar B, Chakravarti S, Prakash C, s00216-019-02209-y
Singh RP et al (2018) Rapid visual isothermal 24. Woźniakowski G et al (2017) Polymerase
nucleic acid-based detection assay of Brucella cross-linking spiral reaction (PCLSR) for
species by polymerase spiral reaction. J Appl detection of African swine fever virus (ASFV)
Microbiol 125:646–654 in pigs and wild boars. Sci. Rep. 7:42903.
https://doi.org/10.1038/srep42903
Chapter 17
Recombinase Polymerase Amplification-Based Diagnostics

of Porcine Viral Diseases
Abstract
Recombinase polymerase amplification (RPA) is a highly sensitive isothermal amplification technique,
operating at 37–42 C, with minimal sample preparation and capable of amplifying as low as 1–10 target
copies in less than 20 min. The recent advent of RPA is enabling the expansion of this technology for
research and diagnostic applications worldwide. By being an affordable, simple, fast, and sensitive method
for the identification of pathogens, RPA has been adopted widely as a molecular tool in many diagnostic
platforms, and has been used to amplify a wide array of organisms and samples. RPA is undoubtedly a
promising isothermal molecular technique for the development of low-cost, rapid, point-of-care diagnostic
assay. It is being successfully used for the detection of many viral pathogens.
Key words Diagnostics, Porcine viruses, Point-of-care, Recombinase polymerase amplification
1 Introduction
In the past few decades, swine production has revolutionized from

traditional husbandry practices that involved a few pigs or small
herds to an intensive concentration of swine raised in multisite
production systems. Although this dramatic change has made the
production of pork very efficient, it has also changed the ecology of
many swine diseases, encouraging the emergence of new infections.
There are many swine viruses that have been infrequently associated
with the disease; sometimes, one or just a few animals are affected,
other times, the disease may spontaneously reach a high incidence
in the herd, and then just as quickly, it dissipates. Some of the
economically important viral pathogens of swine are shown in
Table 1. Early diagnosis of and the immediate establishment of
reliable countermeasures to infectious diseases is essential to limit
severe biophysical and socio-economic consequences. However,
the importance of the many known viral diseases of pigs varies
from country to country.
239
Table 1
Economic and zoonotic viral pathogens of swine. (Reproduced from [1]
Virusa Economicb Vaccinec Zoonoticd

AFSV ++++ No
FMDV ++++ Yes
CSFV +++ Yes
ADV ++ Yes
PRRSV ++++ Yes
IVA-S ++ Yes +++
PCV2 + Yes
PEDV + Yes
SVA + No
JEV + Yes ++
HEV + No ++
Nipah virus + No +++
EMCV + No +
Menangle virus + No +
VSV + No +
VESV + No +
a
ASFV African swine fever virus, FMDV foot and mouth disease virus, CSFV classical swine fever virus, ADV Aujeszky’s
disease virus, PRRSV porcine reproductive and respiratory syndrome virus, IVA-S influenza virus A- swine, PCV2 porcine
circovirus type 2, PEDV porcine epidemic diarrhea virus, SVA Senecavirus A, JEV Japanese encephalitis virus, HEV
Hepatitis E virus, EMCV encephalomyocarditis virus, VSV Vesicular stomatitis virus, VESV Vesicular exanthema of swine
virus
b
Economic impact ranging from + (infrequent/mild) to + + ++ (frequent/severe)
c
Vaccine available to aid in control and prevention: yes or no
d
Zoonotic potential ranging from + (infrequent/mild) to + + ++ (frequent/severe)
Polymerase chain reaction (PCR) thermal cycling methodol-

ogy, and more recently, quantitative real-time PCR (qPCR) are
often viewed as “gold standards” for molecular diagnosis of viral
pathogens. However, these techniques inherently require the use of
thermocycler and a reliable power supply, thus restricting their use
to laboratories. To address requirements of amplification for the
use in low-resource settings or at the point-of-need, isothermal
DNA amplification methods have been developed, including
nucleic acid sequence-based amplification (NASBA), strand dis-
placement amplification (SDA), rolling circle amplification
(RCA), the loop-mediated isothermal amplification (LAMP),
helicase-dependent amplification (HDA), as well as the recombi-
nase polymerase amplification (RPA).
One technology, in particular, RPA, despite its comparatively
late introduction, is experiencing rapid development and popularity
Recombinase Polymerase Amplification-Based Diagnostics of Porcine Viral Diseases 241
due to its simplified equipment requirements and fast reaction

times [2]. RPA was first introduced in 2006 and was initially
demonstrated to be a nucleic acid amplification method for DNA
[3]. Later, RNA was shown to be used as a template by the addition
of reverse transcriptase in the same reaction tube [4]. RPA is
remarkable due to its simplicity, high sensitivity, selectivity, com-
patibility with multiplexing, extremely rapid amplification, as well
as its operation at a low temperature, without the need for the use
of multiple primers or an initial denaturation step. Overall, RPA
positions itself very favorably for widespread exploitation in kits and
assays for use at the point-of-care or point-of-need.
2 RPA Mechanism
The fundamental mechanism of RPA relies on the synthetically

engineered adaptation of a key process in DNA metabolism called
homologous recombination. The standard RPA reaction reagents
comprise of recombinase, recombinase loading factor, and single-
stranded binding protein, which subsequently coordinate with
ancillary components such as DNA polymerase, crowding agent,
energy/fuel components (e.g., adenosine triphosphate, ATP) and
salt molecules to perform the RPA reaction mechanism (Table 2)
[3]. The RPA process starts when a recombinase protein UvsX from
T4-like bacteriophages bind to primers in the presence of ATP and
a crowding agent (a high molecular polyethylene glycol), forming a
recombinase-primer complex. The complex then interrogates
double-stranded DNA seeking a homologous sequence and pro-
motes strand invasion by the primer at the cognate site. In order to
prevent the ejection of the inserted primer by branch migration, the
displaced DNA strand is stabilized by single-stranded binding pro-
teins. Finally, the recombinase disassembles and a strand displacing
DNA polymerase binds to the 30 end of the primer to elongate it in
the presence of dNTPs. The cyclic repetition of this process results
in the achievement of exponential amplification (Fig. 1).
3 RPA Operating Parameters
3.1 Primers The length of RPA primers is relatively long (typically between
32 and 35 nucleotides), unlike PCR primers. However, there are
several reports demonstrating that normal PCR primers can be
used and efficient amplification can be achieved [6, 7]. It is advised
that a large number of small repeats should be avoided, as they
could lead to secondary structures and potential primer artifacts.
Primer dimers can be avoided by employing self-avoiding molecular
recognition (SAMRs) oligonucleotides, where natural bases are
replaced by 2-aminopurine-20 deoxyriboside (A*), 20 -deoxy-2-
Table 2
Summary of RPA reaction components and their functions (Reproduced from [2]
Reaction components Functions

T4 UvsX protein Recombinase that possesses pairing and strand-transfer
activity that is important in genetic recombination,
DNA repair and replication
(or E. coli RecA; recombinase is a central component in the
related processes of recombinational DNA repair and
homologous genetic recombination that is the ortholog of
the UvsX protein)
T4 UvsY protein Recombinase loading factor that is classified as a
recombination-mediator protein that stimulates the single-
stranded DNA-dependent ATPase activity of T4 UvsX and
lowers the critical concentration of T4 UvsX required for
activity
T4 gp32 Single-stranded binding (SSB) protein is involved in DNA
replication, repair and recombination, and binds
preferentially to single-stranded DNA. The T4 UvsX, T4
UvsY, and T4 gp32 proteins work co-operatively to initiate
the RPA reaction via unwinding, D-loop formation and
stabilization of the DNA template
Bacillus subtilis DNA polymerase I DNA polymerase synthesizes new DNA templates
(Bsu) or Staphylococcus aureus homologous to the target nucleic acid by extending
polymerase (Sau) nucleotide building blocks from the bound primers,
complementary to the original target nucleic acid sequence
or “template”
Deoxynucleotide triphosphate (dNTP, An equimolar solution of dATP, dCTP, dGTP, and dTTP are
N ¼ A, T, C, G) building blocks used by the DNA polymerase to synthesize
new templates
Forward and reverse primers Primers are critical to directing the amplification event to the
nucleic acid target of interest through homologous
binding. After binding, the primers provide the essential
30 -OH for the polymerase to perform strand extension
DNA template The oligonucleotide that the primers bind to for the synthesis
of exact new oligonucleotides
Carbowax20M (a high molecular weight The crowding reagent is a good mimic of the real
polyethylene glycol (PEG)) biomacromolecules condition in vivo and facilitates
amplification, as the crowding agents can enhance the
catalytic activity of the enzymes
Dithiothreitol Stabilization of the enzymes by baring free sulfhydryl groups
Phosphocreatine The three components form the energy-supply system for the
Creatine kinase activities of the recombinase and the DNA polymerase
Adenosine triphosphate (ATP)
Tris(hydroxymethyl)aminomethane The two components serve to stabilize and solubilize the
(Tris) DNA in the solution
Potassium acetate
Magnesium acetate Acts as a cofactor for the performance of the enzymes. The
RPA reaction initiates once the magnesium acetate is added
Fig. 1 RPA amplification scheme. Recombinase proteins form complexes with each primer (a), which scans
DNA for homologous sequences (b). The primers are then inserted at the cognate site by the strand-
displacement activity of the recombinase (c) and single-stranded binding proteins stabilize the displaced
DNA chain (d). The recombinase then disassembles, leaving the 30 -end of the primers accessible to a strand
displacing DNA polymerase (e), which elongates the primer (f). Exponential amplification is achieved by the
cyclic repetition of this process. (Reproduced from [5])
thiothymidine (T*), 20 -deoxyinosine (G*), and N4-thyl-2-

0
-deoxycytidine (C*) in the primers [8]. Primers with low or high
GC content (<30% or >70%) must also be avoided, as they can
promote secondary structures or hairpins. RPA can amplify
sequences up to 1.5 kb; however, an optimum amplicon size in
the range of 100–200 bp yields better results. Additionally, RPA
primers and probes have no requirement for melting temperature
because primer annealing and elongation are enzyme-mediated and
not temperature driven.
3.2 Template RPA has successfully been used for different kinds of target organ-
isms: bacteria, virus, protozoa, fungi, animals, and plants, with
diverse samples types, ranging from cultured microorganisms to
body fluids (urine, sputum, respiratory washes, nasal, blood,
plasma, saliva, vaginal, and anal swabs), surgical biopsy specimens,
organ tissues (skin, lymphatic nodes, liver, lungs, stomach, kidney),
as well as animal and plant products (eggs, shrimps, rice, milk, fruit)
[5]. RPA can be used to amplify double-stranded DNA, single-
stranded DNA, methylated DNA [9], cDNA generated through
reverse transcription of RNA or miRNA [10].
3.3 Temperature An important element of isothermal amplification is the incubation

temperature because it is associated with the complexity of instru-
mentation. Isothermal amplification usually operates at lower tem-
peratures (e.g., 30–42 C). RPA reactions can operate at
temperatures ranging from 25 to 42 C without losing reaction
efficiency. However, most published reports are optimized for
temperatures between 37 and 42 C. While a suitable working
environment for RPA enzymes can be provided by the reaction
temperature, agitation can increase the interactions among the
RPA components in a homogenous reaction solution.
3.4 Incubation Time The time required to amplify the DNA to detectable levels inher-
ently depends on the number of starting DNA copies, but 20 min is
usually adequate, although amplification times of as low as 3–4 min
have been observed [11]. Long incubation times are unlikely to be
beneficial in most applications, as for solution phase RPA the
recombinase consumes all the available ATP within 25 min [5].
3.5 Inhibitors RPA has been demonstrated to operate with nucleic acids extracted
from various sample matrices such as blood [12], serum [13], fecal
[14], nasal [15], vaginal swabs [16], plasma [17], foodstuff [18],
plants [19], animal tissues [11], milk [20], stool [21], and urine
[22]. RPA method amplified targets even in the presence of certain
PCR inhibitors like hemoglobin (50 g/L), heparin (0.5 U), urine
(5%), and ethanol (4% v/v) [23, 24]. The robustness of RPA in the
presence of traditional inhibitors facilitates amplification from
crude extracts, which is not achievable using PCR.
3.6 Specificity RPA has been described as highly specific, with 100% specificity for
and Sensitivity the target sequence in most cases. However, a disadvantage of RPA
to discriminate towards closely related species can be observed
when these species share high sequence similarity [25–27]. RPA
has been reported to be dependent on the number and distribution
of mismatches in the sequence of closely related DNA molecules.
Studies also showed that mismatches located at the 30 -end of pri-
mers effectively prevent or reduce amplification, but mismatches at
the 50 -end or center of primers only mildly affect the RPA reaction
[28, 29].
The efficiency of RPA is dependent on the target sequence,
amplicon size, and type of biological sample tested, as the analytical
limit of detection and the turnaround time varied for RNA and
DNA detection. RPA is very sensitive and has been shown to detect
as little as a few copies per reaction of the analyte, which approaches
the analytical sensitivity of PCR. Furthermore, ultra-sensitive

detection down to even a single copy of the analyte can be achieved
in RPA [30, 31].
3.7 Multiplexing Multiplexing with RPA in the same solution is possible but is highly
dependent on target sequences, amplicon size, and primer design
[32]. Several multiplex assays have been reported despite the
requirements for long primers/probes in RPA reactions [3, 13,
16, 20, 32–34]. Multiplex RPA reaction can be performed either
in a single tube (homogenous) or in a parallel fashion (sometimes
refers to heterogeneous). Primer, probe ratios, and concentrations
thus need to be carefully optimized for each multiplexing assay.
However, a much higher assay throughput and multiplex capacity
can be achieved in a parallel fashion as compared to the single tube
multiplex RPA.
4 Detection of Amplicons
RPA enables the amplification of DNA or RNA, and detection of

amplification products can be visualized either by agarose gel elec-
trophoresis, lateral flow strips, or real-time fluorescent probes. With
lateral flow detection, results can be generated on the dipstick strip
within 5 min post-RPA. Three different oligonucleotides (2 primers
and 1 probe) and the TwistAmp® nfo kit are typically used for assay
designs compatible with lateral flow strip detection (Table 3). The
design of a lateral flow probe requires a 50 -fluorophore tag (i.e.,
FAM) and a tetrahydrofuran (THF) residue for nfo nuclease recog-
nition and cleavage, thus serving as a primer. For lateral flow assay,
an opposing amplification primer labeled at the 50 -end with another
label (e.g., biotin) is required. The amplicon produced in the
presence of the probe and the two primers will include the two
labels on one DNA amplicon, ready to be detected in a sandwich
assay format by antibodies.
Agarose gel electrophoresis is a widely used technique for
visualization of amplification products, but post-amplification, it
is necessary to purify the amplicons to avoid smeared bands on the
gel due to the presence of the proteins and the crowding agent
present in the amplification mix.
Bridge flocculation assay is an equipment-free assay that pro-
vides a binary naked eye visual readout, suitable for low-resource
settings. The basic principle of bridging flocculation involves the
use of long polymers to crosslink multiple particles and thus floccu-
late out of solution at a specific buffer condition (e.g., pH and salt
concentration) [35]. To execute the assay, a bead solution is added
to the amplification products and following an ethanol wash, the
beads are re-suspended in a low pH buffer and a positive answer is
obtained if the beads remain flocculated.
Table 3
Commercial RPA reaction kits by TwistDx™ (Reproduced from [2])
Compatible general
Product name Category Nucleic acid detection detection method
TwistAmp® Basic Lyophilized DNA Gel electrophoresis
TwistAmp® Basic RT kit RNA
TwistAmp® Liquid Liquid kit DNA
Basic
TwistAmp® Liquid RNA
Basic RT
TwistAmp® exo Lyophilized DNA Real-time fluorogenic
TwistAmp® exo RT kit RNA probe-based
TwistAmp® Liquid exo Liquid kit DNA
TwistAmp® Liquid exo RNA
RT
TwistAmp® fpg Lyophilized DNA Real-time and end-point
kit fluorogenic probe-based
TwistAmp® nfo Lateral flow strip
TwistAmp® Food safety DNA (Listeria monocytogenes Real-time fluorogenic
exo + ListeriaM lyophilized hly gene) probe-based
TwistAmp® kit DNA (Campylobacter species
exo + Campylobacter including jejuni and coli)
TwistGlow® Salmonella DNA (Salmonella enterica Real-time and end-point
INVA gene) fluorogenic probe-based
TwistFlow® Salmonella Lateral flow strip
Electrochemically active compounds can also be employed as

an alternative strategy for the detection of RPA to produce a signal
in relation to the amplified nucleic acids. Due to the electrochemi-
cally active property of 3,30 ,5,50 -tetramethylbenzidine (TMB),
amperometric signals from RPA-enzyme-linked immunosorbent
(ELISA) assay or RPA-enzymatic assay can be measured for RPA
detection. However, although fastidious, hybridization assays, and
ELISA tests require several optimizations such as probes and anti-
body concentrations, hybridization temperature and time, or reac-
tion volume, etc.
DVDs and low reflectivity DVDs are suitable substrates for the
immobilization of primers for solid-phase or bridge amplification,
facilitating multiplexing through parallelization in individual reac-
tors of the DVD. Once amplification is achieved, a DVD reader can
be used to read out the results in reflection or transmission
mode [36].
Two kinds of probes, RPA-exo or fpg, can be used for real-time
detection of RPA. Nonspecific intercalating fluorophores such as
SYBR Green or Eva Green can be employed for real-time detection,
but, as in the case of real-time PCR, these dyes cannot discriminate
between amplicons and primer-dimer artifacts, thus giving rise to
false-positive results. Whereas PCR Taq polymerases are not
compatible with RPA, conventional probes such as TaqMan cannot

be used either. DNA amplification is inhibited because the 50 –30
exonuclease activity of Taq polymerase progressively digests the
displaced strand during the strand displacement process. Therefore,
strand displacement polymerases that do not have the 50 –30 exonu-
clease activity (e.g., Bsu/Sau DNA polymerase) are used for Taq-
Man probe detection.
RPA reactions can also be detected using silicon microring
resonator (SMR)-based photonic detection. Amplification of the
nucleic acid is performed in an asymmetric manner
(pre-immobilized on one of the primers on the SMR, and all
the other oligonucleotides and reagents are free in the solution)
in the evanescent field of a resonator waveguide [37, 38]. A change
in the refractive index proximal to the waveguide surface is induced
when the nucleic acids bind to the pre-immobilized primers. Real-
time monitoring of the wavelength shift can be monitored on SMR
as the nucleic acid amplification progresses.
Surface-enhanced Raman scattering (SERS) has also been
exploited for the detection of RPA amplicons. This is the phenom-
enon of when a laser excites nanoscale roughened metal surfaces
(e.g., gold or silver), which resonantly drive surface charges, creat-
ing a highly localized (plasmonic) light field. When a molecule is
absorbed or lies close to, the enhanced field at the surface, a large
enhancement in the Raman signal can be observed [39]. SERS is a
highly sensitive spectroscopic detection technique, which shows
narrow and distinct spectral peaks of the detection molecules, and
is particularly prominent for multiple target molecules detection.
5 RPAs for Porcine Viruses
Infectious diseases of food animals have major impacts on eco-

nomic returns and public health. Effective surveillance and control
of animal diseases are very important, in which rapid and accurate
detection of etiological agents play a critical role. Molecular diag-
nostics is considered a powerful tool for detection and identifica-
tion of infectious agents and has been used extensively due to its
high sensitivity, high specificity, high throughput, and short
turnaround time.
RPA has been developed for major swine viral pathogens such
as porcine deltacoronavirus [40, 41], African swine fever virus
[42, 43], porcine circovirus type 2 [27], porcine parvovirus
[44, 45], and porcine reproductive and respiratory syndrome
virus [46, 47]. Almost all of the RPAs developed for viral pathogens
of swine were highly specific and had no cross-reactions with other
porcine associated viruses, including classical swine fever virus
(CSFV), porcine epidemic diarrhea virus (PEDV), transmissible
gastroenteritis virus (TGEV), porcine kobuvirus (PKoV), foot and
mouth disease virus (FMDV), and Seneca valley virus (SVV). The
analytical sensitivity of RPA ranged from 70 copies to 690 copies
per reaction. The positive rate was found to be similar to real-time
PCR, but higher than that of conventional PCR. RPA developed
for porcine viruses were based on either real-time fluorescent detec-
tion or lateral flow dipstick.
6 Conclusion
RPA is a fascinating isothermal amplification technique that has

already garnered a huge amount of attention due to its very attrac-
tive properties, having a widespread application. The short reaction
time (15–30 min) and low incubation temperature make RPA a
suitable assay for quick and sensitive detection of viruses. Another
advantage of RPA is in designing of primers where annealing tem-
perature is not taken into account as primer forms a complex with
the recombinase to target the homologous sequences. RPA is
exploited for laboratory-based analysis, portable analysis in labora-
tory-in-a-suitcase, analysis at the point-of-need/care with biosen-
sors, lateral flow assays and microfluidic devices, and its exploitation
in a range of commercial devices for molecular diagnostics. RPA,
equipped with field-deployable instruments, offers a sensitive and
specific platform for the rapid and reliable point-of-care detection
of porcine viruses, especially in the resource-limited settings. Given
the tremendous advantages of RPA, as well as some of the current
limitations of the technique, it can be expected that there will be
exponential growth in the applications of RPA as well as improving
and extending its performance. Nevertheless, several diagnostic
methods have been developed over the last two decades; seeing
the constant evolution of viruses, newer, sensitive, efficient, and
rapid diagnostics are still warranted for the effective diagnosis of
porcine viruses.
References
1. Lager KM, Buckley AC (2019) Porcine anti- detection of Rift Valley fever virus. J Clin
viral immunity: how important is it? Front Virol 54(4):308–312
Immunol 10:2258 5. Lobato IM, O’Sullivan CK (2018) Recombi-
2. Li J, Macdonald J, von Stetten F (2018) nase polymerase amplification: basics, applica-
Review: a comprehensive summary of a decade tions and recent advances. Trends Analyt Chem
development of the recombinase polymerase 98:19–35
amplification. Analyst 144(1):31–67 6. Martorell S, Palanca S, Maquieira A, Tortajada-
3. Piepenburg O, Williams CH, Stemple DL, Genaro LA (2018) Blocked recombinase poly-
Armes NA (2006) DNA detection using merase amplification for mutation analysis of
recombination proteins. PLoS Biol 4(7):e204 PIK3CA gene. Anal Biochem 544:49–56
4. Euler M, Wang Y, Nentwich O, Piepenburg O, 7. Mayboroda O, Gonzalez Benito A, Sabate del
Hufert FT, Weidmann M (2012) Recombinase Rio J, Svobodova M, Julich S, Tomaso H,
polymerase amplification assay for rapid O’Sullivan CK, Katakis I (2016) Isothermal
solid-phase amplification system for detection
of Yersinia pestis. Anal Bioanal Chem 408(3): Recombinase polymerase and enzyme-linked
671–676 immunosorbent assay as a DNA amplification-
8. Hoshika S, Chen F, Leal NA, Benner SA detection strategy for food analysis. Anal Chim
(2008) Self-avoiding molecular recognition Acta 811:81–87
systems (SAMRS). Nucleic Acids Symp Ser 19. Zhang S, Ravelonandro M, Russell P,
52:129–130 McOwen N, Briard P, Bohannon S, Vrient A
9. Wee EJ, Ngo TH, Trau M (2015b) Colorimet- (2014) Rapid diagnostic detection of plum pox
ric detection of both total genomic and loci- virus in Prunus plants by isothermal AmplifyRP
specific DNA methylation from limited DNA ((R)) using reverse transcription-recombinase
inputs. Clin Epigenetics 7:65 polymerase amplification. J Virol Methods
10. Wee EJH, Trau M (2016) Simple isothermal 207:114–120
strategy for multiplexed, rapid, sensitive, and 20. Santiago-Felipe S, Tortajada-Genaro LA,
accurate miRNA detection. ACS Sens 1(6): Morais S, Puchades R, Maquieira A (2015)
670–675 Isothermal DNA amplification strategies for
11. Xia X, Yu Y, Weidmann M, Pan Y, Yan S, Wang duplex microorganism detection. Food Chem
Y (2014) Rapid detection of shrimp white spot 174:509–515
syndrome virus by real time, isothermal recom- 21. Crannell ZA, Castellanos-Gonzalez A, Irani A,
binase polymerase amplification assay. PLoS Rohrman B, White AC, Richards-Kortum R
One 9(8):e104667 (2014a) Nucleic acid test to diagnose crypto-
12. Aebischer A, Wernike K, Hoffmann B, Beer M sporidiosis: lab assessment in animal and
(2014) Rapid genome detection of Schmallen- patient specimens. Anal Chem 86(5):
berg virus and bovine viral diarrhea virus by use 2565–2571
of isothermal amplification methods and high- 22. Krolov K, Frolova J, Tudoran O,
speed real-time reverse transcriptase PCR. J Suhorutsenko J, Lehto T, Sibul H, Mager I,
Clin Microbiol 52(6):1883–1892 Laanpere M, Tulp I, Langel U (2014) Sensitive
13. Teoh BT, Sam SS, Tan KK, Danlami MB, Shu and rapid detection of chlamydia trachomatis
MH, Johari J, Hooi PS, Brooks D, by recombinase polymerase amplification
Piepenburg O, Nentwich O, Wilder-Smith A, directly from urine samples. J Mol Diagn
Franco L, Tenorio A, AbuBakar S (2015) Early 16(1):127–135
detection of dengue virus by use of reverse 23. Kersting S, Rausch V, Bier FF, von Nickisch-
transcription-recombinase polymerase amplifi- Rosenegk M (2014b) Rapid detection of plas-
cation. J Clin Microbiol 53(3):830–837 modium falciparum with isothermal recombi-
14. Amer HM, Abd El Wahed A, Shalaby MA, nase polymerase amplification and lateral flow
Almajhdi FN, Hufert FT, Weidmann M analysis. Malar J 13:99
(2013) A new approach for diagnosis of bovine 24. Rosser A, Rollinson D, Forrest M, Webster BL
coronavirus using a reverse transcription (2015) Isothermal recombinase polymerase
recombinase polymerase amplification assay. J amplification (RPA) of Schistosoma haemato-
Virol Methods 193(2):337–340 bium DNA and oligochromatographic lateral
15. Boyle DS, McNerney R, Teng Low H, Leader flow detection. Parasit Vectors 8:446
BT, Perez-Osorio AC, Meyer JC, O’Sullivan 25. Moore MD, Jaykus LA (2017) Development
DM, Brooks DG, Piepenburg O, Forrest MS of a recombinase polymerase amplification
(2014) Rapid detection of mycobacterium assay for detection of epidemic human noro-
tuberculosis by recombinase polymerase ampli- viruses. Sci Rep 7:40244
fication. PLoS One 9(8):e103091 26. Patel P, Abd El Wahed A, Faye O, Pruger P,
16. Daher RK, Stewart G, Boissinot M, Bergeron Kaiser M, Thaloengsok S, Ubol S,
MG (2014) Isothermal recombinase polymer- Sakuntabhai A, Leparc-Goffart I, Hufert FT,
ase amplification assay applied to the detection Sall AA, Weidmann M, Niedrig M (2016) A
of group B streptococci in vaginal/anal sam- field-deployable reverse transcription recombi-
ples. Clin Chem 60(4):660–666 nase polymerase amplification assay for rapid
17. Euler M, Wang Y, Heidenreich D, Patel P, detection of the chikungunya virus. PLoS
Strohmeier O, Hakenberg S, Niedrig M, Negl Trop Dis 10(9):e0004953
Hufert FT, Weidmann M (2013) Development 27. Yang Y, Qin X, Sun Y, Cong G, Li Y, Zhang Z
of a panel of recombinase polymerase amplifi- (2017) Development of isothermal recombi-
cation assays for detection of biothreat agents. J nase polymerase amplification assay for rapid
Clin Microbiol 51(4):1110–1117 detection of porcine circovirus type 2. Biomed
18. Santiago-Felipe S, Tortajada-Genaro LA, Res Int 2017:8403642
Puchades R, Maquieira A (2014b)
28. Daher RK, Stewart G, Boissinot M, Boudreau amplification/detection (ISAD) device for
DK, Bergeron MG (2015) Influence of rapid detection of genetic alteration in cancers.
sequence mismatches on the specificity of Lab Chip 13(11):2106–2114
recombinase polymerase amplification technol- 39. Schlucker S (2014) Surface-enhanced Raman
ogy. Mol Cell Probes 29(2):116–121 spectroscopy: concepts and chemical applica-
29. Lillis L, Lehman DA, Siverson JB, Weis J, tions. Angew Chem Int Ed Engl 53(19):
Cantera J, Parker M, Piepenburg O, 4756–4795
Overbaugh J, Boyle DS (2016) Cross-subtype 40. Gao X, Liu X, Zhang Y, Wei Y, Wang Y (2020)
detection of HIV-1 using reverse transcription Rapid and visual detection of porcine deltacor-
and recombinase polymerase amplification. J onavirus by recombinase polymerase amplifica-
Virol Methods 230:28–35 tion combined with a lateral flow dipstick.
30. Lai MY, Ooi CH, Lau YL (2017) Rapid detec- BMC Vet Res 16(1):130
tion of plasmodium knowlesi by isothermal 41. Ma L, Zeng F, Huang B, Zhu Y, Wu M, Xu F,
recombinase polymerase amplification assay. Xiao L, Huang R, Ma J, Cong F, Guo P (2019)
Am J Trop Med Hyg 97(5):1597–1599 Point-of-care diagnostic assay for rapid detec-
31. Mondal D, Ghosh P, Khan MA, Hossain F, tion of porcine deltacoronavirus using the
Bohlken-Fascher S, Matlashewski G, recombinase polymerase amplification method.
Kroeger A, Olliaro P, Abd El Wahed A (2016) Transbound Emerg Dis 66(3):1324–1331
Mobile suitcase laboratory for rapid detection 42. Fan X, Li L, Zhao Y, Liu Y, Liu C, Wang Q,
of Leishmania donovani using recombinase Dong Y, Wang S, Chi T, Song F, Sun C,
polymerase amplification assay. Parasit Vectors Wang Y, Ha D, Zhao Y, Bao J, Wu X, Wang Z
9(1):281 (2020) Clinical validation of two recombinase-
32. Kersting S, Rausch V, Bier FF, von Nickisch- based isothermal amplification assays
Rosenegk M (2014a) Multiplex isothermal (RPA/RAA) for the rapid detection of African
solid-phase recombinase polymerase amplifica- swine fever virus. Front Microbiol 11:1696
tion for the specific and fast DNA-based detec- 43. Miao F, Zhang J, Li N, Chen T, Wang L,
tion of three bacterial pathogens. Mikrochim Zhang F, Mi L, Zhang J, Wang S, Wang Y,
Acta 181(13–14):1715–1723 Zhou X, Zhang Y, Li M, Zhang S, Hu R
33. Crannell ZA, Rohrman B, Richards-Kortum R (2019) Rapid and sensitive recombinase poly-
(2014b) Quantification of HIV-1 DNA using merase amplification combined with lateral
real-time recombinase polymerase amplifica- flow strip for detecting African swine fever
tion. Anal Chem 86(12):5615–5619 virus. Front Microbiol 10:1004
34. Santiago-Felipe S, Tortajada-Genaro LA, 44. Wang JC, Liu LB, Han QA, Wang JF, Yuan WZ
Morais S, Puchades R, Maquieira Á (2014a) (2017a) An exo probe-based recombinase
One-pot isothermal DNA amplification–hybri- polymerase amplification assay for the rapid
disation and detection by a disc-based method. detection of porcine parvovirus. J Virol Meth-
Sens. Actuators B Chem 204:273–281 ods 248:145–147
35. Wee EJ, Lau HY, Botella JR, Trau M (2015a) 45. Yang Y, Qin X, Zhang W, Li Y, Zhang Z
Re-purposing bridging flocculation for on-site, (2016b) Rapid and specific detection of por-
rapid, qualitative DNA detection in resource- cine parvovirus by isothermal recombinase
poor settings. Chem Commun (Camb) polymerase amplification assays. Mol Cell
51(27):5828–5831 Probes 30(5):300–305
36. Tortajada-Genaro LA, Santiago-Felipe S, 46. Wang JC, Yuan WZ, Han QA, Wang JF, Liu LB
Amasia M, Russom A, Maquieira Á (2015) (2017b) Reverse transcription recombinase
Isothermal solid-phase recombinase polymer- polymerase amplification assay for the rapid
ase amplification on microfluidic digital versa- detection of type 2 porcine reproductive and
tile discs (DVDs). RSC Adv 5(38): respiratory syndrome virus. J Virol Methods
29987–29995 243:55–60
37. Koo B, Jin CE, Park SY, Lee TY, Nam J, Jang 47. Yang Y, Qin X, Sun Y, Chen T, Zhang Z
YR, Kim SM, Kim JY, Kim SH, Shin Y (2018) (2016a) Rapid detection of highly pathogenic
A rapid bio-optical sensor for diagnosing Q porcine reproductive and respiratory syndrome
fever in clinical specimens. J Biophotonics virus by a fluorescent probe-based isothermal
11(4):e201700167 recombinase polymerase amplification assay.
38. Shin Y, Perera AP, Kim KW, Park MK (2013) Virus Genes 52(6):883–886
Real-time, label-free isothermal solid-phase
Chapter 18
Cell Culture System for Porcine Virus Isolation

and Propagation
Vishal Rai, Kaushal Kishor Rajak, Kiran, Arfa Fayaz, Monu Karki,
Chris Einstein, Mukesh Bhatt, Ashok Kumar, and Ajay Kumar Yadav
Abstract
Cell culture is an integral part of virology as the viruses are the obligate intracellular parasites that require
replication inside a living cell to produce copies of themselves. Since the introduction of cell culture, a
number of cell culture systems have been developed which are in use to isolate, propagate, and study growth
kinetics vis-à-vis host–virus interactions of a number of virus species affecting diverse range of hosts. These
systems comprise of primary cell culture and cell lines having finite or infinite life span. Owing to their
immortality and other advantages, continuous cell lines are the most frequently used category of cell culture
system for animal viruses. During recent years, traditional cell culture system has changed from the use of
glass bottles/flasks to the use of plastic tissue culture flasks and diverse range of culture media have been
developed to meet the research needs. This chapter elaborates various cell culture systems which have been
developed and are most frequently used for isolation and propagation of common DNA and RNA viruses
affecting pigs.
Key words Primary cell culture, Cell lines, Secondary cell culture, Porcine viruses
1 Introduction
There are three systems for isolation of viruses, viz. animal host
system, embryonated egg system, and cell culture system. Since the
advent of cell culture system, isolation and propagation of viruses
has become relatively simple compared to the olden days, when the
natural or heterologous animal host system was used predomi-
nately. As in case of other viruses, isolation and propagation of
animal viruses in cell culture has manifold applications. For exam-
ple, isolation of viruses serves as the gold standard test for diagnosis
of many viral infections. In addition, cell culture system has been
used extensively for preparation of live attenuated viral vaccines and
production of viral antigen in bulk for the development of different
diagnostics. Like many other fields of biomedical science, the field
251
252 Vishal Rai et al.
of cell culture has also undergone tremendous advancement in the

past few years enabling the establishment of different types of cell
culture system for different viruses, each with its own merits and
demerits based on the virus concerned. In such a situation, it
becomes increasingly important to choose the correct system for a
particular virus. Several swine viruses continue to affect the swine
industry worldwide causing huge economic losses. Swine popula-
tion is susceptible to various DNA and RNA viruses and hence their
detection is pertinent for making accurate diagnosis as well as for
their epidemiological assessment. Virus isolation in susceptible cell
culture system is fundamental and still one of the best methods for
virus identification for laboratory diagnosis. The focus of this chap-
ter lies in the different type of cell culture systems that are available
and used most frequently for the isolation and propagation of the
common DNA and RNA viruses affecting pigs.
2 Types of Cell Culture System
2.1 Primary and Primary cell culture is derived straight from the host. Freshly
Secondary Cell Culture isolated cells derived from animal/human tissue or organ fragments
(either enzymatically or mechanically) that grow successfully, con-
stitute primary cell culture. The cells are mainly heterogenous,
grow slowly, divide only for a limited time and are much identical
with their parental tissue. Primary cell culture is advantageous as
the biochemical dynamics of the cells resembles as in vivo, so
biological response obtained may be much closer to that in vivo.
The cells cannot be transformed and possibility of undergoing
mutation is low. Hence the results obtained with primary cell
cultures can be more relevant. But primary culture is difficult to
establish and have limited life span. Depending on the requirement
for attachment for growth, the primary cells can be classified as
anchorage-dependent or adherent cells and anchorage-
independent or suspension cells.
A culture is considered primary until the cells are passaged or
further subcultured. After first subculture, the primary culture is
called as a secondary culture or a cell line. Sub-culturing prolongs
the lifespan of the cells, but the cells no longer resemble the parent
tissue and chances of mutation increases. The maintenance of sec-
ondary cell culture or cell line is easier as compared to the primary
cell culture.
2.2 Cell Lines A cell line represents a population of cells obtained after subculture
or passaging of a primary culture. It may be of finite or continuous
type. Continuous cell lines are those which comprise of immortal
cells, i.e., they experience indefinite growth upon subsequent sub-
cultures, whereas finite cell lines are those which experience death
of cells upon sub-culturing. Owing to their immortality and other
Cell Culture System for Porcine Virus Isolation and Propagation 253
advantages, continuous cell lines are the most frequently used

category of cell culture system for animal viruses. In the subsequent
subsections, we will discuss the commonly used primary cell culture
and cell lines for common porcine viruses.
3 Cell Culture System for Porcine DNA Viruses
3.1 African Swine ASFV belongs to the genus Asfivirus of the family Asfarviridae.
Fever Virus (ASFV) The virus is the causative agent of a fatal disease called African swine
fever which causes high mortality among infected swine and is a
major concern for swine industries worldwide. It is the only known
arthropod borne DNA virus which is transmitted by the soft ticks of
genus Ornithodoros. African swine fever is an OIE notifiable disease
spreading to newer regions and the current strain reported from
south east Asian countries including India is highly virulent which
caused 100% mortality in domestic pigs [1]. Owing to the interna-
tional importance of the disease the diagnosis becomes crucial and
virus isolation is important for detection of the virus.
Primary swine macrophage culture is used for virus isolation.
Swine bone marrow or peripheral blood leukocyte cultures are also
used. These primary cultures show hemadsorption and CPE occur
within few days of inoculation. The virus can be adapted further in
different cell lines like Vero cells [2].
MA-104 (Microbiological Associates-104, from African green
monkey kidney), a commercially available cell line has been found
to be suitable for the isolation of ASFV from infected field
samples [1].
3.2 Porcine Porcine circoviruses belong to the genus Circovirus of the family
Circovirus Circoviridae. PCV1 and PCV2 are found in both domestic and wild
pigs [3–6]. PCV1 has been isolated from stillborn pigs but is
generally not considered to be pathogenic for swine [2]. PCV2
has worldwide occurrence and causes huge economic losses to
swine industry [7–9]. PCV2 is mainly associated with porcine
circovirus diseases (PCVDs) [10]. In weanling piglets it causes
post-weaning multisystemic wasting syndrome (PMWS). Currently
there is no suitable cytopathogenic cell model for studying the
pathogenesis of PCV2. Some cell lines reported to be useful are
discussed below.
PCV free PK-15 cell line is widely used for virus isolation but it
is not much efficient [11] and presence of virus can only be con-
firmed using immunofluorescence-based assay [3]. Primary porcine
kidney cells are also used for virus isolation. Primary porcine hepa-
tocyte culture was found to be more sensitive for PCV-2 as com-
pared to primary kidney cells [12]. IPEC-J2 (Intestinal Porcine
Epithelial Cell line-J2) and an immortal porcine lymphoblastoid
cell line (lymphoblastoid L35 cell line) have also shown the
replication of PCV2 [13, 14]. An immortalized porcine oral muco-

sal epithelial cell line (hTERT-POMEC) from neonatal unsuckled
piglet produces CPE and can be a better system for PCV
isolation [15].
3.3 Herpesviruses Suid alphaherpesvirus 1, Aujeszky’s disease virus or pseudorabies

of Swine virus (PRV).
Genus: Varicellovirus
Subfamily: Alphaherpesvirinae
Family: Herpesviridae
The virus has a wide-host range from infecting its natural host
pigs to causing fatal disease in cattle, sheep, dogs, rats, etc.
[16]. The terms “pseudorabies” and “mad itch” describe disease
in cattle. In pigs, the PRV is associated with respiratory signs and
CNS involvement in case of neonatal pigs. Since PRV is having a
broad host range, it can be grown in cell lines derived from different
species, viz. RK-13 (rabbit kidney epithelial cells), PK-15 (adult Sus
scrofa kidney epithelial cells), MDBK (Madin–Darby bovine kid-
ney), and Vero cells (African green monkey kidney cells). The virus
isolation can be done in ML (Mink lung) and ST (swine testicle)
cell lines [17]. CPE generally appears within 6–24 h which is
evident as cell lysis and syncytia formation.
Suid betaherpesvirus 2, porcine cytomegalovirus (PCMV).
Genus: Cytomegalovirus
Subfamily: Betaherpesvirinae
Family: Herpesviridae
PCMV is present in nearly all the swine population but it rarely
causes any disease except in young piglets in which the disease is
fatal. Inclusion body rhinitis is an acute or subacute infection of
suckling piglets (4-week-old). In cell culture the virus shows no
CPE, hence further immunostaining is required. Porcine pulmo-
nary macrophages are used for the isolation of PCMV. Primary pig
lung (PL) cells, primary swine testicle (ST), PK-15 cell line and
porcine turbinate (PT) can be used for the propagation of
PCMV [18].
3.4 Porcine Genus: Protoparvovirus

Parvovirus Subfamily: Parvovirinae
Family: Parvoviridae
Porcine parvovirus 1 is the main species of porcine parvoviruses
which is known to cause reproductive failure in sows. In unvacci-
nated herds abortion storms can occur. The virus can be isolated in
primary pig kidney cell cultures. Primary cell cultures are not
typically used as there is a risk of contamination by adventitious

agents. Various continuous cell lines are used, viz. ESK (embryonic
swine kidney), PK-15 (pig kidney), SK6 (swine kidney), ST (pig
testis), STE (swine testicle epithelial cells), and SPEV (embryonic
porcine kidney epithelial cells) [19, 20]. The virus replicates slowly
in cell culture and CPE includes granulations, irregular shape,
intranuclear inclusions, pyknotic nucleus followed by cell death
[19, 21].
3.5 Porcine Porcine adenovirus belongs to the genus Mastadenovirus of the

Adenovirus (PAdV) family Adenoviridae. PAdV is generally present worldwide and has
been isolated from gastrointestinal tract of normal swine [22]. The
virus mainly causes subclinical infections but it has been isolated
from pigs suffering from diarrhea [23–25], pigs with encephalitis
[26], nephritis [27], respiratory disease [28], and reproductive
disorders [29].
The virus is easily cultured from the fecal samples or kidney/
lung homogenates using primary porcine kidney cells, PK-15 cell
line, primary porcine thyroid cells, and primary porcine testicular
cells. Cytopathic effects (intranuclear inclusion bodies) can be seen
2–5 days post infection [30, 31]. PAdV is often an adventitious
agent in the porcine primary cell cultures derived from kidneys,
spleen, or testes [28] creating complications in the virus isolation.
3.6 Swinepox Virus Genus: Suipoxvirus

Subfamily: Chordopoxvirinae
Family: Poxviridae
The virus is present sporadically worldwide and is associated
with poor sanitation. Clinical disease is seen mainly in young pigs
with high morbidity but low mortality. Skin lesions are more pro-
nounced on the ventral abdomen, flanks, legs, inguinal areas, and
ears and less on the face near the eyes [32]. Virus isolation can be
done in primary swine kidney cells, or PK-15 cell line. The charac-
teristic CPEs include cell rounding, vacuolation of nucleus, cell
granulation and fusion followed by detachment. The sample should
be considered negative only after multiple blind passages have been
done in the cell culture.
4 Cell Culture System for Porcine RNA Viruses
4.1 Classical Swine Classical swine fever virus (CSFV), a member of the genus Pesti-
Fever Virus virus in the Flaviviridae family is the causative agent of swine fever
or hog cholera disease of pigs. It is considered as one of the most
economically devastating disease causing huge mortality. Members
of the Suidae family such as domestic and wild pigs are considered
the only natural reservoir of CSFV [33]. Related pestiviruses
causing disease in other animals include border disease virus

(BDV), bovine viral diarrhea virus-1 (BVDV-1), and BVDV-2.
Porcine kidney-15 (PK-15) cells are perhaps the most widely
used cells for isolation and propagation of porcine viruses. It has
been used extensively for the isolation and propagation of CSFV.
PK-15 cells is one amongst the three cell lines recommended for
CSFV isolation/diagnosis, with the other two being swine kidney
6 (SK6) and swine testis endothelial (STE) cell lines [34]. It is
recommended that circovirus-free PK-15 cells be used for CSFV
diagnosis as porcine circovirus 1 (PCV1) is a frequent contaminant
of PK-15 cells. In addition, interference in CSFV diagnosis due
ruminant pestiviruses is also a possibility since porcine cells are
more or less susceptible to these viruses. Hence, it becomes impor-
tant to demonstrate the absence of ruminant pestiviruses in the cell
lines used for CSF diagnosis as well. To minimize the risk of
contamination with ruminant pestiviruses, the aforementioned
cell lines can be adapted to grow in horse serum instead of the
fetal bovine serum or calf serum. Since CSFV are non-cytopathic
viruses, they do not cause any visible alteration in the porcine cell
lines. Consequently, their detection in the cell culture system relies
on indirect techniques such as immunofluorescence or immuno-
peroxidase staining, RT-PCR, etc. [34].
4.2 Foot and Mouth Foot and mouth disease is also one of the most economically
Disease Virus devastating diseases of pigs. However, this disease is not restricted
to swine alone as it affects all cloven-footed animals. The etiological
agent is foot and mouth disease virus (FMDV) of Aphthovirus, a
genus within the Picornaviridae family. Seven different serotypes
including O, A, C, Asia 1, Southern African Territories (SAT)
1, SAT 2, and SAT 3 have been recognized for this virus till date.
The isolation of FMDV in susceptible cell lines has important
benefits; first is the accurate diagnosis of the serotype involved in
an outbreak and second is the production of vaccines, which in turn
are crucial determinants in the control of FMD.
Primary bovine thyroid (BTY) cell culture system has been
demonstrated to be the most sensitive for FMDV isolation
[35]. But this system has several disadvantages such as difficulty in
obtaining thyroid tissue, considerable time and expense required in
the preparation, and the relatively short life span of these primary
cells. So, the isolation and propagation of FMDV is commonly
done using the cell line system in most of the laboratories.
Among the cell lines, baby hamster kidney fibroblasts (BHK-21)
and pig kidney (IB-RS-2/InstitutoBiologico-Rim Suino-2) cells
are the ones which are most frequently used, although they are
less susceptible than BTY cell culture system. Nonetheless, IB-RS-
2 are considered the most susceptible and thus commonly required
for the isolation of pig adapted strains of FMDV as several pig
adapted strains do not replicate in BTY cells [36].
Over the years, novel cell lines expressing the cellular receptor
for FMDV (bovine αVβ6 integrin) have been engineered. It
includes: fetal porcine kidney (LFBK-αVβ6) cells and fetal goat
tongue cells (ZZ-R 127). These cell lines have been shown to
possess equal sensitivity to BTY cells in isolating FMDV. LFBK-α
Vβ6 in addition possesses high sensitivity to pig adapted strains of
FMDV, highlighting its emergence as a novel cell culture system for
diagnosis of FMD in pigs [37].
4.3 Porcine Porcine rotaviruses (PoRV) are one of the major causes of diarrhea
Rotavirus in pigs. They are members of the Rotavirus genus within the
Reoviridae family. Ten groups (A-J) of rotaviruses have been iden-
tified till date based on one of the capsid protein (VP6), out of
which groups A, B, C, E, and H are associated with disease in pigs
[38]. A cell line designated MA-104 (Microbiological Associates-
104), obtained from the kidney of African green monkey is the
most frequently used cell culture system for the isolation and
propagation of porcine rotaviruses.
Permissive cells in addition to MA-104 for propagation of
Group A rotaviruses include colon adenocarcinoma, HepG2 liver
cells, pancreatic islet cells, and other kidney cell lines [39]. Reports
of isolation of group B rotavirus in cell cultures are scarce. A report
of isolation of a group B rotavirus (strain SKA-1) in SKL cells
(swine kidney cells) combined with pancreatin treatment is available
[40]. But, later on it was demonstrated that SKA-1 strain of PoRV
actually belonged to group H rotavirus [41]. In addition to
MA-104, few strains of group C rotaviruses have been cultured in
primary porcine kidney cells, in combination with high pancreatin
or trypsin concentrations [42].
Addition of protease such as trypsin, chymotrypsin, or pancre-
atin in cell culture medium for rotavirus isolation and propagation
is important as proteolytic treatment specifically cleaves the spike
forming outer capsid protein of rotaviruses (VP4) into polypeptides
VP5 and VP8, which in turn enhances the rotavirus infectivity
[43]. As some rotavirus strains are difficult to propagate in cell
culture, the search for a better cell culture system is going on. In
this line, co-cultures of primary porcine intestinal cells (ileocytes
and colonocytes) with myofibroblasts have been developed and
these primary cells were found to be susceptible to different strains
of rotavirus [44]. Whether these cells are more susceptible to
rotavirus isolation remains to be seen.
4.4 Porcine PRRSV belongs to the genus Arterivirus of the family Arterivir-
Reproductive and idae and is an important pathogen of swine population all over the
Respiratory Syndrome globe. There are two distinct genotypes of the virus, type 1 porcine
Virus (PRRSV) reproductive and respiratory syndrome virus (European) and type
2 porcine reproductive and respiratory syndrome virus (North
American). The virus affects all age groups and as the name
suggests, PRRSV affects the reproductive and respiratory system of

the affected pigs. The virus is the cause of SMEDI syndrome in
pregnant sows.
For isolation of PRRSV, primary alveolar macrophage cultures
(PAM) from young pig and monkey kidney cell line MA-104
(African green monkey kidney epithelial cell) are frequently used.
MARC-145 (a derivative of MA-104) can be used preferentially for
isolation of vaccine strains. The cytopathic effects appear as micro-
scopic foci throughout the culture within 72–96 h post inoculation
[45]. In contrast to PRRSV 2 which easily adapt to simian cell
culture, PRRSV 1 isolates are difficult to adapt to simian cells
(like MA-104) but grow readily on porcine macrophages. An effi-
cient MARC-145 cell line expressing porcine CD163 (pCD163)
with higher susceptibility and higher isolation rate of PRRSV has
been established [46].
4.5 Porcine TGEV belongs to the genus Alphacoronavirus of the family Cor-
Coronaviruses onaviridae. It is the cause of highly fatal enteritis in swine with
100% mortality in young piglets. The virus can be isolated from
4.5.1 Transmissible
fecal samples or gut contents from infected pigs in primary and
Gastroenteritis Virus (TGEV)
secondary pig kidney cells (Bohl and [47]), and in ST (swine
testicle) cell line [48].
Additional passages should be done after attempting primary
isolation from field samples as CPE may not appear upon primary
isolation. Infected cells become enlarged, round, and balloon-like
[47]. Pig thyroid cells can be used for initial isolation as they show
progressive cytopathic effect after TGEV inoculation, but they
should be strictly free from adventitious agents like parvoviruses
[49]. Supplementing cell culture media with trypsin or pancreatin
can result in increased susceptibility and better CPE or plaque
detection [50].
4.5.2 Porcine Epidemic PEDV is also a member of the genus Alphavirus under the family
Diarrhea Virus (PEDV) Coronaviridae. It causes porcine epidemic diarrhea which is clini-
cally similar to transmissible gastroenteritis caused by TGEV. Dif-
ferent strains of PEDV are reported to be circulating worldwide.
Neonatal pigs are more susceptible to the infection in which high
mortality occurs [51].
Intestinal contents or fecal samples can be used for virus isola-
tion. Virus isolation can be done using vero cells and different
porcine cell cultures, like porcine bladder and kidney cells
[52]. Trypsin should be added in the cell culture media. The
cytopathic effect can be seen around 30 h post inoculation char-
acterized by cell fusion and large syncytia with appearance of float-
ing cells overtime. In absence of CPE, culture should not be
considered negative upto 5 days post inoculation [53]. Sometimes
blind passages may be required, but for early detection immunoflu-
orescence staining can be done [52, 54].
4.6 Swine Swine influenza virus is associated with outbreaks of acute respira-
Influenza Virus tory disease in pigs [55]. The virus belongs to the genus Influen-
zavirus A of the family Orthomyxoviridae. Common subtypes of the
virus infecting swine are H1N1, H1N2, and H3N2. Pigs are known
to play an important role in the reassortment events as intermediate
hosts, thus leading to the development of subtypes of pandemic
potential [56, 57]. The influenza pandemics of 1918 and 2009
were the results of efficient person-to-person transmission of
swine influenza virus [58].
Currently, MDCK (Madin–Darby Canine Kidney) cell line is
used most frequently for the isolation, propagation, and titration of
swine influenza virus although primary cell cultures from swine
organs (kidney, testicle, lung, and trachea) can also be used
[59]. Addition of trypsin to basal media is recommended
[60]. CPE starts to appear after 24 h of inoculation and is evident
as enlarged focal vacuolation of cells followed by cell
detachment [61].
4.7 Japanese Japanese encephalitis is a zoonotic virus transmitted by mosquitoes

Encephalitis Virus (Culex sp.). Domestic pigs and wild birds are the natural hosts for
the virus and humans are dead-end host. Pigs also act as amplifying
host for JEV.
The virus isolation can be performed in various cells including
primary chicken embryo, Vero cells (African green monkey kidney),
and BHK (baby hamster kidney) cells. The CPE is visible after 24 h
of inoculation. Mosquito cell line, Aedes albopictus C6/36 is also
used but as there is no CPE, further culture in other cells or
detection of viral RNA/ viral antigen for confirmation is
required [62].
4.8 Other Vesicular The VSV virus belongs to the genus Vesiculovirus of the family
Disease Viruses Rhabdoviridae. There are two serotypes of VSV, vesicular stomatitis
New Jersey virus (VSNJV) and vesicular stomatitis Indiana virus
4.8.1 Vesicular
(VSIV) [63]. Swine disease is associated only with VSNJV while for
Stomatitis Virus (VSV)
domestic livestock both the serotypes are pathogenic. Vesicular
stomatitis is clinically indistinguishable from other vesicular dis-
eases of swine (FMD, or swine vesicular disease, or vesicular exan-
thema), therefore laboratory confirmation is imperative for correct
diagnosis. Humans in close contact with the infected animals are
also susceptible to the virus.
The virus can be easily propagated in cell culture. Cytopathic
effects observed in different cell lines such as vero, BHK-21, and
pig kidney IB-RS-2 cell line can distinguish VSV from FMD and
swine vesicular disease [64].
4.8.2 Swine Vesicular SVDV belongs to the species Enterovirus B of the genus Enterovirus
Disease Virus (SVDV) under the family Picornaviridae. Swine vesicular disease was an
OIE-listed disease until 2015, as the disease is clinically similar to
FMD. The disease does not cause much production losses and with
the advent of newer diagnostic techniques it can easily be differ-
entiated from FMD. Also the clinical signs of the disease are milder
than FMD.
Primary or secondary porcine kidney cell cultures are suscepti-
ble to the virus [65]. Cell lines such as IB-RS-2, SK6, and PK-15
are also used. Isolation of virus on IB-RS-2 cells is one of the most
sensitive methods for diagnosis [66].
4.8.3 Swine Vesicular The virus belongs to the genus Vesivirus of the family Caliciviridae.
Exanthema Virus The disease caused by the virus is highly infectious but mortality is
not common. Clinically the disease is indistinguishable from other
vesicular diseases of swine mentioned before. Isolation of the virus
can be done in primary porcine cell cultures such as porcine kidney
cells and in monkey kidney cells (Vero cell line). The virus replicates
rapidly in cell culture resulting in destructive cytopathic
effects [67].
References
1. Rai A, Pruitt S, Ramirez-Medina E, Vuono EA, 7. Novosel D, Lipej Z, Cubric-Curik V, Jungic A
Silva E, Velazquez-Salinas L, Carrillo C, Borca (2012) Presence of torque tenosus virus in
MV, Gladue DP (2020) Identification of a con- porcine circovirus type 2-associated disease in
tinuously stable and commercially available cell Croatia. Vet Rec 17:529
line for the identification of infectious African 8. O’Dea MA, Kabay MJ, Carr J, Wilcox GE,
swine fever virus in clinical samples. Viruses 12 Richards RB (2011) Porcine circovirus-
(8):820 associated disease in weaner pigs in Western
2. Maclachlan NJ, Dubovi EJ, Barthold SW, Australia. Aust Vet J 89(4):122–130
Swayne DF, Winton JR (2017) Fenner’s veter- 9. Wilfred E, Mutebi F, Mwiine FN, James OA,
inary virology. Academic Press, London Lonzy O (2018) Porcine circovirus type 2–sys-
3. Allan GM, Ellis JA (2000) Porcine circoviruses: temic disease on pig farms and associated
a review. J Vet Diagn Investig 12(1):3–14 knowledge of key players in the pig industry
4. Calsamiglia M, Segalés J, Quintana J, Rosell C, in Central Uganda. Int J Vet Sci Med 6
Domingo M (2002) Detection of porcine cir- (2):178–185
covirus types 1 and 2 in serum and tissue sam- 10. Allan GM, McNeilly F, McNair I, Curran MD,
ples of pigs with and without postweaning Walker I, Ellis J, Konoby C, Kennedy S, Mee-
multisystemic wasting syndrome. J Clin Micro- han B (2000) Absence of evidence for porcine
biol 40(5):1848–1850 circovirustype 2 in cattle and humans, and lack
5. Segales J, Domingo M (2002) Postweaning- of seroconversion or lesions in experimentally
mulstisystemic wasting syndrome (PMWS) in infected sheep. Arch Virol 145(4):853–857
pigs.A review. Vet Q 24(3):109–124 11. Zhu Y, Lau A, Lau J, Jia Q, Karuppannan AK,
6. Vicente J, Segalés J, Höfle U, Balasch M, Kwang J (2007) Enhanced replication of por-
Plana-Durán J, Domingo M, Gortázar C cine circovirus type 2 (PCV2) in a homoge-
(2004) Epidemiological study on porcine cir- neous subpopulation of PK15 cell line.
covirus type 2 (PCV2) infection in the Virology 369(2):423–430
European wild boar (Sus scrofa). Vet Res 35 12. Hirai T, Nunoya T, Ihara T, Saitoh T,
(2):243–253 Shibuya K, Nakamura K (2006) Infectivity of
porcine circovirus 1 and circovirus 2 in primary
porcine hepatocyte and kidney cell cultures. J Abstract. In Conference of Research Workers
Vet Med Sci 68(2):179–182 in Animal Diseases, Chicago, IL,
13. Rodriguez-Carino C, Duffy C, Sanchez- 10–11 November
Chardi A, McNeilly F, Allan GM, Segales J 26. Kasza L (1966) Isolation of an adenovirus from
(2011) Porcine circovirus type 2 morphogene- the brain of a pig. Am J Vet Res 27(118):751
sis in a clone derived from the l35 lymphoblas- 27. Nietfeld JC, Leslie-Steen P (1993) Interstitial
toid cell line. J Comp Pathol 144(2–3):91–102 nephritis in pigs with adenovirus infection. J
14. Yan M, Zhu L, Yang Q (2014) Infection of Vet Diagn Investig 5(2):269–273
porcine circovirus 2 (PCV2) in intestinal por- 28. Hirahara T, Yashuhara H, Matsui O,
cine epithelial cell line (IPEC-J2) and interac- Yamanaka M, Tanaka M, Fukuyama S,
tion between PCV2 and IPEC-J2 Izumida A, Yoshiki K, Kodama K, Nakai M
microfilaments. Virol J 11(1):193 et al (1990) Isolation of porcine adenovirus
15. Cui H, Liang W, Wang D, Guo K, Zhang Y from the respiratory tract of pigs in Japan. Jap-
(2019) Establishment and characterization of anese J Vet Sci 52(2):407–409
an immortalized porcine oral mucosal epithe- 29. Dee SA (1995) Viral causes of porcine repro-
lial cell line as a cytopathogenic model for por- ductive failure. 1. Compend Contin Educ Pract
cine circovirus 2 infection. Front Cell Infect Vet 17(7):962–972
Microbiol 9:171 30. Benfield DAH, Richard A (2012) Porcine ade-
16. Pensaert MB, Kluge JP (1989) Pseudorabies noviruses. In: Zimmerman JJ, Karriker LA,
virus (Aujeszky’s disease). In: Pensaert MB Ramirez A, Schwartz KJ, Stevenson GW (eds)
(ed) Virus infections of Porcines. Elsevier, Diseases of Swine, 10th edn. John Wiley &
New York, pp 39–64 Sons, Inc., New York, pp 392–395
17. Onyekaba C, Bueon L, King P, Fahrmann J, 31. Clarke MC, Sharpe HB, Derbyshire JB (1967)
Goyal SM (1987) Susceptibility of various ceil Some characteristics of three porcine adeno-
culture systems to pseudorabies virus. Comp viruses. Arch Gesamte Virusforsch 21
Immunol Microbiol Infect Dis 10 (1):91–97
(3–4):163–166 32. Delhon G, Tulman ER, Afonso CL, Rock DL
18. Yoon KJ, Edington N (2006) Porcine cyto- (2012) Swinepox virus. In: Zimmerman JJ,
megalovirus. In: Straw BE, Zimmerman J, Karriker LA, Ramirez A et al (eds) Diseases of
D’Allaire S et al (eds) Diseases of swine, 9th swine, 10th edn. Wiley, New York, pp 456–460
edn. Blackwell Publishing Company, Ames, IA, 33. Everett H, Crooke H, Gurrala R, Dwarka R,
pp 323–329 Kim J, Botha B, Lubisi A, Pardini A, Gers S,
19. Mengeling WL (1972) Porcine parvovirus: Vosloo W, Drew T (2011) Experimental infec-
properties and prevalence of a strain isolated tion of common warthogs (Phacochoerus afri-
in the United States. Am J Vet Res 33: canus) and bushpigs (Potamochoerus larvatus)
2239–2248 with classical swine fever virus. I: susceptibility
20. Zimmermann P, Ritzmann M, Selbitz HJ, and transmission. Transbound Emerg Dis 58
Heinritzi K, Truyen U (2006) VP1 sequences (2):128–134
of German porcine parvovirus isolates define 34. Ganges L, Crooke HR, Bohórquez JA,
two genetic lineages. J Gen Virol 87 Postel A, Sakoda Y, Becher P, Ruggli N
(2):295–301 (2020) Classical swine fever virus: the past,
21. Cartwright SF, Lucas M, Huck RA (1969) A present and future. Virus Res 289:198151
small haemagglutinating porcine DNA virus: 35. Ferris NP, King DP, Reid SM, Hutchings GH,
I. isolation and properties. J Comp Pathol 79 Shaw AE, Paton DJ, Goris N, Haas B,
(3):371–377 Hoffmann B, Brocchi E, Bugnetti M (2006)
22. Horak S, Killoran K, Leedom Larson KR Foot-and-mouth disease virus: a first inter-
(2016) Vesicular exanthema of swine virus. laboratory comparison trial to evaluate virus
Swine Health Information Center and Center isolation and RT-PCR detection methods. Vet
for Food Security and Public Health Microbiol 117(2–4):130–140
23. Coussement W, Ducatelle R, Charlier G, Hoo- 36. Dunn CS, Donaldson AI (1997) Natural adap-
rens J (1981) Adenovirus enteritis in pigs. Am J tion to pigs of a Taiwanese isolate of foot-and-
Vet Res 42(11):1905–1911 mouth disease virus. Vet Rec 141(7):174–175
24. Haig DA, Clarke MG, Pereira MS (1964) Iso- 37. Gray AR, Wood BA, Henry E, Azhar M, King
lation of an adenovirus from a pig. J Comp DP, Mioulet V (2020) Evaluation of cell lines
Pathol Ther 74:81–IN11 for the isolation of foot-and-mouth disease
25. McAdaragh JP, Eustis S, Benfield DA (1980). virus and other viruses causing vesicular dis-
Adenovirus associated with diarrhea in pigs. ease. Front Vet Sci 7:426
38. Marthaler D, Rossow K, Culhane M, Goyal S, 49. Dulac GC, Ruckerbauer GM, Boulanger P
Collins J, Matthijnssens J, Nelson M, Ciarlet M (1977) Transmissible gastroenteritis: demon-
(2014) Widespread rotavirus H in commer- stration of the virus from field specimens by
cially raised pigs, United States. Emerg Infect means of cell culture and pig inoculation. Can
Dis 20(7):1203 J Comp Med 41(4):357
39. Arnold M, Patton JT, McDonald SM (2009) 50. Bohl E (1979) Diagnosis of diarrhea in pigs
Culturing, storage, and quantification of rota- due to transmissible gastroenteritis virus or
viruses. Curr Protoc Microbiol, Chapter 15: rotavirus. In: Bricout F, Scherrer R (eds) Viral
Unit 15C.3 enteritis in humans and animals. INSERM,
40. Sanekata T, Kuwamoto Y, Akamatsu S, Paris, France, pp 341–343
Sakon N, Oseto M, Taniguchi K, Nakata S, 51. Jung K, Saif LJ, Wang Q (2020) Porcine epi-
Estes MK (1996) Isolation of group B porcine demic diarrhea virus (PEDV): an update on
rotavirus in cell culture. J Clin Microbiol 34 etiology, transmission, pathogenesis, and pre-
(3):759–761 vention and control. Virus Res 286:198045
41. Zimmerman JJ, Karriker LA, Ramirez A, 52. Shibata I, Tsuda T, Mori M, Ono M,
Schwartz KJ, Stevenson GW, Zhang J (2019) Sueyoshi M, Uruno K (2000) Isolation of por-
Diseases of swine (Eleventh Edition). Wiley- cine epidemic diarrhea virus in porcine cell cul-
Blackwell, Hoboken, NJ. Print ISBN: tures and experimental infection of pigs of
9781119350859, Online ISBN: different ages. Vet Microbiol 72
9781119350927. https://doi.org/10.1002/ (3–4):173–182
9781119350927 53. Yang DK, Kim HH, Lee SH, Yoon SS, Park JW,
42. Saif LJ, Terrett LA, Miller KL, Cross RF Cho IS (2018) Isolation and characterization
(1988) Serial propagation of porcine group C of a new porcine epidemic diarrhea virus variant
rotavirus (pararotavirus) in a continuous cell that occurred in Korea in 2014. J Vet Sci 19
line and characterization of the passaged virus. (1):71–78
J Clin Microbiol 26(7):1277–1282 54. Hofmann M, Wyler R (1988) Propagation of
43. Arias CF, Romero P, Alvarez V, Lopez S (1996) the virus of porcine epidemic diarrhea in cell
Trypsin activation pathway of rotavirus infec- culture. J Clin Microbiol 26(11):2235–2239
tivity. J Virol 70(9):5832–5839 55. Terebuh P, Olsen CW, Wright J, Klimov A,
44. Cui T, Theuns S, Desmarets LM, Xie J, De Karasin A, Todd K, Zhou H, Hall H, Xu X,
Gryse GM, Yang B, Van den Broeck W, Nau- Kniffen T, Madsen D (2010) Transmission of
wynck HJ (2018) Establishment of porcine influenza a viruses between pigs and people,
enterocyte/myofibroblast co-cultures for the Iowa, 2002–2004. Influenza Other Respir
growth of porcine Rota-and coronaviruses. Sci Viruses 4(6):387–396
Rep 8(1):1–17 56. Subbarao K, Murphy BR, Fauci AS (2006)
45. De Abin MF, Spronk G, Wagner M, Development of effective vaccines against pan-
Fitzsimmons M, Abrahante JE, Murtaugh MP demic influenza. Immunity 24(1):5–9
(2009) Comparative infection efficiency of por- 57. Webster RG, Bean WJ, Gorman OT, Chambers
cine reproductive and respiratory syndrome TM, Kawaoka Y (1992) Evolution and ecology
virus field isolates on MA104 cells and porcine of influenza a viruses. Microbiol Mol Biol Rev
alveolar macrophages. Can J Vet Res 73(3):200 56(1):152–179
46. Wu X, Qi J, Cong X, Chen L, Hu Y, Yoo D, 58. Kshatriya RM, Khara NV, Ganjiwale J, Lote
Wang G, Tian F, Li F, Sun W, Chen Z (2018) SD, Patel SN, Paliwal RP (2018) Lessons learnt
Establishment and characterization of a high from the Indian H1N1 (swine flu) epidemic:
and stable porcine CD163-expressing MARC- predictors of outcome based on epidemiologi-
145 cell line. BioMed Res Int 2018:4315861 cal and clinical profile. J Family Med Prim Care
47. Bohl EH, Kumagai T (1965) The use of cell 7(6):1506
cultures for the study of swine transmissible 59. Zhang J, Gauger PC (2020) Isolation of swine
gastroenteritis virus. In: Proceedings of 69th influenza a virus in cell cultures and Embryo-
Annual Meeting of United States livestock san- nated chicken eggs. In: Animal Influenza Virus.
itary association meeting. pp 343–350 Humana, New York, NY, pp 281–294
48. McClurkin AW, Norman JO (1966) Studies on 60. Herman M, Haugerud S, Malik YS, Goyal SM
transmissible gastroenteritis of swine: (2005) Improved in vitro cultivation of swine
II. Selected characteristics of a cytopathogenic influenza virus. Int J Appl Res Vet Med 3
virus common to five isolates from transmissi- (2):124–128
ble gastroenteritis. Can J Comp Med Vet Sci 30 61. Zuhairi FR, Maharani TM, TAN M (2012)
(7):190 The role of trypsin in the internalisation
process of influenza H1N1 virus into Vero and Center and Center for Food Security and Pub-
MDCK cell. ITB J Sci 44:297–293 lic Health
62. Williams DT, MacKenzie JS, Bingham J (2019) 65. Nardelli L, Lodetti E, Galandi GL, Burrows R,
Flaviviruses. In: Zimmerman JJ, Karriker LA, Goodridge D, Brown F, Cartwright B (1968)
Ramirez A, Schwartz KJ, Stevenson GW, A foot and mouth disease syndrome in pigs
Zhang J (eds) Diseases of Swine, 11th edn. caused by an enterovirus. Nature 219
Jon Wiley & Sons, Inc., New York, pp (5160):1275–1276
530–544 66. De Castro MP (1964) Behaviour of the foot-
63. Cartwright B, Brown F (1972) Serological and-mouth disease virus in cell cultures: sus-
relationships between different strains of vesic- ceptibility of the IB-RS-2 cell line. Arq Inst
ular stomatis virus. J Gen Virol 16 Biol 31:63–78
(3):391–398. https://doi.org/10.1099/ 67. Horak S, Leedom Larson KR (2016) Porcine
0022-1317-16-3-391. PMID: 4342823 adenovirus. Swine Health Information Center
64. Lambert T, Leedom Larson KR (2016) Vesic- and Center for Food Security and Public
ular stomatitis virus. Swine Health Information Health
Chapter 19
An Overview of Mouse Monoclonal Antibody Production

Kaushal Kishor Rajak, Kiran, Arfa Fayaz, Vishal Rai, Monu Karki,
Chris Einstein, Mukesh Bhatt, Ashok Kumar, Ajay Kumar Yadav,
and R. P. Singh
Abstract
Antibodies are an important tool in the field of diagnostics and therapeutics owing to their affinity to bind
with specific antigen. These can be of two types: polyclonal antibodies that are produced by a mixture of
various B lymphocyte clones, and monoclonal antibodies (mAbs) which are secreted by a single clone of B
lymphocytes. mAbs have a higher affinity to the target protein and are highly selective in nature which
makes them the best choice for specific purposes. Since the development of hybridoma technology in 1975,
a number of mAbs have been developed and are in use for therapeutic and diagnostic purposes. Besides the
recent advances in high throughput mAb generation technologies, hybridoma is the most preferred method
due to its nature to maintain the inherent antibody structure and functional information. This chapter
focuses on the basics of hybridoma technology including various steps involved in production of mAbs and
various critical points which must be considered while generation of monoclonal antibodies.
Key words Hybridoma, Monoclonal antibodies, Therapeutics, Diagnostics, mAb production
1 Monoclonal Antibody (mAb)
Antibodies are the serum immunoglobulins (Igs) that have ability

to bind with a particular antigen. Antibodies are therefore of enor-
mous utility in applications such as experimental biology, medicine,
biomedical research, diagnostic testing, and therapy. Polyclonal
antisera are still used in diagnostic serology and in antigen detec-
tion. However, polyclonal antibodies are often unsuitable for such
assays because they contain immunoglobulins that vary in specific-
ity, affinity, class, and subclass. The sensitivity of immunoassays such
as immunofluorescence antibody test (IFAT) or enzyme-linked
immunosorbent assays (ELISA) is limited largely by antibody affin-
ity to antigen [1, 2]. To this end, high affinity monoclonal anti-
bodies appear to be an attractive option and the antibodies with the
265
266 Kaushal Kishor Rajak et al.
highest affinity for an antigen may be obtained conveniently by

screening among pertinent monoclonal antibodies (mAbs).
[As an aside, mini-antibodies are artificial immunoglobulins
lacking CH1 domain. Both the chains of mini-antibody are
expressed separately in prokaryotic and eukaryotic expression sys-
tem linked to single chain consisting of different specificities. The
decreased size of mini-antibodies helps easy penetration of tissue
hence improved neutralization of the antigen.]
In 1975, Kohler and Milstein bio-engineered the exquisite cells
imitating the role of a single unit with capabilities to function as
natural plasma cell and immortalized myeloma cell [3]. The tech-
nique allows producing monoclonal antibody (mAb) indefinitely
with programmed specificity. However, it can only produce anti-
bodies for the specific epitope. Therefore, to produce different
antibodies targeted against different epitopes, there is a need of
increase in the number of mAb producing lymphocytes to the
desired extent. Consequently, on exposure to certain antigen
there is sensitization of lymphocyte which has the capacity to
recognize the particular antigen, and replicate to create more simi-
lar cells to produce monoclonal antibodies [4].
Myeloma cells are clones of plasma cells, a white type of blood
cell in the bone marrow and important part of immune system. B
cells mature and grow into plasma cells which are produced in large
number when body is under the attack of any microbe to produce
an army of antibodies to fight against the infection and disease
[5]. Sometimes, body loses control on the process of development
of a normal antibody-producing cell and it becomes cancerous
which leads to the formation of a tumor, loses the ability to identify
a particular antigen and causes abnormalities in the immune
response. These cells are called myeloma cells (National Research
Council 1999). Although, plasma cells are highly specialized cells as
they are under the control of biological laws and lack the capacity to
survive indefinitely in vitro. However, this control mechanism
reverses in the myeloma cells and they learn to divide infinitely [6].
2 How Hybridomas Are Produced?
The production of hybridoma is a tedious work. It starts with the

preparation of the antigen and ends with characterization of the
monoclonal antibody. Below, there is a brief description of the steps
involved in the process (Fig. 1).
2.1 Antigen/ Selection of immunizing material is very critical for production of

Immunization mAbs. For production of monoclonal antibodies, various workers
Materials have opted different material as an immunizing agent like ultra-
purified virus antigen and crude virus antigen concentrated with
poly ethylene glycol (PEG) [7, 8]. Even some people have used
An Overview of Mouse Monoclonal Antibody Production 267
Fig. 1 Outline of production of monoclonal antibody
virus infected cell culture lysate for immunization after subjecting it

to sonication and centrifugation [9, 10]. The immunization proto-
col does not show significant variations among various workers.
The immunizing material was inoculated on two, three, or more
occasions. The efficiency of the fusion varied and primarily
depended on immunizing material and the protocol of
immunization.
2.2 Production The most commonly used method for antigen production is by
of Antigen infecting the cell monolayer with virus. The culture is maintained
and checked routinely for cytopathic effect (CPE) specific to virus,
viz. structural changes in the morphology of the host cell, granula-
tion, rounding of the cell, syncytia formation, etc. When the flask
attains 90% of the CPE, the culture is harvested. The antigen
produced in bulk is precipitated using polyethylene glycol (PEG)

with sodium chloride overnight at 37 C. The solution is then
subjected to ultracentrifugation. The antigen is emulsified with
Freund’s adjuvant or other adjuvants used for immunization.
Another method is the production of recombinant antigen in
which the gene of interest is cloned in the bacterium by inserting a
positive plasmid into E. coli. The expression of recombinant protein
is followed by screening of transformant for plasmid construct with
gene of interest. The expression of recombinant protein is then
induced by IPTG (isopropyl-β-D-thiogalactoside). These bacteria
are then harvested and further lysed by sonication. For purification
of the sonicated product, it is centrifuged and washed with PBS
(Phosphate buffered saline) followed by affinity chromatography.
Sometimes, microorganisms, intact cells, and whole membranes are
also chosen for immunization [11].
Mostly, whole virus antigen is preferred for the production of
monoclonal antibodies as they are similar to the shape and structure
which gives us monoclonal antibody with specificity to diverse
epitope. However, this process is cumbersome as it becomes
tedious while screening and expansion for all the positive clones
obtained after fusion. Hence, some researchers prefer recombinant
virus particle as they provide antibodies against specific epitope.
However, it has its own drawback in the sense that it is only
preferred where the antigen is used as a positive control and
required in bulk.
2.3 Mice for mAb Generally, Balb/c mice of 4–6 weeks of age of either sex weighing
Production 16–20 g are used for production of monoclonal antibodies.
Although swiss albino mice can also be used, this may result in
low efficacy of mAb production as compared to Balb/c mice. In
specific cases, immunocompromised (e.g., severe combined immu-
nodeficient [SCID]) mice or other animal species such as the rat
and hamster may also be used [6]. The rationale behind choosing
the BALB/c mice is their capability to induce humoral immune
response and production of plasmacytoma upon injection with
mineral oil which are important for monoclonal antibody produc-
tion. Further, as myeloma cell lines are of BALB/c mice origin and
the ascites fluid from hybridomas is grown in mice which should
share BALB/c histocompatibility, BALB/c mice appear to be the
best choice for immunization to prevent the tumor rejection [12].
2.4 Immunization Firstly, mice are primed with the antigen and then boosters are
of Mice given every 2–3 weeks to produce the desired antibody titer. After
several weeks of immunization, mice are assessed for serum anti-
body titer by collecting the blood samples. Serum antibody titer is
determined by various techniques including serum neutralization
test (SNT), ELISA, flow cytometry, etc. If the antibody titer is high
enough, then mice are boosted 2–3 days before the fusion (Fig. 2).
Fig. 2 Schematic representation of principle steps involved in monoclonal antibody production
2.5 Revival Myeloma cells are revived from liquid nitrogen at the required time
of Myeloma Cells and maintained for fusion. Generally, myeloma cells are grown with
and Feeder Cells 8-azaguanine or 6-thioguanine to ensure their sensitivity for HAT
Preparation (Hypoxanthine Aminopterin Thymidine) media. Myeloma cells
used for fusion could be HGPRT (hypoxanthine-guanine phos-
phoribosyltransferase) negative or TK (thymidine kinase) negative
that means they cannot use the salvage pathway for nucleotide
synthesis [13] as per Table 1.
Additionally, aminopterin in medium blocks the pathway that
allows for nucleotide synthesis via de novo pathway. Hence,
unfused myeloma cell dies because they are unable to synthesize
nucleotide. Although unfused B cells can synthesize nucleotide via
salvage pathway, they die because of the limited life span. Only the
fused cell survives in the HAT medium. The feeder cells are basi-
cally the peritoneal macrophages that are collected from the mice
and seeded approximately 48–72 h before the fusion.
2.6 Fusion The immunized mice are sedated using volatile ether/chloroform
and spleen is taken out from the peritoneal cavity followed by
collection of sensitized splenocytes under aseptic conditions. Both
the myeloma cells and spleen lymphocytes are counted on Neu-
bauer’s chamber followed by dilution in the ratio of 1:10, respec-
tively for fusion. Fusion is completed with both myeloma and
spleen cells in polyethylene glycol (PEG) strictly at 37 C which
helps in cell membrane fusion [14]. Then, fused cells are incubated
in the HAT medium. The fused cells are screened for reactivity with
Table 1
Sensitivity of different cells to HAT media
HAT
Genotype Cell type medium Result
TK or HGPRT Immortal Dies Unable to synthesize nucleotide
cell
TK+TK or Fused Survives Indefinite life span with ability to synthesize
HGPRT+HGPRT hybrid nucleotide
TK +or HGPRT+ Mortal cell Dies Mortal
Note: TK Thymidine Kinase, HGPRT Hypoxanthine-guanine phosphoribosyl transferase
the antigen and selected for expansion in a 25 cm2 flask. Positive

clones are amplified and preserved in liquid nitrogen.
2.7 Single-Cell The positive clones are subjected to single-cell cloning and
Cloning sub-cloning using limiting dilution method in 96-well plate
[15]. Single-cell cloning is done twice to confirm the monoclon-
ality of hybridomas clones. These clones are expanded and pre-
served in liquid nitrogen. An adequate number of monoclonal
antibodies are produced via ascitic fluid and subsequently used for
characterization.
2.8 Bulk Production The mAbs are produced in bulk by mainly two methods, viz. cell
culture and ascites fluid method (National Research Council
1999). Though cell culture method, wherein culture supernatant
is used as mAb, is easy and cheap compared to the other (ascites
fluid), ascites fluid method gives highly concentrated biological
material which are mostly used for development of pen-side diag-
nosis/chromatographic strip tests. Mice ascites are useful in an
experiment where an adequate amount of mAb is required for
improved efficacy or specificity. Mice are injected intraperitoneally
with an immunological adjuvant like pristane (0.5–1 ml per mouse)
prior to the implantation. Clones are suspended in serum free
media (SFM) or PBS and injected into the peritoneal cavity of
mice. Dosage can be increased for subsequent formation of ascites.
Mice are examined daily for the growth of tumor in the belly.
Generally, mice become morbid after 1–4 weeks. Ascitic fluid high
in concentration of mAb is harvested with humane techniques.
Halder and some other scientists quoted that ascitic monoclonal
antibody contains other factors like cytokines, which could render
the use of ascitic fluids “scientifically wrong” [16]. However, where
purity is not a concern this method is widely used.
2.9 Purification The monoclonal antibody produced from mouse ascitic fluid is
subsequently purified. Some ascitic fluid contains blood clots that
should be removed by centrifugation. The whitish lipid layer and
pristane over the ascitic fluid are also removed after centrifugation.
The aliquots are pooled and immunoglobulins are precipitated
using different commercially available kits following the manufac-
turer’s instruction. This method provides antibodies with high
purity. Further antibody purification can be done via an ion
exchange column or affinity chromatography. These purified anti-
bodies are tested for antibody/protein titer by either ELISA or
protein estimation kits (Lawry or Bradford method).
3 Characterization of mAb
After successful selection, monoclonal antibodies are harvested.

The mAbs are characterized for structural characterization (like
amino acid sequence, peptide map, sulfhydryl groups, disulfide
bridges, and carbohydrate structure) and physiochemical character-
ization (like weight, size, isoform patterns, etc.) of antibody. The
main parameters for characterization of monoclonal antibodies
chosen by various workers are class and subclass determination,
protein specificity of mAbs using RIPA and western blot assays,
virus neutralization activity of mAbs and reactivity of mAbs in
ELISA test with various isolates of the same virus and other viruses
of same genus. The class and subclass of the monoclonal antibodies
are determined using commercially available kits. These studies are
helpful when the monoclonal antibodies are subjected to purifica-
tion for the purpose of labeling with enzymes or biotin. Different
classes of antibodies have been shown to have different binding and
elution profiles in protein-A chromatography [17]. Radio immune-
precipitation assay followed by SDS-PAGE and autoradiography
has been extensively used for determining protein/antigenic speci-
ficity of monoclonal antibodies. However, various simple and easy
methods using non-radioactive labels have also been developed for
identifying the specificity of monoclonal antibodies at an early stage
[18, 19]. Immunofluorescence assay has been used to assess the
reactivity of monoclonal antibodies with different viruses and to
determine the virus specificity of mAbs [9, 20].
4 Application of mAbs in Diagnosis
Ever since the development of Hybridoma Technology in 1975 by

Kohler and Milstein, the vision for antibodies as tools for research
in animal disease diagnosis, antigenic characterization of patho-
gens, vaccine production and in the study of genetic regulation of
immune responses has been revolutionized. A very distinctive
advantage in production of hybridomas is generation of specific

antibody using a mixture of antigen. The monoclonal antibodies
directed against single epitopes are highly specific in nature and in
addition, these can be produced in huge quantities without the use
of animals.
4.1 Detection For a sensitive and rapid diagnosis of diseases, monoclonal antibo-
of Antigen dies have been used in different test formats and offer an added
and Antibody advantage of specifically binding with the target. Monoclonal anti-
bodies have been deployed in different tests like ELISA and lateral
flow assays for detection of viral antigen in tissues, secretions (con-
junctival and nasal swabs), lymphocytes, and blood by various
workers in different tests (Potgeiter et al., 1989; [21–23]). Another
approach of using mAb-based ELISA for antigen detection is cell-
ELISA which is a very simple, sensitive, rapid, inexpensive assay that
has been used for detection and quantification of virus infected cells
using monoclonal antibody directed against one of the viral pro-
teins. The cell-ELISA has been used for quantification of molecules
that are being expressed on the surface of cells [24]. The technique
was developed based on two basic immunological methods, i.e.,
(a) Immunohistochemistry: which involves identification/localiza-
tion of antigen in tissue sections and (b) ELISA: which helps in
quantification of soluble antibodies/antigen by making one of the
immune-reactant immobilized on a solid phase. One of the most
important uses of cell-ELISA has been in hybridoma technology for
rapid screening of virus-specific hybridoma clones after fusion.
Several tests have been developed for the detection of antibo-
dies against particular pathogen. The mAb-based detection of anti-
body employing assays like ELISA or chromatographic strip test is
coming in big way for detection of seroconversion due to vaccina-
tion or infection.
4.2 Antigenic Various studies in the past have shown by the use of monoclonal
Profiling of Viruses antibodies that viruses exhibit antigenic differences between the
strains [25–28]. mAbs play crucial role in differentiating viruses of
same family, genus, or even the vaccine and wild type strains of same
virus species. So, antigenic profiling of viruses using mAbs is very
useful to know their antigenic behavior which help in designing the
diagnostics and prophylaxis for better control of the diseases.
5 Conclusion
Monoclonal antibodies have proven to be very useful tool especially

in diagnostic assays. mAbs enable researchers to easily identify and
characterize various pathogens and have therefore revolutionized
the diagnostic science in all fields. The use of monoclonal antibo-
dies in diagnostic platforms like lateral flow assay/chromatographic
strip test has changed the field of diagnosis. Since mAbs are directed
against a single epitope, multiple antigenic determinants can be
identified from a complex mixture by using the approach of multi-
plex LFA tests. One of the blooming topics nowadays is DIVA-
based diagnostic strategy. Using mAbs of distinct epitope can help
in devising an epitope-based marker vaccine. Monoclonal antibo-
dies also find their utility in developing anti-idiotypic vaccines using
an anti-idiotypic mAb which mimics an antigen and can prove to be
more immunogenic as compared to the original one.
References
1. Avrameas S (1983) Enzyme immunoassays and protein. Comp Immunol Microbiol Infect Dis
related techniques: development and 29:157–165
limitations. In: Bachmann PA (ed) New devel- 11. Parray HA, Shukla S, Samal S, Shrivastava T,
opments in diagnostic virology. Springer- Ahmed S, Sharma C, Kumar R (2020) Hybrid-
Verlag, New York, NY, pp 93–99 oma technology a versatile method for isolation
2. Yolken RH (1983) Use of monoclonal antibo- of monoclonal antibodies, its applicability
dies for viral diagnosis. Curr Top Microbial across species, limitations, advancement and
Immunol 104:177–l95 future perspectives. Int Immunopharmacol
3. Köhler G, Milstein C (1975) Continuous cul- 85:106639
tures of fused cells secreting antibody of pre- 12. Lerner EA (1981) How to make a hybridoma.
defined specificity. Nature 256:495–497. Yale J Biol Med 54(5):387–402
https://doi.org/10.1038/256495a0 13. Hundekar YR, Chimkode RM, Salagare MV
4. Pandey S (2010) Hybridoma technology for (2017) Hybridoma technology: a new scientific
production of monoclonal antibodies. Int J tool for AIDS recovery. Int J Pharm Pharm Sci
Pharm Sci Rev Res 1(2):88–94 9(4):1–16
5. Alberts B, Johnson A, Lewis J et al (2002) B 14. Greenfield EA (2018) Screening for good
cells and antibodies. In: Molecular biology of batches of fetal bovine serum for myeloma
the cell, 4th edn. Garland Science, New York and Hybridoma growth. Cold Spring Harb
6. Leenaars M, Hendriksen CF (2005) Critical Protoc 1(3). https://doi.org/10.1101/pdb.
steps in the production of polyclonal and prot103150
monoclonal antibodies: evaluation and recom- 15. Fuller SA, Takahashi M, Hurrell JGR (2001)
mendations. ILAR J 46(3):269–279 Cloning of Hybridoma cell lines by limiting
7. Potgieter LN, Ajidagba PA (1989a) Quantita- dilution. Curr Protoc Mol Biol; Chapter 11:
tion of canine distemper virus and antibodies U n i t 1 1 . 8 . h t t p s : // d o i . o r g / 1 0 . 1 0 0 2 /
by enzyme-linked immunosorbent assays using 0471142727.mb1108s01
protein a and monoclonal antibody capture. J 16. Halder H, Embelton M, Fischer R, de Geus B,
Vet Diagn Investig 1:110–115 Hendriksen C, de Leeuw W, Marx U, Balls M
8. Singh RP, Bandyopadhyay SK, Sreenivasa BP, (1998) Comments on appendix C of the
Dhar P (2004) Production and characteriza- National Institutes of Health response to the
tion of monoclonal antibodies to pests des petition of the American anti-vivisection soci-
Petitis Ruminanats (PPR) virus. Vet Res Com- ety to prohibit the use of animals in the pro-
mun 28(7):623–639 duction of monoclonal antibodies. Altern Lab
9. Liu Y, Hao L, Li X, Wang L, Zhang J, Deng J, Anim 26(4):549–554
Tian K (2017) Development and characteriza- 17. Seppala IJT, Sarvas H, Peterfy H, Makala O
tion of canine distemper virus monoclonal (1981) The four subclasses of IgG can be
antibodies. Monoclon Antib Immunodiagn isolated from mouse serum by using protein
Immunother 36:119–123 A-Sepharose. Scand J Immunol 14:335–342
10. Masuda M, Sato H, Kamata H, Katsuo T, 18. Cole SR, Ashman LK, Ey PL (1987) Biotinyla-
Takenaka A, Miura R, Yoneda M, Tsukiyama- tion: an alternative to radioiodination for the
Kohara K, Mizumoto K, Kai C (2006) Charac- identification of cell surface antigens in immu-
terization of monoclonal antibodies directed noprecipitates. Mol Immunol 24:699
against the canine distemper virus nucleocapsid
19. Schuh R, Kremmer E, Ego E, Wasiliu M, distemper virus. Appl Microbiol Biotechnol
Thierfeldcr S (1992) Determination of mono- 104:10725–10735
clonal antibody specificity by immunoadsorp- 24. Ogino T, Wang X, Ferrone S (2003) Modified
tion and Western blotting. J Immunol flow cytometry and cell-ELISA methodology
Methods 152:9–67 to detect HLA class I antigen processing
20. Bi Z, Xia X, Wang Y, Mei Y (2015) Develop- machinery components in cytoplasm and endo-
ment and characterization of neutralizing plasmic reticulum. J Immunol Methods 278:
monoclonal antibodies against canine distem- 33–44
per virus hemagglutinin protein. Microbiol 25. Giraudon P, Wild TF (1981) Differentiation of
Immunol 59:202–208 measles virus strains and a strain of canine dis-
21. An DJ, Kim TY, Song DS, Kang BK, Park BK temper virus by monoclonal antibodies. J Gen
(2008) An immunochromatography assay for Virol 57:179–183
rapid antemortem diagnosis of dogs suspected 26. Russell PH, Alexander DJ (1983) Antigenic
to have canine distemper. J Virol Methods 147: variation of Newcastle disease virus strains
244–249 detected by monoclonal antibodies. Arch
22. Li Y, Cheng S, Xu H, Wang J, Cheng Y, Yang S, Virol 75(4):243–253
Luo B (2012) Development of a combined 27. Sheshberadaran H, Chen SN, Norrby E (1983)
canine distemper virus specific RT-PCR proto- Monoclonal antibodies against five structural
col for the differentiation of infected and vac- components of measles
cinated animals (DIVA) and genetic virus. I. Characterisation of antigenic determi-
characterization of the hemagglutinin gene of nants on nine strains of measles virus. Virology
seven Chinese strains demonstrated in dogs. J 128:341–353
Virol Methods 179:281–287 28. Ter Meulen V, Loffler S, Carter MJ, Stephen-
23. Zhang Y, Xu G, Zhang L, Zhao J, Ji P, Li Y, son JR (1981) Antigenic characterization of
Liu B, Zhang J, Zhao Q, Sun Y, Zhou E measles and SSPE virus Haemagglutinin by
(2020) Development of a double monoclonal monoclonal antibodies. J Gen Virol 57:357–
antibody–based sandwich enzyme-linked 364
immunosorbent assay for detecting canine
Chapter 20
Nucleic Acid Hybridization Techniques for Viral Disease

Diagnosis: A Detailed Perspective
B. V. Sunil Kumar, Himalaya Bhardwaj, Ankita Gurao, Naveen Kumar,
and Yashpal Singh Malik
Abstract
Despite the newer techniques that offer rapid diagnosis of viral diseases by demonstration of viral antigen or
viral nucleic acid in the specimens, virus isolation in cell culture remains the gold standard against which
newer methods must be compared. But virus isolation requires stringent attention by the clinician and is
time-consuming. Hybridization techniques offer direct detection of viral nucleic acid in the clinical speci-
mens and in tissue sections. However, hybridization techniques are still not widely used in the clinical
laboratory. Recent developments in molecular cloning and the development of non-radioactive probes have
made hybridization techniques more accessible. In this chapter, we present an overview of the various
nucleic acid hybridization techniques used for diagnosis of viral diseases. Emphasis has been made on
optimization of different factors for better results. Southern blotting, northern blotting, and spot hybri-
dization techniques have been detailed with respect to their utility in disease diagnosis. Since a hybridization
experiment depends on various effectors, modification of any one effector usually impacts several others.
Because of this complex interplay of cause and effect, thorough optimization of the experiment is required
to ensure accurate results.
Key words Nucleic acid hybridization, Viral diagnosis, Sensitivity, Specificity, Southern blotting,
Northern blotting, Spot hybridization, Probes, Labeling
1 Introduction
The significance of viral infections has been overwhelmingly recog-

nized in recent years, primarily as a cause of mortality and morbidity
among immunocompromised subjects. The awareness on the part
of people and the medical fraternity of the importance of various
viral infections such as herpes infection or acquired immunodefi-
ciency syndrome (AIDS) or recent coronavirus disease (COVID-
19) has contributed to the spread of viral diagnostic laboratories in
community hospitals and universities [1, 2]. The number of com-
mercial companies dedicated to the manufacture of diagnostic kits
has also increased.
275
276 B. V. Sunil Kumar et al.
To achieve an effective therapy, it must be tailored to the

specific disease and should begun at an earlier stage of the disease
[1, 3]. In order to do this, a prompt and accurate diagnosis of
infection must be made. With the availability of newer antiviral
agents and other therapeutic methods, the physicians also need
precise laboratory diagnosis of their patients’ illnesses which has
led to the burgeoning of viral diagnostic facilities. In addition,
medical professionals themselves should be well acquainted with
the methods for diagnosing viral disease for better
interpretation [1].
Many of the newer viral diagnostic assays are also compared to
the traditional virus isolation technique which remains the “gold
standard” test for most of the newer methods, but the conventional
virus isolation technique is labor-intensive, time-consuming and
requires maintenance of a dedicated cell culture facility. The identi-
fication of viral antigens and nucleic acids is increasingly challenging
the traditional paradigm [4]. Many rapid “same day” diagnostic
methods have been used for viral diagnosis, viz. viral cytopathology,
electron microscopy, immunofluorescence (IF), enzyme immuno-
assay (EIA), polymerase chain reaction (PCR), serology, and
nucleic acid hybridization.
Nucleic acid hybridization is used to identify particular nucleic
acid sequences using specific denatured oligonucleotide probes that
are typically chemically synthesized and fluorescently or radio
labeled [5]. Recent developments in molecular cloning and the
development of non-radioactive probes have made hybridization
techniques more accessible. Availability of extensive cloned viral
DNA has made the nucleic acid hybridization technique more
feasible for the detection of such viruses in clinical specimens
[6]. Various hybridization tests are available commercially for the
identification of herpes simplex virus, cytomegalo virus (CMV),
and human papillomavirus (HPV). Probes for hepatitis B virus,
human immunodeficiency virus (HIV), Epstein–Barr virus (EBV),
adenovirus, and type A rotavirus are also currently available [6]. In
this chapter, our discussion will be limited to nucleic acid hybridi-
zation (NAH) and its role in viral diagnosis. In order to achieve
optimal results in NAH, all the steps from the sample collection to
the analysis of results need to be optimized.
2 Sample Collection
Sample collection still remains a critical step for NAH, as for any
other laboratory diagnostic technique. Ultimate care should be
given to any minute detail such as timing, collection, handling,
etc., otherwise the results may be misinterpreted. In order to get
successful virus isolation, samples should be collected within a few
days after the symptoms begin [6]. Depending on the type of
Nucleic Acid Hybridization Techniques for Viral Disease Diagnosis... 277
infection, different samples, such as swabs of mucosal surfaces, skin

scrapings, tissues, or some other biological fluid, may be collected
for diagnosis. However, sterility should be taken care of for any
sample, and the sample should be transported quickly to the
concerned laboratory at ice-cold temperature (preferably in a
virus transport medium with antibiotics), as any delay in transport
could lead to degradation of the viral nucleic acid or the virus itself.
The type of sample to be collected should be correlated with the
clinical presentation, and in the case of unknown disease, it is often
advised to collect samples from multiple sites. If rhinovirus, para-
influenza, influenza, adenovirus, enterovirus, adenovirus, rotavirus,
norovirus, etc., are suspected, samples of throat swabs, stools,
urine, or serum should be collected, while samples of vesicular
fluid or serum are preferred for suspected infections with varicella-
zoster virus, and herpes simplex virus (HSV-1 and HSV-2) [6].
3 Sample Preparation
The collected sample needs to be processed prior to the hybridiza-

tion step. There are different sample processing methods that
depend on the type of hybridization experiment and the sample
type itself.
3.1 Stool Samples The stool samples should be diluted in 10% phosphate-buffered
saline (PBS) and centrifuged for 10 min at a maximum speed; the
3.1.1 RNA Isolation [7]
supernatant should be stored at 20 C for further use. The
supernatant has to be mixed with equal volume of phenol-
chloroform (3:2 v/v) mixture and mixed well for 1 min. The final
mixture should be centrifuged at 1200 g for 10 min, and the
resulting clear upper aqueous layer should be removed and treated
with 0.5 mL of isopropanol. After incubation at room temperature
for 20 min, the tube should be centrifuged again at 14,000 g for
10 min at 4 C. The supernatant should be discarded and the pellet
containing RNA washed with 0.5 mL of 75% (v/v) ethanol by
centrifuging at 5000 g for 5 min at 4 C. After removal of the
supernatant, the pellet is finally allowed to air-dry briefly and sus-
pended in an appropriate volume of nuclease-free water. To dena-
ture the RNA, the samples should be incubated at 65 C for
10 min, and snap chilled on ice for 2 min.
3.1.2 DNA Isolation Approximately 1–1.5 g of fecal sample is needed for fecal DNA
extraction, which is vortexed and washed using 5 mL ethanol and
centrifuged at 4000 g for 2 min. The pellet should be washed
with 5 mL Tris EDTA (TE) buffer, then 3 μL of TNE buffer
(10 mmol/L Tris-Cl, 0.5% SDS, and 1 mmol/L CaCl2) and
50 μL of Proteinase K (20 mg/mL) are added to the pellet. The
whole mixture must be incubated at 55 C for 1–2 h. Then, the
lysate is centrifuged at 4000 g for 1 min and the supernatant to be

collected. The supernatant is mixed with 3 g of potato starch and
vortexed for 1 min to suspend the particles properly. Further, the
mixture should be incubated at room temperature for 1 min and
centrifuged at 8000 g for 3 min. The supernatant (600 μL)
should be mixed with 150 μL NaCl solution (3.5 mol/L) and
250 μL Cetyl trimethyl ammonium bromide (CTAB) solution
(10% CTAB +0.7 M NaCl). The step is followed by incubation at
70 C for 10 min. The mixture is extracted twice using equal
amount of phenol: chloroform: isoamyl alcohol (25:24:1 v/v)
and equal volume of binding buffer (4 M guanidine hydrochloride,
1 M potassium acetate, pH 5.5). Further, any spin column-based
kit for DNA extraction can be used to process the mixture.
3.2 Nasal Swabs After the collection of nasal swabs in RNAlater, the collection tube
should be briefly vortexed and mixed. Later, after 15 min of centri-
3.2.1 RNA Isolation [8]
fugation at the maximum speed, the RNAlater is decanted and
collected in a microcentrifuge tube. The pellet must then be resus-
pended in 500 μL of TRIzol and incubated for 1 h at room
temperature. Further RNA isolation steps should be carried out
following the TRIzol manufacturer’s instructions. All the centrifu-
gation steps should be performed at 19,000 g at 4 C. RNA is
precipitated with one volume of chilled isopropanol, 10% of 3 M
sodium acetate, and 1 μL of glycogen at 20 C. Extracted RNA is
resuspended in a sufficient volume of nuclease-free water.
3.2.2 DNA Isolation DNA can be isolated from the processed samples using any stan-
dard kit or follow the protocol mentioned in Subheading 3.1.2.
4 Probes
4.1 Preparation A probe is essentially a primer which is chemically bonded to a label

and General at its end. The DNA probes are short, single-stranded, labeled
Considerations DNA sequences used to detect the presence or absence of the
nucleic acid of interest in a sample. The probe corresponding to a
specific gene or part of genome which is highly conserved in the
virus, can be labeled in either ways, radioactive or non-radioactive.
Similarly, synthesis of a probe can be achieved using in vitro synthe-
sis or polymerase chain reaction (PCR) amplification. For preparing
the probe, molecular cloning is an easy yet powerful tool. Viruses
with smaller genome size can be cloned into plasmid vector and
those with a larger genome size using the cosmid or yeast artificial
chromosome (YAC).
Once the viral DNA fragments are cloned, they can be used for
preparing genomic libraries that serve as a potential repository for
choosing the probes. It is possible to obtain DNA probes from
RNA viruses by synthesizing cDNA from the viral RNA [9, 10]. In
general, the gene probes are longer than 500 bases and can be
synthesized by cloning or direct amplification from genes of interest
using PCR. The oligonucleotide probes, on the other hand, are
usually 15–30 bases and offer more precise hybridization with the
target. For both the aforesaid probes, some general requirements,
including GC content of 40–60%, absence of self-complementarity
within the probe, and <70% homologies to non-target sequence
should be met [11]. The labeling protocols are described in depth
below.
4.2 Probe Labeling Several isotopes have been utilized for labeling the probes, which
include 3H, 32P, 35S, and125I, for which detection can be done
4.2.1 Radioactive
using autoradiography or Gieger–Muller counter. In the recent
Labeled Probes
times, however, the use of radioactive labeled probes has drastically
reduced considering their disposal and safety.
Classically, for synthetic oligonucleotides, end labeling is most
widely used, although if 50 overhangs are present, double-stranded
DNA can also be labeled. Using the Maxam–Gilbert chemical
cleavage method, end-labeled oligonucleotides are used for hybri-
dization probes, primer extension analysis, or DNA sequencing.
Bacteriophage T4 polynucleotide kinase could be used for labeling
the 50 ends of DNA (or RNA) with γ[32P]ATP. Polynucleotide
kinase transfers the γ-phosphate group from ATP to the free 50
hydroxyl group of DNA (or RNA). Dephosphorylation of the 50
phosphate group is required prior to labeling using calf intestinal
alkaline phosphatase. The dephosphorylated substrate is then
re-phosphorylated by transfer of γ-phosphate from γ[32P]ATP.
Terminal transferase (terminal deoxynucleotidyl transferase) could
be employed for labeling 30 ends of DNA, with which transfers
dNTPs to free hydroxyl groups at the 30 ends of DNA. End-labeled
probes generated with polynucleotide kinase have relatively low
specific activity because only one mole of 32P is incorporated per
mole of template. Since terminal transferase can add many nucleo-
tides to each molecule of template, this enzyme is frequently pre-
ferred when higher specific activity is required, e.g., in situ
hybridization [12]. Nowadays, many brands use Klenow fragment
for incorporation of radiolabels into the probes.
4.2.2 Non-radioactive The non-radioactive labeling of probes is preferred over the radio-
Labeled Probes active counterpart because they are safer and highly stable. In recent
times, many non-radioactive labeling methods have been devel-
oped including biotin-avidin/streptavidin, enzyme labeling, e.g.,
horseradish peroxidase (HRP), fluorescent labeling of probes,
digoxigenin (DIG) system, etc. The following protocol discusses
30 end labeling with DIG-ddUTP or Biotin-ddUTP.
Digoxigenin is a steroid of plant origin; this molecule has been
widely used as a hapten and can be conjugated with nucleic acids
using conjugation reaction. The protocol for oligonucleotide
30 -end labeling with DIG-ddUTP or Biotin-ddUTP is as follows

[13]. Dissolve the oligonucleotide to be labeled in nuclease-free
water and prepare 1 mM solution of DIG-ddUTP [available com-
mercially as Digoxigenin-3-O-methylcarbonyl-ε-aminocaproyl-{5-
(3-aminopropargyl)-20 ,30 -dideoxyuridine-50 -triphosphate},
Triethylammonium salt]. Next, add 4 μL of 5 concentrated reac-
tion buffer [1 M potassium cacodylate, 0.125 M Tris-HCL,
1.25 mg/mL Bovine Serum Albumin (pH 6.6)]. Add 4 μL of
CoCl2, 100 pmol of oligonucleotide, and 1 μL of DIG-ddUTP.
Further add 1 μL of terminal transferase (50 U) and nuclease-free
water to a final volume of 20 μL. Mix well, centrifuge, and incubate
at 37 C followed by snap chilling. To stop the reaction, add 2 μL of
EDTA-glycogen mixture (200 μL of 0.2 M EDTA (pH 8.0) mixed
in 1 μL of 20 mg/mL glycogen). Finally, precipitate the labeled
oligonucleotide.
5 Optimizing Conditions for Hybridization
The hybridization step is very important for any nucleic acid detec-
tion technique, whether DNA or RNA. However, as it needs a lot
of optimization and standardization, there is no common protocol
for hybridization. Optimization involves playing with the choices of
buffers required in the method: temperature conditions and dura-
tion of the experiment. Another group of effectors that need to be
optimized in a series of experiments for better results are the type of
membranes, probes, and the target constitute [14]. Suboptimal
conditions dramatically decrease the efficacy of all assays by intro-
ducing considerable bias [15].
5.1 Temperature One of the key determinants of a hybridization experiment is the

hybridization temperature, which relies on the melting temperature
(Tm) of the probe, and the nature of the target. Although the Tm
may be determined using different formulas depending on the type
of the probe and the target-probe hybrid, it is often advisable to
obtain it empirically as different variables (type of labels, probes, or
buffer composition) influence the melting temperature and conse-
quently the end point of the experiment. A smaller base modifica-
tion, or non-radioactive tags, for example, can change the
characteristics of the probe and the Tm [16]. In addition, at higher
temperatures, probes with horse radish peroxidase (HRPO) tag are
less stable than those with alkaline phosphatase. Therefore, lower
hybridization temperatures or addition of thermal stabilizers such
as trehalose to the buffers can be considered for improved label
stability when using HRPO tagged probes [14]. The type of probes
also determines its melting temperature, with RNA probes having a
higher Tm as compared to their DNA counterparts. However, due
to the instability of RNA at higher temperatures, the buffers may
alternatively be supplemented with denaturing agents like such as

formamide or urea to allow hybridization at a lower
temperature [14].
Enzyme-coupled probes should be hybridized at the lowest
possible temperature to warrant enzyme stability. However, lower
hybridization temperature compromises with binding specificity
and can lead to background noise, while too higher hybridization
temperature compromises with the strength of end signal
[15]. Between particular signal strength and background levels, a
trade-off often lies. Therefore, for each experiment, the optimum
hybridization temperature should be standardized and the accept-
able limit of background noise should also be determined
accordingly [15].
5.2 Probe The probe concentration is application dependent, but it is usually

Concentration kept higher than the target concentration. The probe concentra-
tion should be selected in such a way that minimum background
noise and optimum specific signal strength are present. In general,
in the absence of any rate enhancers, a probe concentration is
5–10 ng/mL of the hybridization buffer, whereas rate accelerating
buffers require a reduction in the concentration of the probe to
0.1–5 ng/mL of hybridization buffer [14, 17].
5.3 Hybridization For majority of the probe-target complexes, overnight hybridiza-

Time tion should work well. There are some determinants, however,
which determine the optimum duration of hybridization. For
instance, shorter single-stranded probes need less time compared
to longer double-stranded ones for hybridization [17]. Higher
probe concentration buffers (more than 10 ng/mL) and rate
enhancers also facilitate shorter hybridization times [14].
5.4 Buffer Denaturing buffers such as formamide buffers are preferred for
Components thermolabile probes, membranes, or labels over salt/detergent
buffers. Formamide (30% to 80%), urea (3 M to 6 M), ethylene
glycol, sodium perchlorate (2 M to 4 M), or tertiary alkylamine
chloride salts could be used as denaturants [18]. The stability of
probe-target hybrids is influenced by the cations of the salts that
counteract the repulsive forces between the probe phosphodiester
bonds and target and thus stabilize the hybrid formed with the
target. By including sodium chloride upto a final concentration of
1.2 mM, stability may be improved and 80 to 90 mM citrate buffer
or 50 mM sodium phosphate buffer are other alternatives for
improving the stability [14, 19, 20]. Detergents such as 1% to 7%
sodium dodecyl sulfate (SDS), 0.05% to 0.1% Tween-20, 0.1%
N-lauroylsarcosine, or Nonidet P-40, blocking reagents such as
bovine serum albumin (BSA), skimmed milk powder, and genomic
DNA (calf thymus, herring, or salmon sperm), or poly A could be
included in the buffer to prevent non-specific binding on to the
membrane [21].
Hybridization rate enhancers/accelerators such as dextran sul-

fate, ficoll, and polyethylene glycol if included in the hybridization
buffers, significantly shorten the hybridization time. These enhan-
cers work by reducing the free water molecules (volume excluders)
and effectively increasing the probe concentration per mL of the
buffer. Their concentration can be determined empirically, based
on the acceptable background noise limit [14].
6 Nucleic Acid Hybridization Protocol
6.1 Southern Blotting The following buffers have been standardized for Southern blot-
Using Radioactive ting by [22]:
Labeled Probe
A. 20 saline sodium citrate (SSC) C. Neutralizing solution
solution 500 mL of 1 M Tris-HCl
175.3 g NaCl (pH 7.4) solution
88.2 g sodium citrate 300 mL of 5 M NaCl solution
Bring the final volume to 1 L Bring to 1 L with double distilled
with double distilled water. water.
B. Denaturing solution D. Nylon wash solution (pH 7.2)
300 mL of 5 M NaCl solution 40.6 g Na2HPO4
100 mL of 5 M NaOH 18.65 g EDTA
solution 500 g SDS
Bring to 1 L with double Bring the final volume to 3.58 L
distilled water. with double distilled water.
6.1.1 Protocol Digest the extracted DNA with one or multiple restriction
enzymes. Separate the digested DNA on the agarose gel (percent-
age of agarose according to the band size of interest) at 30–50 V
and remove the gel from the electrophoresis tank and incubate for
30 min in the denaturing solution on a platform shaker at approxi-
mately 25 rpm. Rinse the gel twice in double distilled H2O. Again,
incubate the gel for 30 min in neutralizing solution on a platform
shaker at approximately 25 rpm. Re-incubate the gel in fresh neu-
tralizing solution for 30 min. Rinse the gel twice with double
distilled H2O. Before assembling the Southern blot setup, prepare
the SSC buffer. Essentially, the assembly has the neutralized gel
sandwiched in between a nylon membrane and Whatman filter
paper, while the other Whatman filter paper is positioned above
the nylon membrane. Multiple paper towels are stacked and a glass
plate is placed over it topped with 200–500 g of weight (Fig. 1).
The entire setup is kept in a tray filled with 20SSC buffer. Incu-
bate the stacked gel overnight to allow capillary transfer of the
nucleic acid from the gel to the nylon membrane. Next day, disas-
semble the setup and remove the membrane followed by rinsing
with 2SSC and air-dry. The DNA embedded membrane is
Fig. 1 Schematic representation of Southern blotting setup
exposed to UV light to aid cross-linking. Pre-hybridize the mem-

brane in 20 mL 0.75 nylon wash solution (sodium phosphate
dibasic, ethylenediaminetetraacetic acid, sodium dodecyl sulfate)
for 1 h at 65 C. Add the 32P labeled probe to the hybridization
mixture and incubate overnight at 65 C. Wash membrane conse-
quently in 0.5 nylon and 0.1 nylon wash solution for 20 min at
65 C every time. Expose the membrane to the Phospho imager
screen for at least 2 h and record it.
6.2 Northern Blotting The following buffers may be required for Northern blotting.
Using Radioactive
A. 10MOPS (500 mL): B. Loading buffer (1000 μL):
Labeled Probes
41.9 g MOPS 100 μL 10 MOPS
6.8 g NaAc 500 μL Formamide
10 mL 0.5 M EDTA 185 μL Formaldehyde
Store away from light at 40 mg Ficoll 400
4 C Bromophenol blue
215 μL H2O
Store at 20 C
C. Pre-hybridization buffer D. Hybridization buffer:
(100 mL): Pre-hybridization buffer with 5%
25 mL 20 SSC dextran sulfate and without
50 mL Formamide non-homologous DNA
5 mL 100 Denhardt’s
1 g SDS
1 mL 10 mg/mL DNA
E 100 Denhardt’s solution F. 20SSC:
(500 mL): 175.3 g NaCl
10 g Ficoll 400 88.2 g Sodium Citrate
10 g
polyvinylpyrrolidone
MW 360,000
10 g BSA fraction V
Distilled water upto
500 mL
Store at 20 C.
(continued)
G. Strip solution (500 mL):

2.5 mL 1 M Tris
200 μL 0.5 M EDTA
5 mL 5% NaPP
1 mL 50 Denhardt’s
6.2.1 Protocol The RNA (~30 μg) is loaded into the wells of 1.3% agarose-
formaldehyde gel and is run until the RNA is resolved properly.
Examine the gel under UV after staining with ethidium bromide
and rinse gels with 20 SSC buffer (0.03 M sodium acetate,
pH 7.0 and 0.3 M NaCl). Soak gel 3 times 5 min in distilled
water (to remove Formaldehyde) and cut nylon membrane to the
exact gel size. Presoak the membrane. Set up the capillary transfer
stacking as described in Southern blotting protocol. After over-
night blotting, place the membrane on wet Whatman paper and
UV-crosslink damp membrane. Further bake the membrane at
80 C for 1–2 h. Pre-hybridize membrane for 1–4 h @ 42 C with
5–10 mL pre-hybridization buffer. Heat radioactive labeled probe
for 3 min @ 95 C, cool on ice. Discard the pre-hybridization
buffer, add the hybridization buffer and probe, incubate at 42 C.
Wash the membrane once for 15 min and twice with SSC at room
temperature, followed twice with SSC, 0.1% SDS at 65 C until
background is clear. Optionally wash again with 0.1SSC, 0.1
SDS at 65 C. Expose the wet membrane at 80 C under saran
wrap. For stripping, wash membrane for 30 min to 3 h in strip
solution at 75–85 C until no radioactivity can be detected on the
membrane. The membrane can now be air-dried and stored at RT
and the hybridization protocol is followed for re-hybridization.
6.3 Spot For spot hybridization, the following reagents/materials are

Hybridization needed [23]. Proteinase K, Phosphate-buffered saline (PBS),
Sodium chloride (NaCl), sodium citrate, nitrocellulose sheet, form-
amide, N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid
(HEPES), DNA probe, sodium dodecyl sulfate (SDS).
6.3.1 Protocol Pick infected cells and store them at 70 C, if the hybridization
needs to be done at a later time. Take out the cells, thaw and treat
with proteinase K at a concentration of 0.1 mg/mL in PBS. Incu-
bate at 37 C for 1 h, dilute the specimens in stock solution of 20
SSC (l SSC ¼ 0.15 M NaCl, 0.015 M sodium citrate) to a final
concentration of 6 SSC. Divide each sample into two equal parts
and put them separately onto nitrocellulose filters. Then, incubate
the nitrocellulose sheet at 80 C for 2 h and pre-hybridize at 42 C
for 2 h in 50% formamide 50 mM HEPES (pH 7.4). Label the
DNA probe, allow denaturing and addition of a hybridization
solution. Incubate the filters at 42 C overnight for hybridization
with the labeled probe. After hybridization, wash the filters three
times in 2 SSC with 0.5% SDS (w/v) at 42 C. Visualize by

autoradiography overnight for the results.
7 Advantages and Limitations of Nucleic Acid Hybridization Methods [23, 24]
7.1 Advantages 1. Hybridization methods do not require cultivation of the

organisms or any technology-based gene amplification.
2. These techniques have high sensitivity, specificity and rapid
turnover with a high efficiency of hybridization and detection.
3. These methods can be used to study chromosomal aberrations
in non-dividing cells, which are useful for the visualization of
chromosomal aberrations directly in cytological preparations
and tissue sections.
4. They proved to be invaluable in both diagnostics and research.
5. DNA is very stable, so there are less chances of degradation in
tissues compared with protein.
6. Southern blot approach improves the detection resolution to a
level that may be useful for examining specific genomic loci.
7. Northern blot reagents are not too expensive, which allows the
running of many gels at low cost.
8. Northern blot helps in determining transcript size and spliced
transcripts.
9. Spot hybridization makes the maximum use of tissue that is
difficult to obtain (e.g., embryos and clinical biopsies) and
hundreds of different hybridizations can be performed on the
same tissue.
7.2 Limitations 1. Difficulty in identifying targets with low copies of DNA

and RNA.
2. The chemicals used in most northern blots can pose a risk to
the researcher, as formaldehyde, radioactive material, ethidium
bromide, DEPC, and UV light are all dangerous under such
exposure.
3. RNA molecules are often degraded in tissues, and even a slight
degradation of RNA can compromise data quality and thus the
ability to quantify gene expression.
4. Northern blot technique is a labor-intensive technique.
5. Prior knowledge on the nucleic acid sequence of the target
organism is required. These methods are not capable of detect-
ing unknown viruses.
8 Applications of Nucleic Acid Hybridization in Viral Disease Diagnosis
The conventional cell culture-based techniques for virus identifica-

tion need live, infective virus particles to be identified, whereas the
nucleic acid hybridization-based methods can detect both live and
non-infectious viruses [9, 10]. In addition, if the transportation or
delayed sampling causes loss of infectivity of the viruses, such speci-
mens yield negative results on cell culture but may be appropriate
for nucleic acid hybridization techniques. However, nucleic acid
hybridization can only detect viruses with known sequences.
Unknown viruses cannot be detected by this method.
8.1 Applications DNA hybridization technique can be used to detect Herpes Sim-
in Rapid Viral Disease plex virus (HSV) and its major two subtypes, HSV-1 and HSV-2 on
Diagnosis the nitrocellulose membrane [25]. Due to close similarity between
these two subtypes, utmost care should be taken while designing
the probes. Similarly, in the case of Epstein–Barr Virus (EBV) and
rotaviruses infections, in which the infection is either
non-productive or virions are released in low quantity, DOT blot
hybridization is of particular importance [26–28]. Earlier, rota-
viruses were detected by electron microscopy, analysis of RNA on
polyacrylamide gel electrophoresis, RIA, or ELISA. Radioisotope-
labeled probes like 32P for spot and Southern blots, and 3H, 35S, or
125
I for in situ hybridization have also been used. Biotin-labeled
non-radioactive probes have been used to identify viruses such as
human papilloma virus (HPV), human immunodeficiency virus
(HIV), and cytomegalovirus (CMV) [27, 29, 30]. With cytomega-
lovirus, the viral DNA is detected not only in the cytomegalic
inclusion cells, but also in the nucleus and cytoplasm of histologi-
cally unremarkable hepatocytes, pneumocytes, endothelial cells,
and other cells [31]. This approach allows the identification of
viral agents at a much earlier stage than would have been possible
with routine histologic sections.
8.2 Applications Hybridization-based methods often play an importance in their use

in Histopathology in histopathology, mostly when a small proportion of cells in a
population are infected. Therefore, correlated study between hybri-
dization and histology were very helpful in detection of viruses. In
situ hybridization was used to examine the replication of HBV in
liver sections from patients with chronic hepatitis [32]. 3H radioac-
tive probe was used to detect HBV in the liver section. However,
non-radioactive, biotin-labeled DNA/RNA probes may also be
used to detect viral genomes both in tissue culture (parvovirus,
HSV, adenovirus, and retrovirus) and in paraffin sections of tissue
(HSV, and adenovirus) [33]. Biotin-labeled probes decrease hybri-
dization and detection time to less than 24 h, although the sensi-
tivity is lower compared to radiolabeled probes [33].
8.3 Applications Hybridization techniques may be used to study viral epidemiology

in Viral Disease when serological assays distinguishing different strains of virus
Epidemiology (such as CMV) are unavailable. Restriction endonuclease digestion
of CMV from random clinical isolates has shown that no two
viruses generate the same pattern of DNA fragments, when more
than one enzyme is used. Thus, Huano et al. [34] showed that
endogenous virus carried by mothers during pregnancy was the
most frequent cause of recurrent infection, and transmission to
their babies. Re-infection with another strain has rarely occurred.
Spector [35] demonstrated that CMV could be spread from one
child who had viruria to another, in a hospital setting. This finding
has clear consequences for the management of patients in renal and
bone marrow transplant units and in pediatric wards.
HSV isolates have also been typed and classified using restric-
tion endonuclease digestion, as do some adenoviruses, which are
not clearly identified by serological methods [36]. Genome
mapping has provided valuable information about the source of
herpetic infection in nosocomial outbreaks.
9 Conclusion
Conventional methods for diagnosing viral disease are either based

on virus isolation and identification in cell culture, or on the detec-
tion of viral-specific antibody, or on the recognition of viral inclu-
sion bodies by light microscopy. These methods, while tedious,
remain the “gold standard” for the viral diagnostic laboratory.
Hybridization assays offer good sensitivity as diagnostic assays,
but labeling techniques should be refined to make them cost-
effective. While this technique can be applied to identify dead or
non-infective viruses, that are not detectable by cell culture-based
methods, this technique cannot be used to detect those viruses
whose nucleic acid sequence has yet to be identified. In order to
be widely applied, hybridization assays must compete with tradi-
tional and the newer diagnostic laboratory approaches, such as
enzyme immunoassays, in terms of sensitivity, cost, and ease of use.
References
1. Landry ML, Mayo DR, Hsiung GD (1989) 3. Richman DD, Clevland PH, Redfield DC,
Rapid and accurate viral diagnosis. Pharm Oxman MN, Wahl GM (1984) Rapid viral
Ther 40(2):287–328 diagnosis. J Infect Dis 149:298–310
2. Malik YS, Verma AK, Kumar N, Touil N, 4. Landry ML (1990) Nucleic acid hybridization
Karthik K, Tiwari R, Bora DP, Dhama K, in viral diagnosis. Clin Biochem 23:267–277
Ghosh S, Hemida MG, Abdel-Moneim AS, 5. Bhagavan NV, Ha CV (2011) Nucleic acid
Bányai K, Vlasova AN, Kobayashi N, Singh hybridization. In: Bhagavan NV, Ha CV (eds)
RK (2019) Advances in diagnostic approaches Essentials of medical biochemistry with clinical
for viral etiologies of diarrhea: from the lab to cases, 1st edn. Academic. ISBN:
the field. Front Microbial 10:1957–1963 9780120954612
6. Al-Hajjar S (2012) Laboratory diagnosis of mapping homology between heterologous

viral disease. In: Elzouki AY, Harfi HA, Nazer DNAs. Evaluation of polyoma virus genomes.
HM, Stapleton FB, Oh W, Whitley RJ (eds) J Biol Chem 254:4876–4883
Textbook of clinical pediatrics. Springer, Ber- 19. Nakano S, Fujimoto M, Hara H, Sugimoto N
lin. https://doi.org/10.1007/978-3-642- (1999) Nucleic acid duplex stability: influence
02202-9_75 of base composition on cation effects. Nucleic
7. Herring AJ, Inglis NF, Ojeh CK, Snodgrass Acids Res 27:2957–2965
DR, Menzies JD (1982) Rapid diagnosis of 20. Spink CH, Chaires JB (1999) Effects of hydra-
rotavirus infection by direct detection of viral tion, ion release, and excluded volume on the
nucleic acid in silver-stained polyacrylamide melting of triplex and duplex DNA. Biochem-
gels. J Clin Microbiol 16:473–477 ist 38:496–508
8. Espinosa-de Aquino W, Olvera-Ramirez A, 21. Church GM, Gilbert W (1984) Genomic
Arellano-Carbajal F, Lanz-Mendoza H, sequencing. Proc Natl Acad Sci U S A 81:
Villagran-Herrera E, Acevedo-Whitehouse K 1991–1995
(2017) Protein and RNA extraction from 22. Bardelli M, Zarate-Perez F, Agundez L,
mucosal swabs: a minimally invasive source of Jolinon N, Linden RM, Escalante CR, Henck-
ecological datta for studies of natural popula- aerts E (2017) Analysis of replicative inter-
tions. Methods Ecol Evol 8(3):370–378 mediates of adeno-associated virus through
9. Kulski JK, Norval M (1985) Nucleic acid hirt extraction and southern blotting. Bio Pro-
probes in diagnosis of viral diseases of man. toc 7(9):e2271
Brief review. Arch Virol 83:3–15 23. Hyypiä T, Stålhandske P, Vainionpää R, Pet-
10. Kulski JK, Norval M (1985) Nucleic acid tersson U (1984) Detection of enteroviruses
probes in diagnosis of viral diseases of man. by spot hybridization. J Clin Microbiol 19(3):
Arch Virol 83:3–15 436–438
11. de Muro MA (2005) Probe design, produc- 24. Furrer D, Sanschagrin F, Jacob S, Diorio C
tion, and applications. In: Walker JM, Rapley (2015) Advantages and disadvantages of tech-
R (eds) Medical biomethods handbook. nologies for HER2 testing in breast cancer
Springer protocols handbooks. Humana specimens. Am J Clin Pathol 144(5):686–703
Press. https://doi.org/10.1385/1-59259- 25. Singh A, Preiksaitis J, Ferenczy A, Romanowski
870-6:013 B (2005) The laboratory diagnosis of herpes
12. Igarashi P (1999) Radioactive labeling of DNA simplex virus infections. Can J Infect Dis Med
and RNA probes. In: Hildebrandt F, Igarashi P Microbiol 16(2):92–98
(eds) Techniques in molecular medicine. 26. Fleckenstein B, Muller I, Collins J (1982)
Springer lab manual. Springer, Berlin. https:// Cloning of the complete human cytomegalovi-
doi.org/10.1007/978-3-642-59811-1_9 rus genome in cosmids. Gene 18:39–46
13. Eisel D, Grünewald-Janho S, Krushen B (eds) 27. Flores J, Boeggeman E, Purcell RH, Serene M,
(2002) DIG application manual for nonradio- Perez I, White L, Wyatt RG, Chanock RM,
active in situ hybridization, 3rd edn. Roche Kapikian AZ (1983) A dot hybridization assay
Diagnostics, Penzberg for detection of rotavirus. Lancet 1:555–559
14. Herzer S, Englert DF (2001) Nucleic acid 28. Sixbey JW, Nedrud JG, Raab-Traub N, Hanes
hybridization. In: Molecular biology problem RA, Pagano JS (1984) Epstein-Barr virus rep-
solver: a laboratory guide. A.S. Wiley-Liss, Ger- lication in oropharyngeal epithelial cells. N
stein, pp 401–453 Engl J Med 310:1225–1230
15. Sykacek P, Kreil DP, Meadows LA, Auburn RP, 29. Galloway DA, Fenoglio C, Shevchuk M,
Fischer B, Russell S, Micklem G (2011) The McDougall JK (1979) Detection of herpes
impact of quantitative optimization of hybridi- simplex RNA in human sensory ganglia. Virol-
zation conditions on gene expression analysis. ogy 95:265–268
BMC Bioinformatics 12:73. https://doi.org/
10.1186/1471-2105-12-73 30. Myerson D, Hackman RC, Meyers JD (1984)
Diagnosis of cytomegaloviral pneumonia by in
16. Pearlman DA, Kollman PA (1990) The calcu- situ hybridization. J Infect Dis 150:272–277
lated free energy effects of 5-methyl cytosine
on the B to Z transition in DNA. Biopolymers 31. Durst M, Gissmainn L, Ikenberg H, Zur Hau-
29:1193–1209 sen H (1983) A papillomavirus DNA from a
cervical carcinoma and its prevalence in cancer
17. Anderson MLM (1999) Nucleic acid hybridi- biopsy samples from different geographic
zation. Springer, New York regions. Proc Natl Acad Sci U S A 80:3812–
18. Howley PM, Israel MA, Law MF, Martin MA 3815
(1979) A rapid method for detecting and
32. Burrell CJ, Gowans EJ, Jilbert AR, Lake JR, epidemiology of cytomegalovirus infections in
Marmion BP (1982) Hepatitis B virus DNA women and their infants. New Engl J Med 303:
detection by in situ cytohybridization: implica- 958–962
tions for viral replication strategy and patho- 35. Spector SA (1983) Transmission of cytomega-
genesis of chronic hepatitis. Hepatology 2: lovirus among infants in hospital documented
85S–91S by restriction-endonuclease-digestion analyses.
33. Brigati DJ, Myerson D, Leaky JJ, Spaliiolz B, Lancet 1:378–381
Travis SZ, Fong CK, Hsiung GD, Ward DC 36. Waddell G, DeJong JC, Wolontis S (1981)
(1983) Detection of viral genomes in cultured Molecular epidemiology of adenoviruses: alter-
cells and paraffin-embedded tissue sections nating appearance of two different genome
using biotin-labeled hybridization probes. types of adenovirus 7 during epidemic out-
Virology 126:32–50 breaks in Europe from 1958 to 1980. Infect
34. Huano ES, Alford CA, Reynolds DW, Immun 34:368–372
Stango S, Pass RF (1980) Molecular
Chapter 21
Ligase Detection Reaction-Fluorescent Microsphere Assay

A. Raja
Abstract
Disease outbreaks in swine production cause severe economic loss. Conventional diagnostic methods are
laborious and less sensitive or time consuming. Recent molecular methods of diagnosis of diseases especially
genome detection play a major role in the diagnosis and control of diseases. Although several molecular
diagnostic techniques are available, still not all the techniques are employable to detect different pathogens
simultaneously. Most of the recent outbreaks are involved with more than one pathogen. Hence, a simple
and rapid technique which can detect different targets/pathogens simultaneously is in need of time.
LDR-FMA is such an assay where multiplexing is possible. Although LDR-FMA is mainly used to detect
single nucleotide polymorphism, this technique still could be adapted for simultaneous detection of
different pathogens.
Key words Ligase detection reaction, Fluorescent microscopy, MOL-PCR
1 Introduction
Swine disease outbreaks cause serious economical loss worldwide.

Porcine circovirus (PCV), porcine reproductive and respiratory
syndrome virus (PRRSV), classical swine fever virus (CSFV), por-
cine parvovirus (PPV), pseudorabies virus (PRV), and Japanese
encephalitis virus (JEV) are six swine viruses involved in reproduc-
tive and/or respiratory infections which are severe and frequently
reported in swine production. Efficient detection methods are
needed for early diagnosis of these infections. Several methods
and techniques have been proposed for detection of swine viruses,
such as virus isolation, enzyme linked immunosorbent assays (ELI-
SAs), and immunofluorescence assay (IFA). But conventional
methods for viral diagnosis are laborious, less sensitive, or time
consuming. For example, virus isolation is the “gold standard”
for detection but requires 7–10 days and is often insensitive.
DNA ligase catalyzes the annealing of two DNA strands on a
DNA template by the formation of a phosphodiester bond at a nick
junction. DNA ligase is involved in DNA replication, repair, and
291
292 A. Raja
recombination. DNA ligases are classified into two families based

on adenylation cofactor dependence. ATP-dependent ligases are
found in bacterial and eukaryotic viruses, Archaea, yeast, mammals,
and eubacteria, whereas NAD+-dependent ligases are found almost
exclusively in eubacteria with the exception of the sequenced ento-
mopoxvirus genomes Melanoplus sanguinipes and Amsacta moorei.
Some simple eubacteria genomes encode both NAD+- and
ATP-dependent ligases, whereas many eukaryotic organisms
encode multiple ATP-dependent ligases to fulfill diverse biological
functions.
Biotechnological application of DNA ligase started with the
advent of recombinant DNA techniques. The development of the
polymerase chain reaction (PCR) in the mid-1980s enabled genetic
materials to be amplified in vitro [1, 2]. A few years later, a mutation
detection technique based on the use of T4 DNA ligase was
invented, which provided a conceptual foundation for the develop-
ment of many current ligase-based techniques [3]. Similar to PCR,
DNA ligase-mediated strand-joining reactions can be repeated on
product denaturation and primer annealing to achieve signal or
target amplification [4–6] by using thermostable DNA ligase [7].
The LDR was first developed to detect low amounts of patho-
gens [8]. Since even a single base mismatch at the ligation junction
prevents successful ligation, the technique is highly specific [9, 10].
The ligase detection reaction-fluorescent microsphere assay
(LDR-FMA) was designed for multiplex detection of various tar-
gets and for SNP genotyping [11]. This technology has been
applied for the multiplex detection of 15 genetically distinct plant
pathogens including Citrus tristeza virus (CTV), Xanthomonas
genus, and Xylella fastidiosa. LDR-FMA has also been used in the
detection of pathogens along with antibiotic (doxycycline and cip-
rofloxacin) resistance in Yersinia pestis, F. tularensis, and Bacillus
anthracis [11, 12]. LDR-FMA has also been useful in identification
and differentiation of the six main human-associated lineages of
Mycobacterium tuberculosis complex (MTBC) with sensitivity
approaching 99.2% [13]. In 2008, Bruse et al. made few improve-
ments to LDR-FMA assays by using fewer beads and universal
biotinylated oligonucleotides to reduce costs significantly but also
maintaining accuracy [14].
2 Principles of LDR
LDR (Ligase Detection Reaction) is a ligation dependent method-

ology that, unlike LCR (Ligase Chain Reaction), involves only one
pair of probes complementary to one strand of target DNA.
Cycling in LDR results in linear amplification of the ligation prod-
uct. This method can be used to confirm the presence of a particu-
lar SNP in a target sequence that has been amplified by another
Ligase Detection Reaction-Fluorescent Microsphere Assay 293
method (such as PCR). As with LCR, the method uses a high

fidelity thermostable ligase that can discriminate against the ligation
of mismatched probes, such as Taq DNA Ligase.
3 Principles of LDR-Fluorescent Microsphere Assay (LDR-FMA)
Deshpande et al. [15] described the LDR-FMA as multiplex oligo-

nucleotide ligation-PCR (MOL-PCR) where fluorescent micro-
spheres were used to detect different targets. The assay comprises
of three steps: detection, signal amplification and hybridization and
readout.
3.1 Detection Detection is achieved by using 40–70 nucleotide-long, single-

stranded DNA probes called MOLigo pairs. When the target for a
particular MOLigo pair is present in a reaction, the MOLigo pairs
will anneal adjacent to each other on the target. If annealing occurs,
DNA ligase recognizes this structure and covalently links MOLigo1
and MOLigo2. Ligation only occurs if the terminal bases of
MOLigo1 and MOLigo2 at the junction site are complementary
to the target sequence, and this property confers SNP discrimina-
tion. This step creates single-stranded DNA molecules of approxi-
mately 100–120 nucleotides long that serve as PCR templates for
signal amplification and labeling. The ligation reaction can be
multiplexed to allow the simultaneous screening for many signa-
tures in the same reaction tube.
3.2 Signal The ligation products undergo PCR amplification with a universal
Amplification primer pair. If the ligation reaction is multiplex, use of a universal
primer pair allows the amplification of multiple ligated products in
the same reaction tube. In the absence of a target, ligation will not
occur, the forward and reverse primer sites will not be linked, and
the unligated MOLigo pairs will not produce geometrically ampli-
fied and labeled fragments that contain tags.
3.3 Hybridization The reverse PCR primer is tagged with a fluorescent dye (e.g., Alexa
and Readout Fluor 532) at the 50 end, which allows the detection of the ampli-
fied ligation products. The PCR products are hybridized to a
microsphere array using tag sequences complementary to anti-
tags covalently linked to the microspheres. The microspheres are
then analyzed by a flow cytometer, which identifies each bead type
and measures fluorescence intensity emitted by the bead-PCR
product complex. A microsphere in the array will only show fluo-
rescence if it is hybridized to a labeled PCR product amplified from
ligated MOLigo pairs, thus confirming the presence of the target
DNA in the sample.
294 A. Raja
4 Materials
1. Primers and Probes (Sigma).

2. Taq DNA Polymerase (New England Biolab).
3. 96-well PCR plates (Qiagen).
4. MES buffer ((Fisher Bioreagents).
5. 96-well round bottomed plate (Costar).
6. Luminex flow cytometer.
5 Methods
Method for carrying out the LDR-FMA is adopted from

Deshpande et al. [15] and it is as follows:
1. Ligation and PCR is conducted in a single tube as follows:
(a) Final volume of each reaction is 10 μl, consisting of
4 units/μl of thermostable DNA ligase, 5 nM of each
MOLigo pair (50 end of MOLigo 1 is phosphorylated to
ligate with the MOLigo 2), 4 mM Mg2+, 1 Taq buffer,
200 μM dNTPs, 0.04 units of Taq DNA polymerase,
500 μM NAD, 0.125 μM forward PCR primer, 0.5 μM
Alexa Fluor 532 (Life Technologies) labeled reverse PCR
primer and nuclease free water to 9 μl volume, combined
with 1 μl of template DNA.
(b) Ligase-mediated PCR is carried out in a thermal cycler
with the following cycling conditions. Initial denaturation
at 94 C for 1.5 min followed by 30 cycles of 50 C for
30 s, and 94 C for 25 s to complete the ligation process.
(c) Amplification of ligated product is carried by 40 cycles of
94 C for 20 s, 58 C for 20 s, and 72 C for 20 s. At the
end of the reaction, the tubes are stored at 4 C until
further process.
(d) A separate reaction is set for each template/sample. All
reactions are carried out in a 96-well plate. This corre-
sponds to one plate of a singleplex assay per probe. For
simultaneous detection of different targets/SNPs, in the
same tube, different labeled probes are added and the
remaining components are same.
2. Tagging fluorescent microspheres with the LCR products.
(a) Anti-tag-conjugated Luminex xTAG® microspheres of
each set specific to each MOLigo pair is used.
Ligase Detection Reaction-Fluorescent Microsphere Assay 295
(b) Mixture is prepared in such a way that it contained

1000 microspheres/μl of each set, 800 mM NaCl, and
50 mM MES buffer.
(c) 5 μl of this mix is then added to the LCR product (10 μl)
well and gently mixed.
(d) Then the mix is heated to 94 C for 1 min, followed by a
slow ramp down in temperature to 25 C with a hold of
1 min at 5 C decrements in temperature in a thermal
cycler. Samples are stored at 4 C until further process.
(e) Two control tubes are also set up for calculation pur-
pose—a bead-only control and a no template control.
The bead-only control is to report background fluores-
cence obtained from the microspheres alone and the no
template control to report cross-reactivity of MOLigo
pairs in the absence of any template.
3. For analysis on the Luminex flow cytometer, the reaction vol-
ume is increased to 50 μl by adding 35 μl of buffer that contains
10 mM Tris–Cl (pH 8.0), 0.1 mM EDTA, 90 mM NaCl, and
0.02% Tween 20. Then, the reaction is transferred to a 96-well
round bottomed plate and analyzed on a Luminex flow
cytometer.
4. Analysis of the result.
(a) Median fluorescence intensity (MFI) is used to calculate
signal-to-noise. The MFI for this control is also the
“noise” used for calculating the signal-to-noise ratio.
(b) MEI values for all samples and controls are imported into
an Excel file.
(c) The signal-to-noise ratios are calculated by first subtract-
ing the MFI of the bead-only control from all MFIs, and
then dividing the MFI obtained for a sample by the MFI
for the no template control.
(d) A signal-to-noise ratio of 4 and a MFI of 50 are criteria
used to determine “positive samples.”
References
1. Mullis K et al (1986) Specific enzymatic ampli- 4. Barany F (1991) Genetic disease detection and
fication of DNA in vitro:the polymerase chain DNA amplification using cloned thermostable
reaction. Cold Spring Harb Symp Quant Biol ligase. Proc Natl Acad Sci U S A 88:189–193
51:263–273 5. Barany F (1991) The ligase chain reaction in a
2. Saiki RK et al (1988) Primer-directed enzy- PCR world. PCR Methods Appl 1:5–16
matic amplification of DNA with a thermosta- 6. Wu DY, Wallace RB (1989) The ligation ampli-
ble DNA polymerase. Science 239:487–491 fication reaction (LAR)—amplification of spe-
3. Landegren U et al (1988) A ligase-mediated cific DNA sequences using sequential rounds
gene detection technique. Science 241:1077– of template-dependent ligation. Genomics 4:
1080 560–569
296 A. Raja
7. Barany F, Gelfand DH (1991) Cloning, over- Proceedings of SPIE, optics and photonics in
expression and nucleotide sequence of a ther- global homeland security III (65401D)
mostable DNA ligase-encoding gene. Gene 12. Song J, Li PE, Gans J et al (2010) Simulta-
109:1–11 neous pathogen detection and antibiotic resis-
8. Muller R, Ditzen A, Hille K, Stichling M, tance characterization using SNP-based
Ehricht R, Illmer T, Ehninger G, Rohayem J multiplexed oligonucleotide ligation-PCR
(2009) Detection of herpesvirus and adenovi- (MOL-PCR). Adv Exp Med Bio
rus co-infections with diagnostic 680455–680464
DNA-microarrays. J Virol Methods 155:161– 13. Stucki D, Malla B, Hostettler S, Huna T et al
166 (2012) Two new rapid SNP-typing methods
9. Niederhauser C, Kaempf L, Heinzer I (2000) for classifying Mycobacterium tuberculosis
Use of the ligase detection reaction-polymerase complex into the main phylogenetic lineages.
chain reaction to identify point mutations in PLoS One 7:e41253
extended-spectrum beta-lactamases. Eur J 14. Bruse S, Moreau M, Azaro M et al (2008)
Clin Microbiol Infect Dis 19:477–480 Improvements to bead-based oligonucleotide
10. Wang XL, Xie SG, Zhang L, Yang WX, ligation SNP genotyping assays. BioTechni-
Wang X, Jin HZ (2008) Comparison of ligase ques 5:559–571
detection reaction and real-time PCR for 15. Deshpande A, Gans J, Graves SW, Green L,
detection of abundant YMDD mutants in Taylor L, Kunde YA, Leonard PM, Li P-E,
patients with chronic hepatitis B. World J Gas- Mark J, Momchilo V, Scott White P, Song J,
troenterol 14:120–124 Kim HB (2010) A rapid multiplex assay for
11. Mark JA, Green LD, Deshpande A et al (2007) nucleic acid-based diagnostics. J Microbiologic
System integration and development for Methods 80:155–163
biological warfare agent surveillance.
Chapter 22
ELISA as a Diagnostic Weapon

Abstract
The soundness of animal health is gauged by their ability to withstand infection and remain resistant to
reinfection. The robustness of animals can be directly attributed to their sturdy immune system. The
animals are often inflicted with potential infectious agents hindering their growth and productivity and
occasionally succumb to the disease. Hence, diagnosis of diseases at the right stage is a critical step to adapt a
suitable and effective control measures to contain their spread. Scientists rely on different immunological
assays with varying degrees of sensitivity and specificity to determine the immune status of the animal. These
assays are inevitable in detecting either the etiology or the product of an infection manifested as humoral or
cell-mediated immunity. ELISA is a form of immunoassay using an enzyme-linked conjugate and enzyme
substrate producing colored products depending on the concentration of the analyte present in the test
system. It is one of the simple, versatile, reliable technique available in different formats which primarily
detects antibody or antigen with highest level of sensitivity and specificity. This chapter describes the
different types of ELISA procedures available for the diagnosis of foot and mouth disease infection in
swine species.
Key words Enzyme-linked immunosorbent assay (ELISA), Diagnosis, Foot and mouth disease
(FMD), Conjugate, Optical density, Non-structural proteins
1 Introduction
Assays using enzyme-linked conjugate and a substrate to demon-

strate antigen–antibody interactions qualitatively/quantitatively
are generally termed as Enzyme immunoassays (EIA)/
Enzyme-linked immunosorbent assay (ELISA). Enzyme-linked
immunosorbent assay (ELISA) is a kind of immunological assay
used to determine or quantitate the amount of analyte/antigen
present in the biological fluids through the color change obtained
by using an enzyme-linked conjugate and enzyme substrate. The
technique relies on the significant attributes of an immune
response, specificity of the antibody to its antigen. ELISA is classi-
fied as a primary binding assay which directly measures the binding
of antigen to antibody with a sensitivity of detecting 0.0005 μg/
297
298 Ramya Kalaivanan and Sankar Palanisamy
mL. ELISA is a highly versatile, sensitive, and quantitative tech-

nique requiring little equipment and for which critical reagents are
readily available emphasizing its implication as a diagnostic weapon.
1.1 ELISA FMD is an economically significant, acute, contagious disease of

as a Diagnostic cloven-hoofed animals posing severe threat across the globe despite
Weapon for FMD its eradication from few countries. Despite of active vaccination
strategies undertaken by the nations; the disease remains endemic
owing to its quasi-species nature. Although methods based on virus
isolation or the demonstration of FMD viral antigen or nucleic acid
in samples of tissue or fluid or culture products are sufficient for a
positive diagnosis, in general, the ELISA [1, 2] using type-specific
serological reagents is the preferred procedure for the detection of
FMD viral antigen and identification of viral serotype in the early
stages of research. Because it is more specific, sensitive and efficient,
and it is not impacted by pro- or anti-complement factors [3], the
ELISA has access to better development and even replaced comple-
ment fixation (CF) in most laboratories in the early research phase
of FMD. Contrast to complement fixation test and virus isolation,
almost the equivalent, even the higher of sensitivity was achieved in
ELISA [4–6].
Enzyme-linked immunosorbent assay (ELISA) method was
pioneered largely by the Swiss scientists Eva Engvall, and Peter
Perlmann in the year 1971 by modifying the radioimmunoassay
method used by Yalow and Berson in the 1960s. The ELISA
technique was devised by conjugating antibody with enzymes
rather than radioactive iodine 125 radioisotopes in radioimmuno-
assay to determine the levels of IgG in rabbit serum [7]. Van Wee-
man and Schuurs in the same year 1971 [8], a different research
team, succeeded in quantifying human chorionic gonadotropin
amounts in the urine by using horseradish peroxidase enzyme
with the EIA method and also applied for a patent both in the
USA and Europe.
Subsequent to the invention of ELISA, a number of researchers
used it: Carlson and colleagues in 1972 [9], Holmgren and
Svennerholm in diagnostic microbiology in 1973 [10], Ljungstrom
and colleagues to identify the presence of trichinosis in parasitology
in 1974 [11], and Voller et al. to diagnose malaria in 1975
[12]. Bishai and Galli, Leinikki et al., and Ukkonen et al. made
use of the ELISA method to identify infections caused by influenza,
parainfluenza, and mumps viruses in 1978, 1979, and 1981,
respectively [13–16].
In the year 1980, Siegle et al. modified the ELISA test and
incorporated microtitration plates to identify the concentrations of
various hormones, peptides, and proteins apart from its application
in diagnosis of infectious diseases [17]. At present, ELISA has
transcended to newer level and is an indispensable assay in different
formats in several fields of biology in research and diagnosis
laboratories.
ELISA as a Diagnostic Weapon 299
1.2 Timeline 1971—Engvall and Perlmann—Direct ELISA

of Invention 1976—Yorde and his coworkers—Competitive ELISA
of Different Types
1977—Kato and his co-workers—Sandwich ELISA
of ELISA
1978—Lindström and Wager—Indirect ELISA.
The principle of the basic ELISA procedure, direct ELISA is
briefed at this juncture. The antigen utilized in the ELISA method
is bound to a solid phase like tubes or microplates made of rigid
polystyrene, polyvinyl, and polypropylene. These solid phases
appropriately adsorb the antigen and the antibody primarily
through hydrophobic interactions or hydrophobic/ionic interac-
tions between the biomolecules and the surface. The enzymes that
can be employed in ELISA include beta galactosidase, glucose
oxidase, peroxidase, and alkaline phosphatase. Alkaline phosphatase
on interaction with its substrate P-nitro-phenyl phosphate (pNPP)
produces a yellow color in positive reactions at 405 nm. The
horseradish peroxidase on interaction with its substrate 3,30 ,5,5-
0
-tetramethylbenzidine (TMB) or 2,20 -azino-di-[3-ethylbenzthia-
zoline-6-sulfonicacid] (ABTS) or orthophenylenediamine
hydrochloride (OPD) produces a brown color in a positive reaction
at 492 nm. The beta galactosidase on interaction with its substrate
ortho-nitrophenyl- -galactoside (ONPG) produces a yellow color
at 420 nm. The catabolic effects of enzymes determine both the
acceleration and the specificity of the immunological reaction dur-
ing the enzyme-substrate reaction [18]. The enzyme-substrate
reaction is usually completed within 30–60 min. The reaction can
be stopped using sodium hydroxide (NaOH), hydrochloric acid
(HCl), or sulfuric acid (H2SO4) [19].
ELISA is a heterogeneous immunoassay technique involving
washing procedure to remove unbound biomolecules and used to
detect specific antibodies and soluble antigens. ELISA has been
designed in different formats to determine the quantity of sub-
stances to be measured since each of them differs in its structure
and biochemical characteristics. Each of them differs by which
component is immobilized, how it is recognized, and what is
detected (Table 1).
1.3 Direct ELISA Direct ELISA has the simplest format, requiring antigen to be
adsorbed to the plate and then bound by a labeled “detection”
antibody followed by washing which removes the unbound anti-
bodies. Subsequent addition of appropriate substrate produces a
signal through color development depending on the concentration
of analyte. “Direct” refers to the first and only antibody acting as
both the antigen recognition molecule and signal delivery mole-
cule. It is suitable for determining the amount of high molecular
weight antigens.
Table 1
300
Features of and the differences between ELISA types
S. no. Types of ELISA Infographics Detects Advantages Disadvantage

1. Direct ELISA Antibodies l Faster than other l False-positive.
ELISA. l Antigen immobilization
l Less prone to error. is not specific: may cause
higher background
noise.
l Less flexible: each target
protein needs a specific

conjugated primary
antibody.
l No signal amplification:
reduces assay sensitivity.

2. Indirect ELISA (iELISA) Antigen/antibodies l Highly sensitive. l Possibility of background
l Economical. noise: secondary
l Greater flexibility: antibody may be cross-

different primary reactive.
antibodies can be used l Longer procedure than
with a single labeled direct ELISA technique:

secondary antibody. additional incubation
step for secondary
antibody needed.
3. Competitive/blocking/ Antigen/antibodies l Very highly sensitive.
inhibition ELISA l No sample processing is
(cELISA) required and crude or
impure samples can
be used.
l More robust—less
sensitive to sample
dilution and sample
matrix effects than the
sandwich ELISA.
S. no. Types of ELISA Infographics Detects Advantages Disadvantage
l More consistent—less
variability between
duplicate samples and
assays.
l Maximum flexibility—
it can be based on
direct, indirect, or
sandwich ELISA.
l Suitable for detecting
small antigens.
4. Sandwich ELISA Antigens l High sensitive: 2–5
times more sensitive
than direct or indirect
ELISA.
l High specificity: two
antibodies are involved

in capture and
detection.
l Analysis of complex
samples possible: the

antigen does not need
to be purified prior to
measurement.
l Flexibility: both direct
and indirect detection

can be used.
Antigen Unknown Antibody Primary antibody Labeled antigen Conjugate (Enzyme-
Substrate specific antibody labeled detection
antibody)
ELISA as a Diagnostic Weapon
301
1.4 Indirect ELISA Indirect ELISA as the name implies allows for the amplification of
signal by using a secondary antibody. The indirect ELISA proce-
dure starts with adsorption of antigen to the well of an ELISA plate.
Following standard blocking and washing steps an unlabeled pri-
mary antibody (test serum sample to determine the concentration)
binds to the specific antigen. The antigen and antibody complex
formed during incubation is recognized by addition of an enzyme
conjugated species-specific secondary antibody. The color develop-
ment upon suitable substrate addition depends on the concentra-
tion of the primary antibody present in the serum sample.
1.5 Sandwich ELISA The distinguishing feature of a sandwich ELISA is the adsorption of
a “capture” antibody to the plate. Antigen is bound or captured by
the plated antibody and then “sandwiched” between the capture
and a detecting antibody which recognizes a distinctly different
epitope on the antigen. Each antibody is therefore specific for a
different and non-overlapping region or epitope of the same anti-
gen. A major benefit of a sandwich ELISA is the ability to specifi-
cally measure antigen from impure samples. The capture antibodies
provide the assay specificity by adsorbing the antigen of interest on
the contrary the crude sample to the plate. The opportunity for
indirect detection is also available in a sandwich ELISA, wherein the
detection antibody would not carry the signal but rather be tar-
geted by yet a third antibody which would impart the signal to the
assay.
The procedure for sandwich ELISA involves coating the well of
an ELISA plate with a capture antibody. The analyte or sample is
then added, followed by a detection antibody. The detection anti-
body can be enzyme conjugated, which is referred to as a direct
sandwich ELISA. In contrast, in an indirect sandwich ELISA a
secondary enzyme-conjugated detection antibody is needed if the
detection antibody is unlabeled.
1.6 Competitive/ The competitive/inhibition ELISA is predominantly used to mea-

Inhibition ELISA sure the concentration of an antigen or antibody in a sample by
detecting interference in an expected signal output. In this format,
the sample antigen or antibody competes with a reference for
binding to a limited amount of labeled antibody or antigen, respec-
tively. The higher the sample antigen concentration, the weaker the
output signal, indicating that the signal output inversely correlates
with the amount of antigen in the sample.
The procedure involves coating a known antigen to a multiwell
plate. Following standard blocking and washing steps, samples
containing unknown antigen are added. Labeled detection anti-
body is then applied for detection using appropriate substrates.
The intensity of color development is inversely related to the con-
centration of antigen in the sample.
2 Materials
2.1 Solid-Phase Solid-phase competitive ELISA (SPCE) has replaced complement

Competitive ELISA fixation in most laboratories as it is more specific and sensitive
for the Diagnosis [21]. The method described by Paiba et al. [21, 22] can be used
of FMD [20] for the detection of antibodies against each of the seven serotypes
of FMDV. Moreover, SPCE has the advantage that the test is rapid
(the result can be read in 1 day vs. waiting 3 days in VNT) and easier
to perform [23].
1. Trapping/Capture Antibody: Serotype-specific antiserum
raised in rabbit/guinea pig/monoclonal antibody.
2. Coating buffer: Carbonate/Bicarbonate buffer:
Sol. A: 0.2 M sodium carbonate solution:

Na2CO3 21.198 g
Distilled water 1000 ml
Sol. B: 0.2 M sodium bicarbonate solution
NaHCO3 16.8 g
Working solution Store at 4 C
Solution A 16 ml
Solution B 32 ml
Triple distilled water up to 200 ml
3. Washing Buffer: Phosphate buffer saline (PBS)—10:
NaCl 80 g
KCl 2g
Na2HPO4 14.4 g
KH2PO4 2.45 g
Triple distilled water to 1000 ml
Adjust pH of the solution to 7.4 with HCl and sterilize by

autoclaving for 20 min at 15 psi.
4. Blocking buffer:
PBS Volume required

Tween 20 0.05% [w/v]
NBS—New born serum/Fetal calf serum 10% [v/v]
Normal rabbit serum 5% [v/v]
5. Antigen: 146S antigen (see Note 1).

6. Diluent: Blocking buffer with phenol red indicator.
7. Test sera: Diluted in PBST blocking buffer.
8. Detection antibody: Guinea-pig antisera (see Note 2).
9. Rabbit (or sheep) anti-guinea-pig immunoglobulin conjugated
to horseradish peroxidase at a predetermined optimum con-
centration in PBSTM blocking buffer.
10. Substrate solution: Citrate buffer (pH 5.0). Store at 4 C.
Citric acid 7.30 g

Na2HPO4∙2H2O 11.87 g
Distilled water up to 1000 ml
11. Stopping solution: 1 M sulfuric acid.
Conc. H2SO4 5.56 ml

Stored at room temperature.

12. Multichannel pipette and disposable pipette tips.
13. 96-well ELISA plates (polystyrene, medium to high binding).
14. ELISA reader.
2.2 Liquid Phase l Antigen: 146S antigen (see Note 3).

Blocking ELISA l Diluent.
for Diagnosis of FMD l Rabbit antiserum homologous to the antigen.
[20]
l Coating buffer: Carbonate/Bicarbonate buffer (see
Subheading 2.1).
l Blocking buffer: (see Subheading 2.1).
l Test sera: Diluted in PBST blocking buffer.
l Detection antibody: Guinea-pig antisera against 146S antigen of
one of the seven serotypes of FMDV.
l Rabbit (or sheep) anti-guinea-pig immunoglobulin conjugated
to horseradish peroxidase at a predetermined optimum concen-
tration in PBSTM blocking buffer (see Subheading 2.1).
l Substrate solution: Citrate buffer (pH 5.0) (see Subheading 2.1).
l Stopping solution: 1 M sulfuric acid (see Subheading 2.1).
2.3 Indirect ELISA The non-structural proteins of foot and mouth disease virus are
to Detect nothing but the enzymes involved in the viral replication. The
Non-structural Protein presences of non-structural proteins or antibodies to non-structural
(NSP ELISA) [20] proteins in the clinical specimens are clear indication of the animal
being infected or exposed to the live virus. The NSPs, unlike

structural proteins, are highly conserved and therefore are not
serotype specific and as a consequence, the detection of these anti-
bodies is not serotype restricted. Hence, demonstrations of anti-
bodies to non-structural proteins (Lpro, 2A, 2B, 2C, 3A, 3B, 3Cpro
and 3D) [24, 25] are yardstick to differentiate clinically infected
animals from the vaccinated ones (DIVA—Differentiation of
infected from vaccinated animals) since animals are vaccinated
only with inactivated viruses.
Enzyme-linked immunosorbent assay (ELISA) based assays can
be used to detect anti-NSPs antibodies in carrier cattle [26]. Several
recombinant NSPs of FMDV viz. 2B, 2C, 3A, 3AB, 3B, 3ABC, and
3D have been used in NSP ELISAs [27, 28]. These ELISAs either
use purified antigens absorbed directly to microplates or use PAbs
or MAbs to trap specific antigens from semi-purified preparations
[29–32]. NSP ELISA although not considered as a suitable test for
certifying animals prior to movement, NSP ELISAs may be a
valuable adjunct in circumstances where the serotype or subtype
of virus in the originating country is not known.
l Recombinant 3ABC antigen [33] (see Note 4).
l Coating buffer: Carbonate/Bicarbonate buffer (see
Subheading 2.1).
l Washing buffer.
PBS (pH 7.2) Volume required

Tween 20 0.05% [w/v]
l Blocking buffer.
PBS Volume required

Tween 20 0.05% [w/v]
Nonfat dry milk 5% [w/v]
Equine 10% [v/v]
Escherichia coli lysate 0.1%
l Serum from suspected swine.

l Conjugate: Anti-porcine enzyme-linked IgG dilution as per the
supplier instruction.
l Substrate solution: Citrate buffer (pH 5.0) (see Subheading 2.1).
l Stopping solution: 0.5 M Sulfuric acid (see Subheading 2.1).
Conc. H2SO4 2.78 ml

Stored at room temperature.

l See Note 5.
3 Methods [20]
3.1 Solid-Phase l Coat the ELISA plates with 50 μl/well rabbit antiserum homol-
Competitive ELISA ogous to the antigen being used, diluted in carbonate/bicar-
for the Diagnosis bonate buffer, pH 9.6, and leave it overnight in a humid
of FMD chamber at 4 C.
l Wash the ELISA plates thrice with PBS.
l Add 50 μl of the FMDV antigen diluted in blocking buffer to
each well of the ELISA plates. Cover the plates and place it on an
orbital shaker at 37 C for 1 h, with continuous shaking.
l After washing thrice with PBS, add 40 μl of blocking buffer to
each well, followed by 10 μl of test sera (or control sera), giving
an initial serum dilution of 1/5.
l Add 50 μl of guinea-pig serotype specific anti-FMDV antiserum
diluted in blocking buffer immediately to give a final serum
dilution of 1/10.
l Cover the plates and incubate on an orbital shaker at 37 C for
1 h.
l After washing thrice with PBS, add 50 μl of anti-guinea-pig
immunoglobulin conjugate (preblocked by incubation for 1 h
at room temperature with an equal volume of NBS) diluted in
blocking buffer. Cover the plates and incubate for 1 h at 37 C
on an orbital shaker.
l After washing thrice with PBS, add 50 μl of substrate solution
containing 0.05% H2O2 plus orthophenylene diamine or a suit-
able alternative chromogen to each well.
l Stop the reaction after 10 min by adding 50 μl of 1 M
sulfuric acid.
l Read the plates at 492 nm on a spectrophotometer linked to a
computer.
l Controls: On each plate.
– Conjugate control (two wells)—No guinea-pig serum.
– Strong and weak positive sera (four wells each).
– Negative sera (two wells).

– 0% competition (four wells)—Without test sera.
l Interpretation of the results:
The percentage of inhibition is calculated for each well
either manually or using a suitable computer program.
100Optical density of each test or control value

Percentage of inhibition ¼ 100
Mean optical density of the 0% competition
It represents the competition between the test sera and the
guinea-pig anti-FMDV antisera for the FMDV antigen on the
ELISA plate (see Note 6).
3.2 Liquid Phase l Coat the ELISA plates with 50 μl/well rabbit antiserum homol-
Blocking ELISA (LPBE) ogous to the antigen being used and leave overnight in a humid
for Diagnosis of FMD chamber at room temperature.
l Wash the ELISA plates thrice with PBS.
l Prepare 50 μl of duplicate, twofold series of each test serum in
U-bottomed multiwell plates (carrier plates) starting at 1/8.
Add 50 μl of a constant dose of viral antigen that is homologous
to the rabbit antisera used to coat the plates to each well and
leave the mixtures overnight at 4 C, or incubated at 37 C for
1 h. The addition of the antigen increases the final serum dilu-
tion to 1/16.
l Transfer 50 μl of serum/antigen mixtures from the carrier plates
to the rabbit serum coated ELISA plates and incubate at 37 C
for 1 h in an orbital shaker.
l After washing thrice with PBS, add 50 μl of guinea-pig antise-
rum homologous to the viral antigen used in the previous step
(preblocked with NBS and diluted in PBST containing 5%
skimmed milk powder) to each well and incubate at 37 C for
1 h on a rotary shaker.
l Wash the plates thrice and add 50 μl of rabbit anti-guinea-pig
immunoglobulin conjugated to horseradish peroxidase (pre-
blocked with NBS and diluted in PBST containing 5% skimmed
milk powder) to each well and incubate at 37 C for 1 h in an
orbital shaker.
l Wash the plates again thrice and add 50 μl of substrate solution,
containing 0.05% H2O2 plus orthophenylene diamine or a suit-
able alternative chromogen to each well.
l Stop the reaction after 15 min by adding 50 μl of 1 M
sulfuric acid.
l Read the plates at 492 nm on a spectrophotometer linked to a

computer.
l Controls: Include on each plate a minimum of four wells each of
bovine reference sera at a final dilution of 1/32.
– strong positive.
– weak positive.
– negative.
– Reaction (antigen) control wells containing antigen in dilu-
ent alone without serum.
– For end-point titration tests, duplicate twofold dilution series
of positive and negative homologous reference sera should be
included on at least one plate of every run.
l Interpretation of the results:
Antibody titers are expressed as the 50% end-point titer, i.e.,
the dilution at which the reaction of the test sera results in an
optical density equal to 50% inhibition of the median optical
density of the reaction (antigen) control wells [33]. The median
is calculated as the mean of two mid-values of the reaction
control wells, eliminating from the calculation the highest and
lowest values (see Note 7).
3.3 Indirect ELISA l Coat microplates with 1 μg/ml of the recombinant fusion anti-
to Detect gen 3ABC in carbonate/bicarbonate buffer, pH 9.6 (100 μl per
Non-structural Protein well) for overnight at 4 C.
(NSP ELISA) l Wash the plates six times with washing buffer (PBST).
l Add 100 μl of 1/20 dilution test sera in blocking buffer per well
and incubate the plates for 30 min at 37 C and wash six times
in PBST.
l Add 100 μl optimally diluted horseradish-peroxidase-conju-
gated rabbit anti-species (swine) IgG in the blocking buffer per
well and incubate the plates for 30 min at 37 C.
l After six washings, fill each well with 100 μl of 30 30 , 50 5-
0
-tetramethylbenzidine plus 0.004% (w/v) H2O2 in phos-
phate/citrate buffer, pH 5.5.
l Stop the reaction after 15 min of incubation at room tempera-
ture by adding 100 μl of 0.5 M H2SO4.
l Read the absorbance at 450 nm and at 620 nm for background
correction.
l Interpreting the results:
Test results are expressed as percent positivity relative to the
strong positive control
Optical Density of Testor Control wells

Percent positivity ¼ 100
Optical Density of Strong Positive Control
or alternatively as a test to control (T/C) index relative to a cut-off
(i.e., threshold positive) control. Profiling the NSP antibody reac-
tivity levels in herds along with age/vaccination stratification aids
interpretation of herd infection status in vaccinated
populations [34].
4 Notes
1. The 146S antigen needs to be prepared by propagating the

serotype of interest in cell culture and inactivating viruses with
ethyleneimine as described for vaccine manufacture (The final
dilution chosen is that which, after addition of an equal volume
of diluent, gives an absorbance on the upper part of the linear
region of the titration curve) (optical density
approximately 1.5).
2. Prepared by inoculating guinea-pigs with FMDV 146S antigen
of serotype of interest. Predetermined optimal concentrations
to be prepared in blocking buffer PBS containing 0.05% Tween
20, and 5% dried, nonfat skimmed milk (PBSTM).
3. Antigens are prepared from selected strains of FMDV grown
on monolayers of BHK-21 cells. The unpurified supernatants
are used and pretitrated in a twofold dilution series but without
serum. The final dilution chosen is that which, after addition of
an equal volume of diluent (see below), gives an absorbance on
the upper part of the linear region of the titration curve (optical
density approximately 1.5).
4. (a) The five bioengineered FMDV NSPs 3A, 3B, 2C, 3D, and
3ABC are expressed in E. coli C600 by thermo-induction.
The 3D polypeptide is expressed in its complete form
[35], whereas the rest of the proteins are obtained as
fusions to the N-terminal part of the MS-2 polymerase
gene [36].
(b) The expressed polymerase is purified over phosphocellu-
lose, followed by poly(U) Sepharose columns. The fused
proteins 3A, 3B, 2C, and 3ABC are purified by sequential
extraction of the bacterial extracts with increasing concen-
trations of urea. The 7M fraction containing the fusion
proteins is further purified on a preparative 10%
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel
electrophoresis). The fusion protein band is excised from
the gel and electroeluted [35].
5. (a) Includes each plate a set of strong and weak positive and
negative controls calibrated against the International
Standard Sera
(b) Test cut-off values, with or without suspicious zones, need
to be determined with consideration to the purpose of
testing and the intended target population. Inconclusive
results may be followed up using confirmatory tests, retest-
ing with EITB or a second NSP ELISA (taking account of
the conditional dependence of the two tests). Although not
a suitable test for certifying animals prior to movement,
NSP ELISAs may be a valuable adjunct in circumstances
where the serotype or subtype of virus in the originating
country is not known.
6. The assay should be validated in terms of the cut-off value
above which sera should be considered positive in relation to.
(a) the particular serotypes and strains of virus under
investigation
(b) the purpose of testing
(c) the population under test
The reference values may be as per the OIE Reference
Laboratory at Pirbright, for serotype O, for all species, for the
purposes of demonstrating freedom from infection in a naı̈ve
population, greater than 60% inhibition is considered positive
[21]. For maximum sensitivity, for example when certifying
individual animals for international trade, an inconclusive
range may be set between 40 and 60%.
7. In general sera with titers greater than or equal to 1/90 are
considered to be positive. A titer of less than 1/40 is consid-
ered to be negative. For certification of individual animals for
the purposes of international trade, titers of greater than 1/40,
but less than 1/90 are considered to be doubtful, and further
serum samples may be requested for testing; results are consid-
ered to be positive if the second sample has a titer of 1/40 or
greater. For the purposes of herd-based serosurveillance as part
of a statistically valid serological survey, a cut-off of 1/90 may
be appropriate. Cut-off titers for evaluating immunological
protection afforded by vaccination have to be established
from experience of potency test results with the relevant vac-
cine and target species.
References
1. Ferris NP, Donaldson AI (1992) The World 2. Roeder PL, Smith PLB (1987) Detection and
Reference Laboratory for Foot and Mouth typing of foot-and-mouth disease virus by
Disease: a review of thirty-three years of activity enzyme-linked immunosorbent assay: a
(1958-1991). Rev Sci Tech 11(3):657–684
sensitive, rapid and reliable technique for pri- mumps virus antibodies. Proc Soc Exp Biol
mary diagnosis. Res Vet Sci 43(2):225–232 Med 160(3):363–367
3. Ferris NP, Dawson M (1988) Routine applica- 15. Ukkonen P, Penttinen K, Granström ML
tion of enzyme-linked immunosorbent assay in (1981) Mumps-specific immunoglobulin M
comparison with complement fixation for the and G antibodies in natural mumps infection
diagnosis of foot-and-mouth and swine vesicu- as measured by enzyme-linked immunosorbent
lar diseases. Vet Microbiol 16(3):201–209 assay. J Med Virol 8(2):131–142
4. Hamblin C, Armstrong RM, Hedger RS 16. Siegle RL, Jennings BR, Adams PL, King LP
(1984) A rapid enzyme-linked immunosorbent (1980) Development of a model using poly-
assay for the detection of foot-and-mouth dis- peptide antibodies for scintigraphy of the pan-
ease virus in epithelial tissues. Vet Microbiol creas. Investig Radiol 15(5):457–461
9(5):435–443 17. Engvall E (2010) The ELISA, enzyme-linked
5. Have P, Lei JC, Schjerning-Thiesen K (1984) immunosorbent assay. Clin Chem 56(2):
An Enzyme-Linked Immunosorbent Assay 319–320
(ELISA) for the primary diagnosis of foot- 18. Hornbeck P (1992) Enzyme-linked immuno-
and-mouth disease characterization and com- sorbent assays. Curr Protoc Immunol 1(1):2-1.
parison with complement fixation. Acta Vet https://doi.org/10.1002/0471142735.
Scand 25(2):280–296 im0201s01. (Chapter 2: Unit 2.1)
6. Smitsaart EN, Saiz JC, Yedloutschnig RJ, Mor- 19. Ma LN, Zhang J, Chen HT, Zhou JH, Ding
gan DO (1990) Detection of foot-and-mouth YZ, Liu YS (2011) An overview on ELISA
disease virus by competitive ELISA using a techniques for FMD. Virol J 8(1):1–9
monoclonal antibody specific for the 12S pro- 20. OIE Terrestrial Manual (2018) Chapter.3.1.8.
tein subunit from six of the seven serotypes. Vet Foot and mouth disease (infection with Foot
Immunol Immunopathol 26(3):251–265. and Mouth disease virus). OIE
https://doi.org/10.1016/0165-2427(90)
90095-A 21. Paiba GA, Anderson J, Paton DJ, Soldan AW,
Alexandersen S, Corteyn M, Donaldson AI
7. Engvall E, Perlmann P (1971) Enzyme-linked (2004) Validation of a foot-and-mouth disease
immunosorbent assay (ELISA) quantitative antibody screening solid-phase competition
assay of immunoglobulin ELISA (SPCE). J Virol Methods 115(2):
G. Immunochemistry 8(9):871–874 145–158
8. Van Weemen B, Schuurs AHWM (1971) 22. Sevik M, Ozturk FF (2013) Comparative eval-
Immunoassay using antigen—enzyme conju- uation of liquid-phase blocking ELISA and
gates. FEBS Lett 15(3):232–236 solid-phase competition ELISA methods for
9. Carlsson HE, Lindberg AA, Hammarström S the detection of antibodies to the structural
(1972) Titration of antibodies to Salmonella O proteins of foot-and-mouth disease types O
antigens by enzyme-linked immunosorbent and A viruses. Turk J Vet Animal Sci 37(5):
assay. Infect Immun 6(5):703–708 523–528
10. Holmgren J, Svennerholm AM (1973) 23. Belsham GJ (1993) Distinctive features of
Enzyme-linked immunosorbent assays for foot-and-mouth disease virus, a member of
cholera serology. Infect Immun 7(5):759–763 the picornavirus family; aspects of virus protein
11. Ljungström I, Engvall E, Ruitenberg EJ synthesis, protein processing and structure.
(1974) Proceedings: ELISA, enzyme linked Prog Biophys Mol Biol 60(3):241–260
immunosorbent assay-a new technique for 24. Klump W, Marquardt O, Hofschneider PH
sero-diagnosis of trichinosis. Parasitology (1984) Biologically active protease of foot and
69(2):xxiv mouth disease virus is expressed from cloned
12. Voller A, Huldt G, Thors C, Engvall E (1975) viral cDNA in Escherichia coli. Proc Natl Acad
New serological test for malaria antibodies. Br Sci 81(11):3351–3355. https://doi.org/10.
Med J 1(5959):659–661 1073/pnas.81.11.3351
13. Bishai FR, Galli R (1978) Enzyme-linked 25. Moonen P, van der Linde E, Chénard G, Dek-
immunosorbent assay for detection of antibo- ker A (2004) Comparable sensitivity and speci-
dies to influenza A and B and parainfluenza ficity in three commercially available ELISAs to
type 1 in sera of patients. J Clin Microbiol 8 differentiate between cattle infected with or
(6):648–656 vaccinated against foot-and-mouth disease
14. Leinikki PO, Shekarchi I, Tzan N, Madden virus. Vet Microbiol 99(2):93–101
DL, Sever JL (1979) Evaluation of enzyme- 26. Clavijo A, Wright P, Kitching P (2004) Devel-
linked immunosorbent assay (ELISA) for opments in diagnostic techniques for
differentiating infection from vaccination in mouth disease by the detection of antibodies

foot-and-mouth disease. Vet J 167(1):9–22 to the non-structural proteins 3D, 3AB and
27. Brocchi E, Bergmann IE, Dekker A, Paton DJ, 3ABC in ELISA using antigens expressed in
Sammin DJ, Greiner M et al (2006) Compara- baculovirus. Arch Virol 143(8):1461–1476
tive evaluation of six ELISAs for the detection 32. Neitzert E, Beck E, de Mello PA, Gomes I,
of antibodies to the non-structural proteins of Bergmann IE (1991) Expression of the
foot-and-mouth disease virus. Vaccine aphthovirus RNA polymerase gene in Escher-
24(47–48):6966–6979 ichia coli and its use together with other bioen-
28. Bergmann IE, Malirat V, Neitzert E, Beck E, gineered nonstructural antigens in detection of
Panizzutti N, Sanchez C, Falczuk A (2000) late persistent infections. Virology 184(2):
Improvement of a serodiagnostic strategy for 799–804
foot-and-mouth disease virus surveillance in 33. Karber G (1931) 50% end point calculation.
cattle under systematic vaccination: a com- Archiv Exp Pathol Pharma 162:480–483
bined system of an indirect ELISA-3ABC with 34. Bergmann IE, Neitzert E, Malirat V, Ortiz S,
an enzyme-linked immunoelectrotransfer blot Colling A, Sanchez C, Melo EC (2003) Rapid
assay. Arch Virol 145(3):473–489 serological profiling by enzyme-linked immu-
29. De Diego M, Brocchi E, Mackay D, De Simone nosorbent assay and its use as an epidemiologi-
F (1997) The non-structural polyprotein cal indicator of foot-and-mouth disease viral
3ABC of foot-and-mouth disease virus as a activity. Arch Virol 148(5):891–901
diagnostic antigen in ELISA to differentiate 35. McCullough KC, Bruckner L, Schaffner R,
infected from vaccinated cattle. Arch Virol Fraefel W, Müller HK, Kihm U (1992) Rela-
142(10):2021–2033 tionship between the anti-FMD virus antibody
30. Mackay DKJ, Forsyth MA, Davies PR, reaction as measured by different assays, and
Berlinzani A, Belsham GJ, Flint M, Ryan MD protection in vivo against challenge infection.
(1998) Differentiating infection from vaccina- Vet Microbiol 30(2–3):99–112
tion in foot-and-mouth disease using a panel of 36. Strebel K, Beck E, Strohmaier K, Schaller H
recombinant, non-structural proteins in (1986) Characterization of foot-and-mouth
ELISA. Vaccine 16(5):446–459 disease virus gene products with antisera
31. Sørensen KJ, Madsen KG, Madsen ES, Salt JS, against bacterially synthesized fusion proteins.
Nqindi J, Mackay DKJ (1998) Differentiation J Virol 57(3):983–991
of infection from vaccination in foot-and-
Chapter 23
SDS-PAGE and Western Blotting: Basic Principles and

Protocol
Mukesh Bhatt, Vishal Rai, Ashok Kumar, Kiran, Ajay Kumar Yadav,
Kaushal Kishor Rajak, Vikas Gupta, Vishal Chander, and R. K. Avasthe
Abstract
Western blotting is an important analytical technique used in cell and molecular biology for last four
decades. It involves separation of proteins in SDS-PAGE and then transfer of proteins to a membrane
followed by detection. By using a western blot, one can identify specific protein from a complex mixture of
proteins. Along with its use as a diagnostic aid, it can also be used to verify proteins of interest in exploratory
proteomic studies to identify different disease mechanisms. The ease of performing the technique, low cost,
and accessibility further support the use of western blot in proteomic research. However, a good under-
standing, initial training, and optimization are of utmost importance because being a multi-step technique,
it is prone to false results and incorrect interpretation. This chapter attempts to explain the technique and
theory behind western blot along with some ways to troubleshoot.
Key words SDS-PAGE, Western blot, Protein blotting, Immunoblot
1 Introduction
The term “blotting” refers to the transfer of biological samples

from a gel on to a membrane and their subsequent detection on
the surface of the membrane. The western blot (often called as the
protein immunoblot because an antibody is used to specifically
detect the antigen) is a widely used analytical technique to detect
specific proteins in a given sample of tissue homogenate or extract.
The technique was initially described by Towbin et al. in 1979 [1]
and the name was given by Burnette in 1981 [2] to match similar
techniques used for detection of DNA in Southern blotting [3] and
RNA in Northern blotting [4].
The first step in a Western blotting procedure is to separate the
macromolecules using SDS-polyacrylamide gel electrophoresis
(SDS-PAGE). Following electrophoresis, the separated molecules
313
are transferred or blotted onto a second matrix, generally a nitro-

cellulose or polyvinylidene difluoride (PVDF) membrane
[5]. Next, the membrane is blocked to prevent any nonspecific
binding of antibodies to the surface of the membrane. The trans-
ferred protein is then complexed with an enzyme-labeled antibody
as a probe. An appropriate substrate is then added to the enzyme
and together they produce a detectable product such as a chromo-
genic or fluorogenic precipitate on the membrane for colorimetric
or fluorometric detection, respectively. This technique is very useful
to detect the presence of a given protein in a sample, to assess
posttranslational modifications such as phosphorylation and to
characterize protein complexes. Although the western blotting
protocol for specific purpose can be optimized, here we discuss
step-by-step, the general principle and protocol followed for west-
ern blotting procedure in virology.
2 Sample Preparation
The first step for western blotting is sample preparation. Protein

samples from cultured cells can be extracted either by direct disso-
lution in a denaturing buffer or by homogenization in a buffer
containing protease inhibitors. Sample denaturing buffers contain
the powerful anionic detergent sodium dodecyl sulfate (SDS),
which linearizes the proteins, and a reducing agent such as
2-mercaptoethanol to break disulfide bonds. The ionic buffers are
supposed to provide higher yield of proteins as compared to the
nonionic buffers; however, this may lead to the loss of some func-
tional properties of proteins [5]. The viscosity of the sample treated
with ionic buffers is also higher due to the release of the chromatin
and thus may need sonication.
The other agents used for sample preparation are nonionic
detergents which generally include Triton X-100, Nonidet P-40,
Tween, etc. and zwitter-ionic (CHAPS) detergents. Although these
agents are less harsh and less denaturing and keep the proteins
relatively intact, the protein yield is comparatively low especially
from subcellular organelles and membranes [6]. As the cell lysis
leads to the release of proteases and phosphatases, the samples must
be kept at 4 C and protease inhibitor cocktail must be used to
prevent any further protein degradation. It is useful to know the
protein concentration in the lysates to control the amount loaded
on the gel and to allow comparison between experiments. It is also
important to have an idea of the protein concentration in the
extracted sample using a suitable method [7–9] so as to ensure
that the total protein amount in each sample under analysis is
equivalent.
The final step is to mix the samples with a loading buffer or
sample treatment buffer composed of (a) glycerol to increase the
SDS-PAGE and Western Blotting: Basic Principles and Protocol 315
density of the sample facilitating its loading on the gel, (b) a color-
ing dye to make the sample visible (bromophenol or Coomassie
Blue), (c) the agents reducing disulfide bonds (β-mercaptoethanol
or dithiothreitol, DTT), (d) SDS in case of denaturing conditions,
and (e) Tris-HCl. To ensure complete denaturation of the proteins,
samples are usually boiled at 95 C for 5–10 min before loading.
3 Electrophoresis
The electrophoresis can be optimized for the specific proteins and

based on the purpose of the study. The two common methods used
for electrophoresis are discussed here.
4 SDS-PAGE
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is the most

widely used method for analyzing protein mixtures qualitatively
[10]. Denatured proteins in individual samples are separated by
SDS-PAGE. Polyacrylamide gels are formed by the polymerization
of two compounds, acrylamide and the crosslinking agent N,
N-methylene bisacrylamide, in a buffered solution containing 1%
SDS in presence of TEMED (tetramethylethylenediamine) and
APS (ammonium persulfate). The polymerized gels form a 3-D
network of long hydrocarbons cross-linked by methylene groups
forming a sieve of pores that allows the movement of proteins. The
pore size is inversely proportional to the concentration of the
acrylamide in the gel (i.e., higher percentage gel allows better
resolution of smaller proteins while the low conc. Gels allow better
resolution of high molecular weight proteins) (Table 1). The nega-
tive charge to the proteins is rendered by binding of SDS molecule
to the denatured proteins, thereby swamping the original charge on
protein molecules. Generally, one molecule of SDS binds every two
amino acids in a protein. Two vertical gels are used in SDS-PAGE
procedure, i.e., stacking gel, which is having lower pH (6.8) and
larger pore size and the resolving gel which is having a pH of 8.8
and different pore size depending on the target protein size and
acrylamide concentration [11]. The composition of stacking and
resolving gels is given in Table 2.
In stacking gel, proteins move at a faster speed sandwiched
between chloride ions at top and glycinate ions at bottom to form
a layer at the junction of stacking and resolving gel. After reaching
the resolving gel, the glycinate becomes fully ionized and moves at
a faster speed leaving behind the protein-SDS complexes which
move towards the anode [11]. As protein-SDS complexes have
the same charge per unit length, they travel into the separating
gel under the applied electric field with the same mobility.
Table 1
Suggested acrylamide concentration in resolving gel for separation of proteins based on molecular
weight
Protein mol. weight (kDA) Acrylamide conc. in resolving gel (%)

10–43 15
12–60 12
20–80 10
30–95 8
50–200 6
Table 2
Composition of stacking and resolving gel for SDS-PAGE
Resolving gel (10 ml)
Component Stacking gel (5 ml) 15% 12% 10%

Water 3.4 2.3 3.3 4.0
Acrylamide: bisacrylamide (29:1) 0.83 5.0 4.0 3.3
Stacking gel buffer (1.0 M pH 6.8) 0.63 –
Resolving gel buffer (1.5 M pH 8.8) – 2.5 2.5 2.5
10% SDS 0.05 0.1 0.1 0.1
10% APS 0.05 0.1 0.1 0.1
TEMED 0.005 0.004 0.004 0.004
5 Native PAGE
Although SDS-PAGE is the most frequently used gel system for

studying proteins, it is not a suitable method to study the proteins
(enzymes) based on their biological activity as the proteins are
denatured by the SDS–PAGE procedure [11]. In such cases, the
non-denaturing conditions are used where the polyacrylamide gels
but without SDS are used and no sample treatment is done before
sample loading. Since all the proteins carry their native charge at the
gel pH (8.7), proteins are separated based on their electrophoretic
mobility and difference in charge and size of the proteins in a given
mixture. The biological properties of proteins under study can be
measured using a suitable substrate solution which may produce
detectable signal at the position of protein in the gel.
6 Protocol for SDS-PAGE
6.1 Materials l Acrylamide:bisacrylamide (29:1).

Required l SDS (10%).
l Ammonium persulfate (10%).
l TEMED.
l SDS-PAGE apparatus.
l Resolving and stacking gel buffers.
l Gel loading buffer.
l Electrophoresis buffer.
l 2 gel loading buffer.
l Staining solution.
l Destaining solution.
6.2 Procedure Determine the volume of the gel to be prepared. Prepare the
required volume of solution containing the desired concentration
6.2.1 Casting of Gel
of acrylamide for resolving gel as given in table (Table 1).
6.2.2 Details of Gel 1. Mix the components in the order shown. Polymerization will
Preparation begin as soon as the TEMED has been added. Without delay,
swirl the mixture rapidly and proceed to next step.
2. Pour the acrylamide solution between the glass plates using a
pipette. Overlay with water or water saturated butanol.
3. After polymerization is complete, pour off the overlay and wash
the top of gel several times with deionized water to remove any
unpolymerized acrylamide.
6.2.3 Stacking Gel Pour the stacking gel solution directly on the surface of the poly-
merized resolving gel. Immediately insert a clean Teflon comb in
the stacking gel solution being careful to avoid trapping air bubble.
6.2.4 Sample 1. While the stacking gel is polymerizing, prepare the samples by
Preparation and Loading boiling for 3 min in 1 SDS gel loading buffer to denature the
protein.
2. After polymerization is complete, remove the Teflon comb
carefully and mount the gel in the electrophoresis apparatus.
Add Tris-glycine electrophoresis buffer to the top and bottom
reservoirs.
3. Load upto 15 μl of each of the sample in a predetermined
order.
4. Attach the electrophoresis apparatus to an electric power sup-
ply and run the gel for 1–2 h at 100 V constant voltage.
5. After completion of run, take apart the glass plates and carefully
remove the gel into a staining tray.
6.2.5 Staining of Proteins separated by SDS-PAGE can be simultaneously fixed with

Polyacrylamide Gel with methanol: glacial acetic acid and stained with Coomassie brilliant
Coomassie Brilliant Blue blue.
1. Dissolve 0.25 g of Commassie Brilliant Blue R250 in 90 ml of
methanol: H2O (1:1 v/v) and 10 ml of glacial acetic acid. Filter
the solution through a Whatman No.1 filter.
2. Immerse the gel in 5 volume of staining solution, keep at room
temperature for 4 h.
3. Remove the stain.
4. Destain the gel in destaining solution for 12 h (Methanol:
Acetic Acid:Water ¼ 30:10:60).
After destaining, gels may be stored indefinitely in water.
In case, the gel is to be used for western blotting, the staining
step is not performed and the gel is used for transfer of separated
proteins on to a nitrocellulose (NCM) of PVDF (polyvinylidene
difluoride) membrane.
6.2.6 Electrotransfer For transferring the separated proteins, gel is kept over either the
nitrocellulose or PVDF membrane and sandwiched between the
tissue papers followed by application of electromagnetic filed in
perpendicular direction (Fig. 1). The efficient transfer of protein
bands can be confirmed by using the protein-staining dyes like
Ponceau S which do not interfere with the subsequent immuno-
logical detection [6]. Additionally, the prestained protein molecular
weight markers also facilitate the monitoring of transfer of proteins
on the membrane. The efficiency of transfer depends on a number
of factors including size of protein, method of transfer, buffer
composition, and type of membrane used. Generally, the transfer
buffer is a Tris and glycine solutions supplemented with methanol
and SDS (Table 3). The balance between SDS and methanol is
crucial for efficient transfer of proteins as SDS improves the solu-
bility and the migration of proteins but reduces their binding to
membranes, while methanol precipitates proteins reducing their
migration but improves protein-binding to membranes.
There are two methods for transfer of separated proteins: wet
and semidry [12]. For a wet transfer, the gel-membrane sandwich
system is kept submerged in the transfer buffer for the entire
duration and is more favorable for larger proteins of size more
than 100 kDa. However, this method is time-consuming and
requires more volume of buffers. On the other hand, in a semidry
transfer (Fig. 1), the gel-membrane sandwich is placed directly
between electrodes and only the filter paper is soaked with the
transfer buffer. This method is comparatively fast which completes
between 10 min to 1 h and requires less volume of buffer. However,
the transfer is sometimes less effective and poses difficulty in visua-
lizing the transferred proteins especially when the proteins are of
Apparatus Cover
Cathode (-)
Filter Papers
Gel
Transfer
Direction Membrane
Filter Papers
Anode (+)
Apparatus Cover
Fig. 1 Representation of semidry system for transfer of proteins from gel to membrane
Table 3
Transfer buffer components and their uses
Component Example Use

Buffer Tris, CAPS, Carbonate Support conduction, maintain appropriate pH
Alcohol Methanol, ethanol Increase binding of proteins to membrane
Detergent SDS Promote migration of proteins out of gel
Tris-tricine Tris-tricine up to 9.5 Tricine separates low MW proteins from free SDS
high molecular weight. Nevertheless, due to its convenience, semi-

dry method is the most commonly used method for the transfer of
separated proteins.
Out of the nitrocellulose and PVDF membranes, nitrocellulose
membrane is charged and binds with the hydrophilic part of pro-
teins while the PVDF membrane binds with hydrophobic part.
Although the nitrocellulose membrane binds very quickly and effi-
ciently with the proteins, the bonding is mechanically weaker than
that of PVDF membrane and hence the NCM is not suitable when
re-probing is required. In contrast to NCM, the PVDF membrane
produces high background noise and requires activation with
methanol.
6.2.7 Blocking As the membranes used for the transfer of proteins have character-
istics for nonspecific protein binding, it may lead to background
development in subsequent processing. Therefore, blocking is
done by using the solution containing irrelevant proteins such as
1–5% nonfat dry milk or bovine serum albumin. The nonionic
detergent like 0.01% Tween 20 can also be included to reduce
nonspecific binding without disrupting specific binding of antibody
to the target proteins [5].
6.2.8 Probing After blocking, the protein of interest is probed with an antibody
specific to that protein which may require extensive optimization
owing to the variable strength and specificity of different antibodies
to proteins. The optimum antibody dilution must be determined
using different conc. of detector antibody so as to obtain better
sensitivity in the assay. Probing can be performed in two ways:
one-step or two-step probing. In one-step probing, the antibody
conjugated to an enzyme [such as horseradish peroxidase (HRP),
or alkaline phosphatase (AP)], radio-isotope or fluorophore is used
to detect the target protein [6]. In contrast, in two-step probing,
two different antibodies are used; one against the protein of interest
(primary antibody) and secondly the secondary anti-species anti-
body against the primary antibody conjugated with an enzyme or
fluorophore. In addition to anti-species antibodies, other proteins
like protein A and G, which have the affinity for immunoglobulin
heavy chains, can also be used as secondary reagents. These
reagents can be radiolabeled (e.g., with 125I) or, more commonly,
conjugated to biotin, fluorescein, or to an enzyme such as horse-
radish peroxidase (HRP) [5, 6]. As more than one secondary anti-
bodies can bind to the one primary antibody leading to higher
signal production and sensitivity, the two-step procedure is the
most commonly used method for probing of target proteins in
western blot. In some cases, the antibody can be replaced by
other proteins like avidin and streptavidin which can be used to
detect biotinylated proteins on the membrane.
The excess unbound antibodies may produce the background
during development and hence must be removed through washing
step. Generally, a mild detergent solution like phosphate buffered
saline with 0.05% Tween 20 (PBST) is used for washing of
unbound antibody on the membrane before addition of substrate
solution.
6.2.9 Detection of Bound The bound primary antibody on the membrane can be quantified
Antibody by three means: colorimetry, chemiluminescence, and fluorescence
[1, 5, 6]. Out of these three, colorimetry is the cost-effective
though less sensitive than other two methods and can be used for
the procedures where the amount of target protein is sufficiently
large to be detected. Colorimetric detection is based on the gener-
ation of colored product that is formed by the reaction of enzyme
on conjugated antibody and the substrate which is deposited on the
membrane. No specialized equipment is required in colorimetry for
visualization of colored product and the color produced is also
quite stable.
Fluorometric detection (Fig. 2b) is based on the use of an
antibody which is labeled with a fluorophore. A light source is
then used to excite the fluorophore, which subsequently produces
a transient light emission as it returns to its ground state. The light
Fig. 2 Detection of target protein. (a) Chemiluminescence method and (b) Fluorescent detection
emitted at a higher wavelength than that was used for excitation is

detected by a fluorescence reader. The advantage of this method is
that it can be used for detection of more than one protein at the
same time using fluorophores that produce different color signal.
In chemiluminescence method (Fig. 2a), the enzyme labeled
secondary antibodies (e.g., HRP labeled antibody) are visualized
using a substrate solution containing luminol. The results are
recorded in a digital imager. Although chemiluminescence method
of detection is quite sensitive with an ability to detect proteins in
femtolitres, substrate acts as a limiting reagent and as the substrate
gets exhausted, the light production decreases and eventually stops.
However, a well-optimized tests are reported to produce a stable
light output for several hours, allowing consistent and sensitive
protein detection.
6.2.10 Stripping and Stripping is an optional step in the western blotting when more
Re-probing than one protein is analyzed by different antibodies. The procedure
of removing the bound antibody is called stripping and is per-
formed before the re-probing. In mild stripping, generally low
pH buffers (e.g., pH ¼ 2.2) with low concentration of detergent
(e.g., 0.1% SDS) are used while for harsh stripping, a high incuba-
tion temperature (e.g., 50 C) with high concentration of deter-
gent (e.g., 2% SDS) is used [5]. After stripping, the membrane is
blocked again and subjected to probing as per standard protocol.
However, it is to mention that the stripping is not consistent
throughout the membrane and hence the downstream results
must be used with caution for semiquantification.
7 Protocol for Western Blot
7.1 Reagents and 1. Electroblotting transfer buffer for 1000 ml.

Materials Required for
Blotting Tris Base 25 mM 3g
Glycine 192 mM 14.4 g
Methanol 20% (v/v) 20 ml
SDS (sodium dodecyl sulfate) 0.1% 1g
2. Phosphate buffered saline solution (PBS) pH 7.4 for 1000 ml.
KH2PO4 (anhydrous) 1.4 mM 0.20 g

Na2HPO4 (anhydrous) 8 mM 1.14 g
NaCl 140 mM 8.0 g
KCl 2.7 mM 0.2 g
3. Phosphate buffered saline with 0.05% Tween 20 (PBST).

4. Bovine serum albumin (BSA)—3% in PBST (Blocking
solution).
5. Nitrocellulose (NC) membrane 0.45 μm pore size. 0.2 μm pore
size nitrocellulose membrane may be used for low molecular
weight molecules.
6. Primary antibody.
7. HRPO-conjugated secondary antibody.
8. Minigel system.
9. Power pack.
10. Semidry blotter.
11. DAB (3,30 Diaminobenzidine) solution (prepared freshly
before use with 0.05% DAB, 0.05% Nickel Chloride and
0.015% H2O2 in PBS, pH 7.2).
7.2 Procedure Carry out SDS-PAGE (cassette size approx. 100 100 mm). Load
5–20 μg of protein per well. Apply constant voltage at 50–100 V.
7.2.1 Protein Blotting 1. Build the transfer “sandwich” onto the anode (+) plate as
(Semidry Method) and follows: 3–4 sheets blotting papers soaked in blotting buffer
Developing (roll out air bubbles), NC membrane pre-wet with deionized
water, Slab gel, 3–4 sheets blotting papers soaked in blotting
buffer (avoid trapping air bubbles between gel and membrane).
2. Carry out the transfer at a constant current of 1.9–2.5 mA per
cm2 of the gel area for 1.5 h at room temperature.
3. Remove the NC membrane from the apparatus and air-dry the

NC blot thoroughly.
4. NC membrane is then incubated in 3% w/v BSA in PBST
(blocking solution) at room temperature for 2 h or overnight
at 4 C. The choice of blocking reagent depends on the type of
probe that will be subsequently used in the overlay procedure
and should be chosen accordingly.
5. Remove the blocking buffer by decantation and wash the NC
membrane 3–4 times with 10–15 ml of PBST for 5 min each.
6. Overlay the blot with 10 ml of primary antibody at an appro-
priate dilution (generally 1:50–1:500). Incubate for 1–3 h at
37 C.
7. Wash the NC membrane 3–4 times for 5 min each, with
sufficient PBST.
8. Incubate the NC membrane for 1 h at 37 C in HRPO-
conjugated secondary antibody at an appropriate dilution (1:
2000 to 1:10,000) in PBST.
9. Wash the NC membrane 3–4 times for 5 min each, with
sufficient PBST.
10. The membrane is developed by adding 10 ml of DAB solution
for 5–10 min or until the color development.
11. Stop the reaction by washing the NC membrane in several
changes of distilled water.
12. Air-dry the strips and store in the dark in a plastic sleeve
between two sheets of blotting paper.
7.2.2 Precautions l Avoid touching the surface of the membrane. Wear clean gloves
and handle the blot only with clean forceps.
l Do not use skimmed milk in blocking when using avidin/biotin
detection systems. Endogenous biotin in milk can cause high
background.
l Do not use sodium azide as a preservative for buffers when using
an HRP detection system. Sodium azide inhibits HRPO enzyme
activity.
8 Applications of PAGE and Western Blotting in Virology
1. Native PAGE can be employed to study the electrophoretic

mobility of segmented viruses like porcine rotaviruses. For this,
the extracted RNA is loaded into the wells of a polyacrylamide
gel and the genomic segment migration can be visualized by
silver staining procedure [13]. In some cases, the animal species
may be carrying more than one species of rotaviruses which can
be differentiated based on the electropherotype pattern of

RNA isolated from fecal samples [14].
2. Polyacrylamide gel electrophoresis can be employed to study
the differential protein expression in virus-infected and
non-infected cells [15].
3. Western blotting procedure using virus specific antiserum can
be used to study the difference in protein migration between
the parental and mutant viruses in which some part of the
genome is missing or deleted [16].
4. The expression of different viral proteins intended for develop-
ment of vaccines and diagnostics can be studied with the help
of western blotting.
5. Western blot can be utilized to assess the posttranslational
modifications. The target protein is first immunoprecipitated
and then probed with antibodies against ubiquitin or small
ubiquitin-like modifier (SUMO) or vice versa. Similarly, west-
ern blot can also be used to study the protein–protein interac-
tions by testing the presence of a potential interactor using
co-immunoprecipitation. Here, one protein (the bait) is immu-
noprecipitated using a specific antibody against the bait and the
immunocomplex is allowed to run onto an SDS-PAGE. The
membrane carrying the immobilized immunocomplexes is
then subjected to WB using antibodies specific to potential
interactors (targets) [5].
6. Western blot can also be used in diagnostic virology for detec-
tion of expressed proteins in body fluids [17] especially in the
prion diseases to detect prion proteins in body tissues.
Although there are no reports of prion disease in pigs following
natural infection, the western blot is commonly used to study
the prion proteins in experimentally infected pigs [17, 18].
7. Western blotting is also a useful tool in analytical virology to
study the antibodies in the serum of infected animals or to
assess the diagnostic potential of expressed proteins for devel-
opment of different immunoassays [19–21].
9 Limitations of Western Blot
Although western blot is a useful analytic technique, there are some

caveats which are discussed here that must be considered.
1. Quantification: Western blot does not provide the quantitative
results for the protein measurements but can be used with
caution to study the relative expression of proteins. Although
the digital imaging devices like charged coupled devices (CCD)
have an advantage over the X-ray imaging techniques, it is
always recommended to use an internal control to improve the

accuracy. In such cases, the immunoreactivity of the target
antigen is normalized to the immunoreactivity of the
housekeeping protein in each sample for quantitative analysis.
This makes it important to carefully determine the equivalent
protein loading volume in each well so as to increase the
accuracy which makes the test time-consuming and more
laborious.
2. Specificity: The specificity of the primary antibody should be
meticulously characterized to rule out the concerns over cross-
reactivity with nontarget proteins. The structurally similar pro-
teins of similar size may move to the same position in the gel
and the cross-reactive antibody may result in false results.
Therefore, control experiments should always be run and fur-
ther the membrane should also be probed with secondary
antibody alone to ensure that the immunoreactive bands are
specific for primary antibody only.
3. Sensitivity: Although the western blot is quite sensitive and the
detection methods like chemiluminescence can detect protein
levels up to femtograms, due to some intrinsic factors like
insufficient transfer, decreased affinity to antibodies, changes
in buffer conditions, etc. the detection limit is generally higher.
Therefore, the proteins in very low concentration are generally
missed and hence the negative results cannot be interpreted as
complete lack of target protein in the sample.
10 Troubleshooting
Sr.
no. Problem Possible causes Interventions needed
1. No signal Insufficient amount of a sample Make sure to measure the
concentration of lysates to ensure
that the proper amount of total
proteins is loaded on the gel
(20–50 μg of cell lysate per lane or
25–100 ng purified proteins). If the
target protein is expected to be at low
level, increase the amount of lysate
loaded to the gel.
Incomplete transfer Make sure that the transfer is uniform
and complete. A prestained protein
ladder is an easy way to assess protein
transfer but staining the membrane
with Ponceau S or the gel with
Coomassie Blue is more thorough. If
(continued)
Sr.
the transfer is incomplete, extend the
time and/or increase
electromagnetic field (voltage or
current) or consider performing a
wet transfer. Buffer composition
should also be checked to meet the
requirements.
Poor detection There are two main reasons for poor
detection: (1) weak recognition
and/or binding of a target protein by
the antibody or (2) problems with
detection system. Validate the
antibody by taking along a purified
target protein or a sample with
heterologously expressed target
protein. Carefully standardize the
optimum concentration of the
antibody or extend the incubation
time (overnight at 4 C). Monitor
the wash buffer composition also.
Use less stringent washing
conditions. In case of a
chemiluminescent detection system,
use a more sensitive substrate (e.g.,
ECL femto) and increase the
exposure time.
2. High back The high background usually comes 1. Decrease the amount of proteins
ground from the (a) binding of the antibody to loaded per lane.
nonspecific targets, (b) overloaded 2. Optimize the number and/or extend
samples, (c) insufficient washings, or time of washings after antibody
(d) poor selectivity of the incubation.
antibody used. 3. Ensure sufficient blocking of the
membrane.
4. Increase stringency of an antibody
incubation by using a blocking solution
or increase detergent concentration.
5. Optimize the concentration of the
antibody.
6. Dry the membrane before imaging.
Degradation or posttranslational Minimize degradation by using
modifications of the target protein appropriate inhibitors and handle
may result in the detection of multiple samples at 4 C.
bands.
Membrane not fully stripped Ensure adequate buffer volume and
stripping time
3. Misshaped or Usually this indicates problems with the Run the gel and transfer on ice or
missing electrophoresis or the transfer due to decrease the voltage/current. Make
bands excessive heat or uneven transfer. sure that all the air is “rolled out”
from the blot sandwich.
(continued)
Sr.
Bulky bands and migration artifacts may Decrease loading or optimize the
indicate gel overloading or buffer loading protein concentration.
problems (“smiling gel”).
Cellular debris in samples Sonicate and filter to remove the debris.
For DNA in samples, shear samples
with syringe or add DNase
11 Conclusion
Western blot has been in practice for more than four decades now
and has proved to be a very useful analytic method to study the
relative protein expression, posttranslational modifications, and
protein–protein interactions. Although it is not a quantitative tech-
nique, it has been adapted for semiquantitative analysis of proteins.
The low cost, ease to perform and accessibility has made this
method a widely used technique in research labs for studying the
proteins. Western blotting can also be used to verify proteins of
interest in exploratory proteomic techniques such as
two-dimensional gel electrophoresis as well as to identify the poten-
tial mechanisms underlying aberrant tissue functions or disease. It is
also used routinely to help in diagnosis of some important viral
diseases especially the prion diseases. However, a good understand-
ing, initial training, and optimization are of utmost importance
because being a multi-step technique, it is prone to false results
and incorrect interpretation.
References
1. Towbin H, Staehelin T, Gordon J (1979) Elec- 5. Gwozdz T, Dorey K (2017) Western blot. In:
trophoretic transfer of proteins from polyacryl- Basic science methods for clinical researchers.
amide gels to nitrocellulose sheets: procedure Academic, Boca Raton, pp 99–117
and some applications. Proc Natl Acad Sci U S 6. Counts SE (2010) Western blot. In: Encyclo-
A 76(9):4350 pedia of movement disorders, vol 2010. Aca-
2. Burnette WN (1981) “Western blotting”: elec- demic, pp 323–326 . ISBN 9780123741059.
trophoretic transfer of proteins from sodium https://do i.org/10.1016/B978-0-12-
dodecyl sulfate—polyacrylamide gels to 374105-9.00297-5
unmodified nitrocellulose and radiographic 7. Bradford MM (1976) A rapid and sensitive
detection with antibody and radioiodinated method for the quantitation of microgram
protein A. Anal Biochem 112(2):195–203 quantities of protein utilizing the principle of
3. Southern EM (1975) Detection of specific protein-dye binding. Anal Biochem 72:
sequences among DNA fragments separated 248–254
by gel electrophoresis. J Mol Biol 98 8. Lowry OH, Rosebrough NJ, Farr AL, Randall
(3):503–517 RJ (1951) Protein measurement with the Folin
4. Alwine JC, Kemp DJ, Stark GR (1977) phenol reagent. J Biol Chem 193(1):265–275
Method for detection of specific RNAs in aga- 9. Smith PK, Krohn RI, Hermanson GT, Mallia
rose gels by transfer to diazobenzyloxymethyl- AK, Gartner FH, Provenzano MD et al (1985)
paper and hybridization with DNA probes. Measurement of protein using bicinchoninic
Proc Natl Acad Sci U S A 74(12):5350–5354 acid. Anal Biochem 150(1):76–85
10. Kurien BT, Scofield RH (2015) Western blot- virus nonstructural polyprotein 3AB-coding
ting: an introduction. Methods Mol Biol 1312: region to design a negative marker virus.
17–30. https://doi.org/10.1007/978-1- Virus Res 243:36–43. https://doi.org/10.
4939-2694-7_5. PMID: 26043986; PMCID: 1016/j.virusres.2017.10.010
PMC7304528 17. Greenlee JJ, Kunkle RA, Smith JD, Greenlee
11. Wilson K, Walker JM, Hofmann A, Clokie S MHW (2016) Scrapie in swine: a diagnostic
(2018) Wilson and Walker’s principles and challenge. Food Saf 4(4):110–114. https://
techniques of biochemistry and molecular biol- doi.org/10.14252/foodsafetyfscj.2016019.
ogy. Cambridge University Press, PMID: 32231914; PMCID: PMC6989210
New York, NY 18. Hedman C, Otero A, Douet JY, Lacroux C,
12. Kurien BT, Scofield RH (2009) Protein blot- Lugan S, Filali H, Corbière F, Aron N, Badiola
ting and detection: methods and protocols. JJ, Andréoletti O, Bolea R (2018) Detection of
Humana Press, New York PrPres in peripheral tissue in pigs with clinical
13. Kattoor JJ, Saurabh S, Malik YS, Sircar S, disease induced by intracerebral challenge with
Dhama K, Ghosh S, Bányai K, Kobayashi N, sheep-passaged bovine spongiform encepha-
Singh RK (2017) Unexpected detection of lopathy agent. PLoS One 13(7):e0199914.
porcine rotavirus C strains carrying human ori- https://doi.org/10.1371/journal.pone.
gin VP6 gene. Vet Q 37(1):252–261. https:// 0199914
doi.org/10.1080/01652176.2017.1346849 19. Ameri M, Zhou EM, Hsu WH (2006) Western
14. Todd D, McNulty MS, Allan GM (1984) The blot immunoassay as a confirmatory test for the
use of polyacrylamide gel electrophoresis of presence of anti-mycoplasma hyopneumoniae
virus RNA in the study of rotavirus infections. antibodies in swine serum. J Vet Diagn Investig
In: McNulty MS, McFerran JB (eds) Recent 18(2):198–201. https://doi.org/10.1177/
advances in virus diagnosis. Current topics in 104063870601800210
veterinary medicine and animal science, vol 29. 20. Plotzki E, Keller M, Ivanusic D, Denner J
Springer, Dordrecht. https://doi.org/10. (2016) A new western blot assay for the detec-
1007/978-94-009-6039-8_11 tion of porcine cytomegalovirus (PCMV). J
15. Urzainqui A, Tabarés E, Carrasco L (1987) Immunol Methods 437:37–42. https://doi.
Proteins synthesized in African swine fever org/10.1016/j.jim.2016.08.001
virus-infected cells analyzed by 21. Yang S, Li L, Yin S et al (2018) Single-domain
two-dimensional gel electrophoresis. Virology antibodies as promising experimental tools in
160(1):286–291. https://doi.org/10.1016/ imaging and isolation of porcine epidemic diar-
0042-6822(87)90076-6 rhea virus. Appl Microbiol Biotechnol 102:
16. Bhatt M, Mohapatra JK, Pandey LK, Mohanty 8931–8942. https://doi.org/10.1007/
NN, Das B, Prusty BR, Pattnaik B (2018) s00253-018-9324-7
Mutational analysis of foot and mouth disease
Chapter 24
Immune Assays as Diagnostic for Pig Viral Diseases

Prabhakar Maurya, Jupi Talukdar, Sarmistha Debbarma,
Monuj Kumar Doley, and Luit Barkalita
Abstract
In the present era of globalization, there is a constant threat of infectious diseases with the potential of
cross-species transmission, hence mitigation of infectious diseases is a one-health problem which needs to
be addressed using collaborative research and resources. Correct and quick diagnosis is central to any
infectious disease management strategy. Among different available diagnostic options, immunological and
new generation molecular biology-based tests have become more popular than the rest. Immuno-
diagnostic assays are widely used for routine diagnosis and surveillance of viral and bacterial infections in
human and animals. Immunoassays are frequently used in various livestock diseases, including diseases of
pig because of its specificity, sensitivity, ease of operation, relatively low-cost involvement, and advantage of
being usable for population screening. In recent decades, increased popularity of pork in different cuisines
has contributed to growth of organized swine farming in many states in India. As a collateral effect,
increased swine population and human–pig interaction have exacerbated the circulation of viruses and
challenged our ability to prevent, control, and/or eliminate impactful pig diseases, making early and
efficient diagnosis of pig viral disease a need of the day. Here, we highlight different methods for diagnosis
of pig viral diseases with a special focus on immuno-diagnostics.
Key words Pig, Infectious disease, One-health, Immuno-diagnostic assays, Fluid-phase immunoassay
1 Introduction
The occurrence of diseases puts one to think of the state of health

and how being healthy is different from being diseases. At initial
thoughts the answer to “What is a disease?” is simple but a look
through any medical dictionary soon shows that articulating a
satisfactory definition of disease is surprisingly difficult. Not only
there are various types of diseases in various species but the reasons
of occurrence of diseases are different with different manifestations
in different species and even may differ from individuals within the
same species though the etiology may be same. The World Health
Organization’s claim that health is “a state of complete physical,
mental and social well-being, not merely the absence of disease or
329
330 Prabhakar Maurya et al.
infirmity” [1] has been praised for embracing a holistic viewpoint,

and equally strongly condemned for being wildly utopian: the
historian Robert Hughes remarked that it was “more realistic for
a bovine than a human state of existence” [2, 3].
The emergence of sudden diseases, escalating the spread to a
small geographical region or over the whole world, now considered
as possible events especially after the present pandemic of COVID-
19, but a peek in the history reveals that such events have been
reported since antiquity. In modern times we have had the devas-
tating pandemics of influenza, cholera, Ebola, SARS, and recent
COVID-19. The source of infection has always been regarded as an
utmost factor in epidemiology. Although many diseases are species
specific, meaning that they can only occur in one animal species,
many other diseases can be spread between different animal species.
These are infectious diseases, caused by bacteria, viruses, or other
disease causing organisms that can live as well in humans as in other
animals. There are different methods of transmission for different
diseases.
Communicable diseases can be classified according to the
source of infection as anthroponoses (when the source is an infec-
tious human; interhuman transfer is typical), zoonoses (the source
is an infectious animal; interhuman transfer is uncommon), and
sapronoses (the source is an abiotic substrate, nonliving environ-
ment; interhuman transfer is exceptional) [4]. Many of these dis-
eases are caused by viruses, particularly those with RNA genomes
[5]. In some cases, zoonotic diseases are transferred by direct
contact with infected animals, much as being near an infected
human can cause the spread of an infectious disease. The source
of infection is often the reservoir or, in ecologic terms, the habitat
where the etiologic agent of the disease normally thrives, grows,
and replicates. A characteristic feature of most zoonoses and sapro-
noses is that once transmitted to humans, the epidemic chain is
usually aborted, but the clinical course might be sometimes quite
severe, even fatal. An ecologic rule specifies that an obligatory
parasite should not kill its host to benefit from the adapted long-
term symbiosis, whereas an occasionally attacked alien host, such as
a human, might be subjected to a severe disease or even killed
rapidly by the parasite because no evolutionary adaptation to that
host exists [4].
When considering the factors that lead to the emergence of
viral diseases, it becomes very apparent that there is a high degree of
interrelatedness. Human encroachment in a virgin ecosystem pro-
vides the opportunity for a previously unencountered virus to infect
human beings or their domestic animals. Viral mutability could
enable a virus to adapt to the new host. Subsequent trade and
migration could allow spread of the new virus to susceptible indi-
viduals and the large populations found in urban centers could
ensure that the virus is maintained. The interplay of all these factors
Immune Assays as Diagnostic for Pig Viral Diseases 331
provides a rational framework and explanation for virus emergence.

However, to gain an appreciation of the components of this process
it is necessary to focus on each in itself before linking them together
[6]. By its nature, the emergence of unknown diseases is impossible
to predict [7].
2 Viral Diseases of Pigs and Their Public Health Importance
Between 1968 and 2018, the worldwide swine inventory increased

from 550 to 981 million pigs (+78%), with the most marked
growth in the developing regions of the world, i.e., Africa + 504%,
Asia + 137%, and South America + 59% [8]. Over the same period,
albeit with regional variations, the majority of pig production
moved from smaller, farrow-to-finish enterprises into larger, multi-
site production systems that are highly dependent upon the inter-
change of animals, people, equipment, and sundries between
production sites; a process that connects farms and moves infec-
tious agents between them [9, 10].
The majority of emerging human pathogens are zoonotic.
Frequently changing husbandry practices and environmental fac-
tors (e.g., large-scale domestic animal production, urbanization,
interaction between wild and domestic swine populations with
humans, population increases, etc.) may predispose humans and
pigs to pathogens common to other species, or may allow for the
adaptation of these organisms to humans or swine. Being omnivo-
rous and having the anatomy and physiology similar to that of man,
pigs are a good medium for the adaptation and increase in virulence
of organisms that have so far not been identified as human
pathogens [11].
During recent times, much emphasis has been focused on
human emerging infectious diseases (EID) caused by pathogens
of animal origin. All these zoonotic threats and events have empha-
sized the need for a “One Health” approach, which has been
summarized in the so-called 12 Manhattan principles. The “One
Health” approach integrates communication, collaboration, and
coordination between public health, animal health, and other com-
munities at multiple levels to prevent, detect, and control emerging
or re-emerging infectious diseases at the animal–human–environ-
ment interface [12].
Given the sheer number of swine present worldwide, and the
large percentage of the population that consume pork, swine rep-
resent a significant reservoir of confirmed or potential zoonoses.
Swine represent a potential reservoir for many novel pathogens and
may transmit these to humans via direct contact with live animals
(such as swine farmers and large animal veterinarians), or to the
general human population via contaminated meat [13]. Over the
last 30 years, diseases caused by emerging swine viruses (ESV) have
acquired great relevance, more than in other species [14].
Major viral disease outbreaks occur continually in the pig

industry (Table 1) [17]. The global understanding of viral disease
dynamics requires to account for all interactions at all levels, from
within-host to between-herd, to have all the keys for development
of control measures [18]. The intensification and globalization of
the swine industry has contributed to the emergence and global
spread of pathogens of swine, driven in part by frequent move-
ments of pigs, feed, and pork products at local, national, and
international scales [19]. Effective prevention and clinical manage-
ment of infectious diseases are intimately linked to early and accu-
rate screening of pathogens, not only by detecting the infectious
particles in the organism but also by elucidating the aspects that
confer resistance to therapy and immune escape profiles, including
mutations and genotype disparity. Therefore, rapid diagnosis ben-
efits in allowing timely therapy to prevent complications; and ben-
efits public health by collecting data for epidemiological studies, to
prevent outbreaks and spreading of diseases [20].
3 Diagnostic Methods for Viral Diseases: Needs and Challenges
Infectious diseases represent a global threat to humans, livestock

and wildlife animals, and plants with potential cross-species trans-
mission. Mitigating infection is therefore a one-health problem,
which needs to be addressed using all materials in hands, both in
terms of research and resources. When referring to such problems,
one commonly thinks about biological analysis, i.e., virological and
immunological diagnostics, which are essential for the understand-
ing of host–pathogen interactions (Table 2) [18]. Major viral dis-
ease outbreaks occur continually in the pig industry [17]. From
serious pandemics and highly contagious infections to common
influenza episodes, clinical prognosis often relies on early detection
of the infectious agent. Thus, effective identification of viral patho-
gens is needed to help prevent transmission, set up appropriate
therapy, monitor response to treatment and lead to efficient disease
management and control [20].
Diagnostic assays for detection and monitoring of virus infec-
tions have become more and more important and are widely used in
routine diagnostics. In particular, the detection of newly emerging
infectious diseases is challenging; therapeutic treatment regimens
and prevention strategies can also influence the need for diagnostic
assays [21]. An important first step in choosing an appropriate
diagnostic approach is to decide the objectives of the tests to be
performed. These objectives might include detection of infection in
at least one animal in a herd, determining the prevalence of a virus
within the herd, confirming exposure to virus or vaccine, or asses-
sing the timing of an infection. These factors will determine which
animals to test, the number of samples required, and the
Table 1
Major viral diseases of pigs (Adapted from [15, 16])
Sl. no. Virus Disease(s) caused Zoonotic concern

1 African swine fever virus African swine fever No
2 Aujeszky’s disease virus Aujeszky’s disease, also known as pseudorabies No
3 Classical swine fever virus Classical swine fever, also known as hog cholera No
4 Coronaviruses Primarily causing diarrhoea with similar treatment and control Not established properly
but immunologically are very distinct from each other
(i.e. no cross-protection between these different viruses)
5 Foot-and-mouth disease virus Foot-and-mouth disease No
6 Influenza A virus Respiratory infections Yes
7 Porcine circovirus type 2 virus Cause variety of systemic diseases in pigs including wasting, Not established properly
pneumonia, late-term abortions, stillbirths, porcine dermatitis,
nephropathy syndrome and diarrhoea
8 Porcine reproductive and Primarily causing diarrhoea with similar treatment and control but No
respiratory syndrome virus immunologically are very distinct from each other (i.e. no
cross-protection between these different viruses).
9 Rotaviruses Major cause of diarrhoea in neonatal and young pigs No
10 Swine vesicular disease virus Swine vesicular disease No
Immune Assays as Diagnostic for Pig Viral Diseases
333
Table 2
Summary of main viral diagnostic methods (Adapted from [20])
Diagnostic
technique Principle Variants
Immunoassay Formation of Ag–Ab through recognition and binding RIA
EIA (FPIA, MEIA,
CLIA)
Nucleic Acid Amplification and detection of Sequences from the viral RT-PCR
Amplification genome (DNA or RNA) qPCR
Tests (NAAT) NASBA
TMA
NGS: Next- Polymerization of DNA template by incorporation of Pyrosequencing
Generation labeled dNTPs, and terminate the extension Fluorescently labelled
Sequencing dNTP
Detection of released
hydrogenion (H+)
MS: Mass Ionization of the sample, then separation and detection MALDI-TOF MS
Spectrometry of the particles according to their mass-to charge ESI MS
ratio (m/z) Often combined with
other methods:
PCRMS
CLIA chemiluminescent immunoassay, dNTP deoxyribonucleotide triphosphate, EIA enzyme immunoassay, ESI elec-
trospray ionization, FPIA fluorescence polarization immunoassay, MALDI-TOF matrix-assisted laser desorption ioniza-
tion time-of-flight, MEIA micro-particle enzyme immunoassay, MS mass spectrometry, NGS next-generation
sequencing, NAAT nucleic acid amplification test, NASBA nucleic acid sequence-based amplification, qPCR quantitative
polymerase chain reaction, RIA radio-immunoassay, RT-PCR real-time polymerase chain reaction, TMA transcription-
mediated amplification
appropriate tissues to use. Furthermore, interpreting individual test

results may not reflect the status of the entire herd, as the timeline
of infection will vary for each individual animal—so appropriate
measures need to be taken to compensate for the cases of delay in
diagnostic procedure or statistically insignificant results.
Molecular and serological techniques should be used in a com-
plimentary fashion for the diagnoses of virus infections. Knowledge
of the stage of infection, viral pathogenesis, and epidemiology help
to make decisions regarding the correct test to choose for appro-
priate diagnosis. Advances in specificity and sensitivity and capacity
to test for a range of viruses through multiple platforms make
accurate diagnosis and rapid identification of circulating viruses
for real-time clinical relevant data more feasible. Virus isolation
and collaboration with specialist laboratories make newer techni-
ques for identification of emerging and re-emerging viruses
possible [22].
Prominent swine viral diseases such as swine fever, porcine
reproductive and respiratory syndrome, classical swine fever, food
and mouth disease, swine vesicular disease, influenza A virus
disease, African swine fever, porcine respiratory disease complex,

etc. have not only clinical importance but also great economic
importance [23]. Therefore, the primary requirement of diagnosis
of suspected viral diseases in pigs is of rapid diagnosis and with
maximum accuracy. Most of the pig farms are commercial or indus-
trial in nature and are intensive farms mostly located at a certain
distance from the populated areas [24], hence the access of veteri-
nary and laboratory care is not immediate. Currently, there are
many diagnostic tests for the detection of viral diseases, but many
challenges are faced on the field in the diagnostic procedures:
l Sample collection and transportation from field to laboratory:
Most of the diagnostic tests use serum samples which require
blood collection and then centrifugation. Such facilities are not
available at the farm and require preservation and then transport
of sample to appropriate laboratory settings which increases the
time duration from sample collection to diagnostic results.
l Sample processing: Complex diagnostic tests require exhaustive
sample processing procedures which can further increase the
duration for diagnostic results. Improper sample processing
can provide inaccurate results.
l Location and capacity of laboratories: Specialized laboratory
settings are required for viral diagnostics and such laboratory
settings are not readily accessible to farm settings in semi-urban
or rural areas.
l False positive of false negative results: Many diagnostic tests can
display false positive or false negative results due to external or
technical factors.
l Cost benefit ratio: Pig farming is carried out on a commercial
scale in most places, but for accurate diagnostic results, a large
number of samples are to be processed from suspected farm,
which increases the cost of diagnostics.
Citing the above factors, efficient but cost-effective and rapid
diagnostic tests are required for detection of viral diseases of swine.
Blood collection from multiple animals is itself a challenging task in
the field setting which further increases the chances of exposure of
research personnel to susceptible animals. Saliva-based viral diag-
nostic tests are most practical in terms of logistics as well as time
required for collection, oral fluid-based surveillance reflects the
adaptation of conventional testing methods to an alternative diag-
nostic specimen [25]. Most of the diagnostic tests discussed above
do not effectively meet the cost benefit requirements of pig farmers
expecting large commercial farms or research studies. Hence, the
most effective and cost-effective diagnostic tests are radio-
immunoassay and enzyme immunoassays.
4 Recent Methods in the Diagnosis of Viral Infections
In a rapidly growing world of technology, the industry is continu-

ously delivering up-to-date instruments but many factors are limit-
ing their implementation in healthcare and field settings which
unfortunately delays global benefit. Diagnostic tests are direct
measures of exposure or vaccination, and indirect measures of
immunity. Depending on the specific diagnostic assay, laboratory
tests detect antibodies or proteins that are produced in large quan-
tities by the pig in an immune response to an infection or the
presence of an infectious agent [26]. Broadly the following diag-
nostic methods are available for detection of viral infections
[20, 27].
4.1 Immunoassay- Immunoassays are bioanalytical methods in which the quantitation

based Tests of the analyte depends on the reaction of an antigen (analyte) and
an antibody (Table 3). Automated immunoassay-based methods
are among the most frequently used for testing, and are effective
because of the high specificity and binding affinity between antigen
and antibody. Therefore, the principle of the test relies in the
formation of an immuno-complex between antibody present in
Table 3
Classification of various immunoassays and their characteristics (Adapted from [27])
Diagnostic Bound vs free

technique Labels (Reporter Groups) separation Signal detection Sensitivity
Precipitation Not required Not required Naked eye 10 μg/
immunoassays mL
Turbidity
Nephelometry
Particle Artificial particles (gelatin, Not required Naked eye 5 ng/
immunoassays particles, latex, etc.) Pattern analyzer mL
Spectrophotometry
Particle counting
Radioimmunoassays Radioisotopes (I 125, H 3) Required Photon counting 5 pg/
mL
Enzyme Enzymes Required Spectrophotometry 0.1 pg/
immunoassays mL
Fluorometry
Photon counting
Fluorescent Fluorophores Required Photon counting 5 pg/
immunoassays mL
Chemiluminescent Chemiluminescent Required Photon counting 5 pg/
immunoassays compounds mL
the sample and synthetic antigen present in the reagent or vice

versa, to generate a measurable signal.
Immunoassays use labels conjugated to synthetic antibodies or
antigens which are linked to a solid phase, and used to capture
corresponding antigens or antibodies present in sera samples. These
labels could be radioactive isotopes, enzymes that cause change in
color or light-generating substances. Consequently, this principle
has generated several methodologies for the testing.
4.1.1 Radio- It uses radioisotopes (such as Iodine 125) to label antigen or

immunoassay (RIA) antibody. The amount of substance to analyze is determined by
the amount of the generated radioactivity. RIA is a highly sensitive
method but the main drawback is the handling and disposal of
hazardous radioactive substances. Fluid-phase radio binding assays
(RBAs) provide a more efficient alternative. In this method, the
autoantigens are tested in solution rather than being immobilized
on a solid surface.
Various competitive RIA methods have been developed to
measure a plethora of different biological compounds. Initially, a
known amount of labeled antigen and an antigen from a biological
specimen are combined and reacted with a known amount of
antibody that is usually coated on a solid phase such as sepharose
beads or on the inner wall of plastic tubes. After the mixture
equilibrates, it is washed to remove unreacted antigens, and the
immune complex containing both labeled and unlabeled antigen is
trapped in the solid phase. The washing step is referred to as B/F
(bound versus free) separation.
RIAs can offer a number of advantages over other immunoas-
says in that they are highly sensitive and precise, although the latest
chemiluminescent-based methods typically surpass RIA limits of
detection. Disadvantages include the fact that radioisotopes are
highly regulated, may have a short half-life, and that RIAs are
heterogeneous assays.
4.1.2 Enzyme Immunoassays employing enzyme-mediated reactions as labels

Immunoassays were developed as alternatives to RIAs. Commonly used techniques
include the enzyme-linked immunosorbent assay (ELISA), the
EIA, and the enzyme-multiplied immunoassay (EMIT).
Heterogenous EIAs are essentially the same as RIAs except that
enzymes are used as labels rather than radioisotopes. Unlike RIAs,
however, homogenous assays can be developed that eliminate
washing steps for separation of bound and free molecules. EIAs
have a number of advantages compared to other immunoassay
methodologies in that highly sensitive assays can be developed
because of the ability of enzymatic reactions to amplify antigen–
antibody interactions. In addition, reagents are cheap and have a
longer shelf life than RIAs. Further, no radiation hazards exist.
Finally, a number of different types of assays have been developed.
Disadvantages of EIAs include the fact that assays can be more

complex, and enzyme activity may be affected by various substances
in biological fluids.
An important advantage of EIAs over RIAs is that the former
can be developed as homogenous assays in which the tedious
washing step to remove free antigen is eliminated, although
homogenous EIAs are frequently less sensitive than RIAs or
heterogeneous EIAs.
The main variants of EIA are as follows:
1. Fluorescence polarization immunoassay (FPIA): uses fluores-
cent label and polarized light.
2. Micro-particle enzyme immunoassay (MEIA): widely used and
relies on alkaline phosphatase enzyme and a corresponding
fluorescent substrate.
3. Chemiluminescent immunoassay (CLIA): uses chemilumines-
cent or light-emitting labels.
4.1.3 Precipitation and These methods, unlike other immunoassays, are often qualitative in
Particle Immunoassays nature. They have also been in use longer than other assay types.
Precipitation assays are used to measure immunoprecipitation reac-
tions, which form when large complexes of antigens and antibodies
combine to generate insoluble complexes. Detection of complexes
can be afforded using light scattering instrumentation and is
termed nephelometry. The lower limit of sensitivity using these
methods is about 10 mg/mL. Common assays that use these
techniques include the measurement of many major serum
proteins.
A related method of immunoassay is particle agglutination in
which either antibodies or antigens are detected in biological fluids
using corresponding antigens or antibodies, respectively, bound to
various particles. Commonly used particles are latex beads and
gelatin particles. These assays have wide applicability and can mea-
sure biological molecules as diverse as human chorionic gonadotro-
pin or antibodies to HIV.
4.1.4 Multiplex Technological advances in the analysis of materials have created a

Immunoassays situation where hundreds of candidate biomarkers can be generated
in the discovery phases of biomarker research. Each of the candi-
date markers needs to be confirmed in the verification/validation
phase.
4.2 Amplification- The most widely used variants of conventional amplification are
based Assays real-time PCR (quantitative PCR) and reverse transcription-PCR
(RT-PCR). Other amplification-based tests such as nucleic acid
sequence-based amplification (NASBA) and transcription-
mediated amplification (TMA) are suited for detection of RNA
viruses by amplification of the mRNA instead of conversion
to cDNA.
4.3 Next-Generation Next-generation sequencing (NGS) is one of the greatest achieve-

Sequencing ments of the modern era. Beyond genome sequencing from known
organisms, it allowed discovery of novel viruses responsible for
unknown human diseases, and tracking of outbreaks and pan-
demics such as influenza to understand their emergence and trans-
mission profiles.
4.4 Mass Mass spectrometry (MS) is nowadays a benchmark of laboratory

Spectrometry qualitative and quantitative investigation, particularly in bacteriol-
ogy. The principle of MS relies on converting the sample into
charged particles (ions) by ionization process. These ions are sepa-
rated according to their mass-to-charge ratio (m/z) and analyzed
by a detector. The result obtained is compared to a reference
database (library), existing within the system and delivered as an
interpretive spectrum.
In clinical laboratories, matrix-assisted laser desorption ioniza-
tion (MALDI) and electrospray (ES) are the most used ionization
methods because they allow processing of considerable amounts of
analyte. These approaches have extensively been evaluated experi-
mentally and provided excellent results, either used alone or com-
bined with other molecular methods, such as PCR, in order to
enhance sensitivity. The combination (RT-PCR/ESI-MS) was
able to detect viral pathogens usually undetected by regular testing
methods, and provided rapid and detailed data (types and subtypes)
within a short time.
5 Immune Assays in Swine Viral Diseases
Diagnostic assays either measure the immune response induced by

infection or detect the pathogen itself. Most common diagnostic
assays are based on detection of antibodies specific to the pathogen.
Typically, serum is used as the sample, although thoracic fluid and
colostrum are also used. Diagnostic assays or tests are tools used by
producers and veterinarians to assess the disease and immune status
of pigs. Immunity is essentially the pig’s resistance to infection.
Diagnostic assays do not measure immunity. Rather, they look for
evidence of disease exposure in response to vaccination, antibodies,
or infectious agents. In terms of immunity, the underlying assump-
tion is that prior infection or vaccination has stimulated an immune
response that will provide some level of protection from disease
symptoms in the future. For these reasons, diagnostic tests are
direct measures of exposure or vaccination, and indirect measures
of immunity [26].
Immunoassays represent the most frequently used and perhaps
simplest approach to the analysis of biological materials for the
translational researcher. Enzyme immunoassay (EIA) and enzyme-
linked immunoassay formats are the easiest to utilize and are widely
commercially available. More sensitive methods of immunoassay

(electrochemiluminescence, RIA, fluorescence polarization immu-
noassay) require specialized instrumentation for use [27]. Antibody
detection assays have distinct advantages in detection of a patho-
gen. The sample (usually blood) is easy to collect, handle, and store;
antibodies are abundant in blood and can usually be detected for a
long time after infection. With most diseases, antibodies are detect-
able long after the pathogen is not. The pathogen may be detected
by isolation (growing it in the laboratory), staining and visualiza-
tion by microscopy [26].
6 Immunoassays and Immunochemistry
Immunoassay methodologies represent, perhaps, the most fre-

quently used approach to measure biological compounds in trans-
lational and clinical research. Assays exist, from either commercial
or research sources, for both the qualitative and quantitative mea-
surement of a plethora of naturally occurring small molecules such
as lipid mediators and hormones as well as larger peptides and
proteins that are present in human body fluids and tissues
[28]. In addition, a number of therapeutic agents can be measured
by immunoassays. It is important to note that immunoassays can
not only measure antigens but antibodies as well. Many immunoas-
says are extremely sensitive and can detect as little as 0.1 pg of
compound per milliliter of body fluid [29].
The first immunoassays resulted from the pioneering work of
Yalow and Berson in the late 1950s and utilized antibodies labeled
with a radioisotope like 125I. Regardless of the method used, all
immunoassays rely upon the interaction of an antigen with an
antibody [28]. The extent to which this interaction occurs (the
amount of antigen that is bound to antibody versus free) allows
one to measure, either qualitatively or quantitatively, the amount of
that particular antigen that is present in a biological fluid or tissue.
Detection methods for particular assays vary and depend on the
approach used to detect the antigen-antibody complex.
Antigens are defined as any substance that possesses antigenic
sites (epitopes) which produce corresponding antibodies
[28]. Antigens can be small molecules such as haptens, hormones,
etc. or they can be very large compounds such as glycolipids and
proteins. Antibodies that are generated in response to antigens can
be one of the five types and include IgG, IgM, IgA, IgE, and IgD.
Antibodies consist of heavy chains and either k or l light chains and
possess constant and variable regions. The hypervariable region can
be assembled to recognize a wide variety of epitopes [29].
Although antibodies can serve as antigens, for purposes of
immunoassays, they are reactants used to detect antigens. Different
types of antibodies can be obtained from several sources. Polyclonal
antibodies are generated by immunizing an animal with an antigen.
In this case, multiple antibodies are generated, which recognize

different epitopes. As a consequence, the affinity of polyclonal
antibodies for a complex antigen is usually stronger than that of a
monoclonal antibody. Monoclonal antibodies are generated using
somatic cell fusion and hybridoma selection [30].
The resulting established cell line generates a homogeneous
antibody population that represents a single epitope [29]. While
specific for a certain epitope, it should be kept in mind that mono-
clonal antibodies may cross-react with different antigens that pos-
sess the same epitope. Nonetheless, the development of
monoclonal antibodies has revolutionized immunoassay methodol-
ogies because monoclonal antibodies are well defined, and specific
reagents and their production can yield a nearly limitless supply of
an antibody [31]. Further, they can be prepared through immuni-
zation of a nonpurified antigen. A more recent approach to the
development of antibodies for use in immunoassays is phage dis-
play, in which antibody fragments of predetermined binding speci-
ficity are encoded in a phage and expressed in bacteria [32].
7 Swine Viral Diseases and Major Diagnostics Immunoassays
7.1 African Swine African swine fever is one of the most important viral diseases in
Fever pigs caused by an Asfivirus. There are different strains with different
virulence. It is a systemic disease and is notifiable on most countries.
Alternative names: ASF.
Diagnosis:
l It presents postmortem changes with hemorrhagic lymph
nodes, necrotic areas in the spleen, multiple small hemorrhages
in kidneys, and button ulcers in the intestine.
l In all suspected cases, the diagnosis should be confirmed by
laboratory analysis.
l Laboratory analysis include the identification of virus via PCR,
isolation of the virus and the presence of antibodies in serum. In
most countries, the ASF is a notifiable disease.
7.2 Aujeszky’s The Aujeszky’s disease is caused by a herpesvirus that can remain
Disease latent and causes respiratory, reproductive, and nervous problems.
Alternative names: Pseudorabies (PRV).
Diagnosis:
Serology analysis confirms the diagnosis.
7.3 Blue Eye Disease It is a viral disease that produces nervous symptoms, reproductive
failure, and corneal opacity that develops a bluish color caused by a
paramyxovirus.
Alternative names: blue eye disease, BE, paramyxovirus.
Diagnosis:
l In severe/acute outbreaks, a presumptive diagnosis can be made
based on clinical signs.
l Confirm diagnosis by serological tests.
l Isolation of virus from brain tissue.
l Histopathologic lesions especially in the brain are highly
suggestive.
7.4 Bovine Viral Disease caused by two pestiviruses in the same group as the swine
Diarrhea Virus fever virus. These viruses mainly affect cattle and sheep, but can
enter pig farms causing reproductive problems.
Alternative names: Border Disease Virus, BDV, Border disease.
Diagnosis:
Laboratory analysis. A cross reaction (false positive) with anti-
bodies against classical swine fever is possible, which may cause
problems for classical swine fever surveillance in epidemiology and
eradication.
7.5 Classical Swine Classical swine fever is one of the most important viral diseases in
Fever pigs caused by a pestivirus related with bovine viral diarrhea and
with border disease. There are several strains with different viru-
lence. It is a systemic disease and it is notifiable in most countries.
Alternative names: CSF.
Diagnosis:
l They present typical postmortem changes with hemorrhagic
lymph nodes, dead zones in the spleen, multiple small hemor-
rhages in kidneys and so-called “button ulcers” in the intestine.
l In all suspicious cases, the diagnosis should be confirmed by
laboratory analysis.
l Laboratory analysis include the identification of viral antigen,
virus isolation, and the presence of antibodies in serum. In most
countries, CSF is reportable.
l Infections with bovine viral diarrhea and with border disease
may give false positive.
7.6 Delta The diarrhea caused by deltacoronavirus is similar to porcine epi-

Coronavirus demic diarrhea (PED) but at a very low severity.
Alternative names: Deltacoroavirus, Delta corona virus.
Diagnosis:
Suspected by clinical signs, but cannot be differentiated from
TGE and PED. The presence of the organism is confirmed by PCR.
7.7 Ebola Reston Ebola is a very significant infection in humans. Of the five species of
Virus Ebola virus, Ebola Reston virus does not infect humans but can
infect pigs.
Alternative names: Ebola, Ebola Reston, Ebolavirus, REBOV.
Diagnosis:
Serological tests or identification of the virus by PCR.
7.8 Encephalo- Infection caused by Encephalomyocarditis virus, found globally.

myocarditis Usually it is of no clinical importance, but there are some myocar-
ditis cases with high mortality or reproductive problems.
Alternative names: EMC.
Diagnosis:
To make a definitive diagnosis, the virus must be isolated and
identified or an increase in blood antibodies must be demonstrated
in two samples taken in 2 weeks apart. Encephalomyocarditis can be
confused with Aujeszky, parvovirus, and PRRS virus, although
there are symptoms that differentiate these four infections.
7.9 Enteroviruses Swine enteroviruses are found in the intestine, although their clini-
cal significance is questionable.
Alternative names: picornavirus, SMEDI.
Diagnosis:
It has no clinical importance. Virus isolation and serology must
be performed to diagnose.
7.10 Foot-and- Foot-and-mouth disease is one of the most important vesicular

Mouth Disease diseases. Foot-and-mouth disease belongs to the Picornaviridae
virus family of which there are more than 60 strains classified into
seven serotypes.
Diagnosis:
Serological and PCR tests are needed. Foot-and-mouth disease
does not differentiate clinically from the rest of the vesicular dis-
eases. Laboratory samples must include blood, vesicular tissue and
fluid, when possible.
7.11 Hepatitis E Hepatitis E has been identified in pigs with uncertain clinical signif-
Virus icance, but it is a very important viral infection in humans, mostly
seen in third world countries of Asia and Africa. Hepatitis E virus is
prevalent in pigs worldwide.
Alternative names: HEV.
Diagnosis:
l Identification of histological lesions in the liver.
l Identification of the virus by PCR.
l Serology by ELISA.
7.12 Influenza Influenza is a respiratory disease of high importance due to its fast
transmission and zoonotic potential. Porcine influenza is caused by
several Influenza Type A viruses closely related, characterized to
have the ability to change the antigenic structure and create new
strains.
Each serotype is identified through surface proteins called “H”
and “N”. The three most common serotypes affecting pigs are H1
N1, H1 N2, and H3 N2.
Alternative names: Porcine Influenza, influenza type A.
Diagnosis:
In the acute disease, a reliable diagnosis can be done based on
clinical signs, due to the fact that there are no other diseases with
such a dramatically quick transmission and clinical effects. Blood
samples taken from sows at the beginning of the disease and
2–3 weeks after, show an increase in antibody titers. Influenza
virus can be identified from nasal swabs analyzed by PCR.
7.13 Japanese B Japanese B encephalitis is caused by a virus found in South Asia. It is

Encephalitis transmitted by mosquitos and presents as reproductive problems.
Pigs are a significant source of infection.
Diagnosis:
l Lab tests of stillbirths and of testes from affected boars.
l Definitive diagnosis requires tissue culture virus isolation, and
serum antibodies from stillbirths, usually using ELISA.
7.14 Nipah Virus The Nipah virus is zoonotic causing respiratory disease in pigs and
Disease mild to severe symptoms or even death in humans caused by
paramyxovirus.
Alternative names: paramyxovirus.
Diagnosis:
l In the postmortem examination, the predominant sign is the
consolidation of the lungs.
l The diagnosis is made by serological testing, virus isolation and
identification. On infected farms, high levels of antibodies are
detected in the sows.
7.15 PRRS The Porcine Reproductive and Respiratory Syndrome (PRRS) is

the viral infection caused by an arterivirus. The virus causes repro-
ductive problems and affects the respiratory system.
Alternative names: Porcine respiratory and reproductive syn-
drome, Blue ear disease.
Diagnosis:
This is based on the clinical signs, postmortem examinations,
and the known presence of the virus by PCR. There are several tests
available, but ELISA is the standard test used. Currently, oral fluids
are used to monitor farms. Virus sequencing can only be used

epidemiologically to investigate the presence of a new strain and
possibly its origin.
7.16 Parainfluenza Porcine Parainfluenza virus type-1 of the Paramyxoviridae family is

a novel agent suspected of causing mild respiratory disease in pigs.
Alternative names: PPIV1.
Diagnosis:
l PCR is currently used to confirm the presence of the virus in
trachea, lung, nasal swabs, and nasal turbinates. ELISA serology
can also be used to confirm exposure to the virus.
7.17 Porcine Porcine circovirus is caused by porcine circovirus type 2 (PCV2).

Circovirosis Clinically it is presented as a disease which deteriorates animals from
the weaning to the finishing period producing a high mortality rate.
Alternative names: Porcine circovirosis type 2 (PCV2), PMWS.
Diagnosis:
l Due to the fact that most farms have antibodies for porcine
circovirus, tests to detect antibodies in blood do not help with
the diagnosis.
l Clinical signs are not specific, so in order to have a diagnosis, it is
often necessary to perform postmortem examinations in
several pigs.
l The diagnosis is based on three factors:
– Lymph depletion.
– High amount of PCV2 present in tissue (immunohistochem-
ical tests).
– The clinical picture corresponds to PCV2 infections.
7.18 Porcine Cytomegalovirus is a herpes virus common and its consequences

Cytomegalovirus are often insignificant; it is characterized by rhinitis which causes
sneezing.
Alternative names: Porcine cytomegalovirus infection, PCMV,
inclusion bodies rhinitis.
Diagnosis:
It can be confirmed by serology, fluorescence antibody tests,
and demonstrating the presence of inclusion bodies in histological
sections of tissue.
7.19 Porcine Porcine epidemic diarrhea is caused by a coronavirus leading to

Epidemic Diarrhea vomiting and diarrhea with mortality up to 100% within susceptible
piglets under 2 weeks of age.
Alternative names: PED.
Diagnosis:
Clinical signs might help, but cannot be differentiated from

TGE. The presence of the organism is confirmed by PCR. Histo-
logical lesions are characteristic of PED and TGE, thus immuno-
chemistry (IHC) or PCR is used to confirm the disease.
7.20 Porcine Parvovirus affects mainly non-vaccinated primiparous sows, causing

Parvovirus Infection reproductive problems such as mummies.
Alternative names: PPV.
Diagnosis:
Antibodies fluorescent tests and PCR can be performed to
mummified piglets in order to confirm the parvovirus infection.
Serology with very high titers is an indicative of exposition to the
field virus because vaccination does not produce high titers.
7.21 Rotavirus Rotavirus infections present clinically as diarrhea in nursing piglets

Infection or in the first 2 weeks after weaning.
Alternative names: Rotavirus, rotavirosis.
Diagnosis:
As with any diarrhea problem in pigs 10–14 days old, it is
important to make sure—through histological changes and
PCR—that rotavirus is the main factor causing the diarrhea.
7.22 Senecavirus A Senecavirus A (SVA) is a non-enveloped RNA virus from genus

Senecavirus and family Picornaviridae which also includes foot-
and-mouth disease (FMD) virus and swine vesicular disease virus.
Vesicular disease is indistinguishable from foot-and-mouth disease.
Senecavirus A can also cause high transient mortality in piglets up
to 7 days of age.
Diagnosis:
l Timely confirmation of diagnosis is critical to rule out FMD.
l Presence of vesicles especially on snout and feet.
l Detection of virus via PCR from vesicular fluid.
l New ELISA assays have been developed to confirm exposure.
7.23 Teschen Commonly Teschovirus does not produce clinical cases, but there
Disease are very virulent strains that affect the central nervous system.
Alternative names: Teschen disease, Talfan disease, benign
enzootic paresis, poliomyelitis suum, Teschovirus encephalomyeli-
tis, Teschen/Talfan.
Diagnosis:
Identification of the virus is by isolation or by PCR.
7.24 Transmissible Transmissible gastroenteritis is a very important and highly infec-

Gastroenteritis tious disease in pigs caused by a coronavirus, with severe impact in
reproduction, where diarrhea can cause a 100% mortality in piglets
younger than 2 weeks of age.
Alternative names: TGE.

Diagnosis:
The clinical picture of the acute disease is almost pathogno-
monic. Except for the epidemic porcine diarrhea, there are no other
enteric diseases so rapidly transmitted, and that cause high mortal-
ity in piglets. Final diagnosis must be made in the lab from samples
of the intestine of a recently death pig using fluorescent antibodies
test. PCR in feces can also be used. Serology interpretation is
complicated due to a crossed reaction with respiratory coronavirus
antibodies, which is a common disease that only affects the respira-
tory system.
7.25 Vesicular Vesicular exanthema is clinically indistinguishable from foot-and-

Exanthema mouth disease and therefore is of great importance.
Diagnosis:
Same diagnosis as for suspected FMD and swine vesicular
disease and requires laboratory tests for its identification. Serologi-
cal and PCR tests are needed.
7.26 Vesicular Vesicular stomatitis produces a disease clinically indistinguishable

Stomatitis from foot-and-mouth disease (FMD) and therefore is of great
importance.
Alternative names: VSVs.
Diagnosis:
Paired blood samples can be taken (i.e., a sample during the
initial phase of the disease and at 10–14 days). Virus neutralization,
complement fixation, and ELISA are the usual tests used. In pigs,
isolated positive samples would be strongly indicative of active
infection. The disadvantage of blood tests and serology is that it
involves a long delay of at least two weeks to get results.
8 Conclusion
From an animal health perspective, larger pig populations com-

bined with the extensive movement of pigs, people, and material
between production sites produce conditions that promote the
circulation of infectious diseases within/between farms. These cir-
cumstances challenge our ability to prevent, control, and/or elimi-
nate impactful swine pathogens. Disease management based on
clinical observations is neither timely nor accurate; we require an
active and systematic process that achieves the timely detection of
pathogens and produces useful data that can guide management
decisions. Viral infectious diseases represent an important portion
of global public health concerns with thousands of deaths annually.
From serious pandemics and highly contagious infections to com-
mon influenza episodes, clinical prognosis often relies on early
detection of the infectious agent. Thus, effective identification of
viral pathogens is needed to help prevent transmission, set up

appropriate therapy, monitor response to treatment, and lead to
efficient disease management and control. Recent technological
advances in viral identification, including polymerase chain reac-
tion, mass spectrometry, and next-generation sequencing are
applied in the diagnosis and management of viral infections.
These powerful tools combine rapidity and efficiency in detecting
viral pathogens and have revolutionized the field of clinical diag-
nostics. However, a number of drawbacks such as high cost have
limited their use in many laboratories, particularly in resource-
limited settings. On the contrary, the advent of microfluidic tech-
nology coupled with immunoassay has attracted increasing interest
from biomedical research groups, and could represent a challenging
alternative to diagnose viral infections at lower cost.
References
1. WHO (1946) Preamble to the constitution of the spread of classical swine fever during the
the World Health Organization. WHO, 1997-1998 epidemic in the Netherlands. PLoS
New York One 9:95278
2. Hudson RP (1993) In: Kiple KF (ed) The 10. Kinsley AC, Perez AM, Craft ME, Vanderwaal
Cambridge world history of human disease. KL (2019) Characterization of swine move-
Cambridge, Cambridge University Press, pp ments in the United States and implications
45–52 for disease control. Prev Vet Med 164:1–9
3. Scully JR (2004) What is a disease? EMBO Rep 11. Khan SU, Atanasova KR, Krueger WS,
5(7):650–653. https://doi.org/10.1038/sj. Ramirez A, Gray GC (2013) Epidemiology,
embor.7400195 geographical distribution, and economic con-
4. Hubálek Z (2003) Emerging human infectious sequences of swine zoonoses: a narrative
diseases: anthroponoses, zoonoses, and sapro- review. Emerg Microb Infect 2(1):1–11.
noses. Emerg Infect Dis 9(3):403–404. https://doi.org/10.1038/emi.2013.87
https://doi.org/10.3201/eid0903.020208 12. Segalez J (2015) Safepork 2015 conference;
5. Taylor LH, Latham SM, Woolhouse ME keynote lecture: emerging swine diseases and
(2001) Risk factors for human disease emer- infections: an increasing zoonotic threat. pp
gence. Philos Trans R Soc Lond Ser B Biol Sci 97–100
356(1411):983–989. https://doi.org/10. 13. Smith TC, Harper AL, Nair R, Ferguson DD,
1098/rstb.2001.0888 Wardyn SE, Hanson BM, Dressler AE (2011)
6. Johnson N (2014) A short introduction to Emerging swine zoonoses. Vector Borne Zoo-
disease emergence. In: The role of animals in notic Dis 11:9. https://doi.org/10.1089/vbz.
emerging viral diseases. Academic, pp 1–19. 2010.018
https://do i.org/10.1016/B978-0-12- 14. Perfumo CJ, Pereda A, Jongkaewwattanna A,
405191-1.00001-6 Chen Z, Perez DR, Ma J (2020) Editorial:
7. Tesh R (1994) The emerging epidemiology of emerging swine viruses. Front Vet Sci 7:132.
Venezuelan haemorrhagic fever and Oro- https://doi.org/10.3389/fvets.2020.00132
pouche fever in tropical South America. Ann 15. Murcia P, Donachie W, Palmarini M (2009)
N Y Acad Sci. https://doi.org/10.1111/j. Viral pathogens of domestic animals and their
1749-6632.1994.tb19863.x impact on biology. Medicine and Agriculture.
8. FAO (2020) Food and Agriculture Organiza- Encyclopedia of Microbiology 80–819.
tion of the United Nations. FAOSTAT statisti- https://doi.org/10.1016/B978-012373944-
cal database. FAO. http://www.fao.org/ 5.00368-0
faostat/en/#data/QA 16. Ramirez (2018) Chapter-1: diseases
9. Boender GJ, Van Den Hengel R, Van Roer- affecting pigs: an overview of common bacte-
mund HJW, Hagenaars TJ (2014) The influ- rial, viral and parasitic pathogens of pigs. In:
ence of between-farm distance and farm size on Wiseman J (ed) Achieving sustainable produc-
tion of pig meat: animal health and welfare, vol
3. Burleigh Dodds Science Publishing, Cam- 24. Zanon S (2020) Research links industrial pig
bridge, pp 1–29. https://doi.org/10.19103/ farming and virus outbreaks. Mongabay
AS.2017.0013.14 25. Henao-Diaz A, Giménez-Lirola L, Baum DH,
17. Kedkovid R, Sirisereewan C, Thanawongnu- Zimmerman J (2020) Guidelines for oral fluid-
wech R (2020) Major swine viral diseases: an based surveillance of viral pathogens in swine.
Asian perspective after the African swine fever Porcine Health Manag 6:28, pp 1–12. https://
introduction. Porcine Health Manag 6:20. doi.org/10.1186/s40813-020-00168-w
https://doi.org/10.1186/s40813-020- 26. Zimmerman J, Thacker E (2003) Monitoring
00159-x immunity through diagnostics. National Hog
18. Andraud M, Rose N (2020) Modelling infec- Farmer
tious viral diseases in swine populations: a state 27. Kellogg MD (2017) Chapter 8: measurement
of the art. Porcine Health Manag 6:22. of biological materials. Clin
https://doi.org/10.1186/s40813-020- Transl Sci:137–155. https://doi.org/10.
00160-4 1016/B978-0-12-802101-9.00008-9
19. VanderWaal T, Deen J (2018) Global trends in 28. Davies C (1994) Principles. In: Wild D
infectious diseases of swine. PNAS 115(45): (ed) The immunoassay handbook. Stockton
11495–11500 Press, New York, pp 3–47
20. Souf S (2016) Recent advances in diagnostic 29. Ashihara Y, Kasahara Y, Nakamura RM (2001)
testing for viral infections. Biosci Horizon 9: Immunoassays and immunochemistry. In:
1 – 1 1 . h t t p s : // d o i . o r g / 1 0 . 1 0 9 3 / Henry JB (ed) Clinical diagnosis and manage-
biohorizons/hzw010 ment by laboratory methods. W.B. Saunders,
21. Verheyen J (2015) Challenges in the diagnosis Philadelphia, pp 821–849
and prevention of viral infections. Lab Med 30. Koehler G, Milstein C (1975) Continuous cul-
38(2). https://doi.org/10.1515/labmed- tures of fused cells secreting antibody of pre-
2014-0020 defined specificity. Nature 256:495–497
22. Pretorius M, Venter M (2017) Diagnosis of 31. Zola H (1987) Monoclonal antibodies: a man-
viral infections. Viral Infect Child 1:151–182. ual of techniques. CRC Press, Boca Raton, FL
h t t p s : //d oi . o r g / 1 0. 1 0 07 / 9 78 - 3 - 3 1 9- 32. Winter G (1994) Making antibodies by phage
54033-7_6 display technology. Annu Rev Immunol 12:
23. Straub OC (1993) The important viral infec- 433–455
tions of pig. Swine Health Product 2(4):15–18
Chapter 25
Production of Virus-like Particles Using the Baculovirus

Expression System and Their Application in Vaccines
and Viral Disease Diagnosis
Hemanta Kumar Maity, Rajib Deb, Sinéad Lyons, and Ian M. Jones
Abstract
Over many years, the baculovirus expression vector system (BEVS) has grown in popularity as a platform for
expressing recombinant proteins. In addition to high levels of expression, the recombinant protein is near
authentic as the insect cell is capable of posttranslational modifications such as glycosylation, oligomeriza-
tion, disulfide bond formation, acylation, and phosphorylation.
The BEVS method has been particularly successfully employed to produce virus-like particles (VLP) from
a variety of enveloped and non-enveloped viruses. VLPs are recombinant viral structural proteins which
assemble spontaneously into a virus-like structure which is structurally similar to the authentic virus but
devoid of any genomic material, providing a safe choice compared to the risk associated with the handling of
live virus. Currently, many experimental vaccine prototypes have adopted the BEVS as their choice
production VLP based platform as well as for the development of diagnostics for viral diseases. Following
immunization of laboratory animals, insect cell generated VLPs have produced antigen-specific antibody
consistent with a protective capacity equivalent to commercially available live or inactivated vaccines.
In this chapter we provide a detailed protocol for the purification of VLPs of an FMDV O serotype using
the baculovirus expression vector system. The process illustrated by this method may be effectively used for
the development of vaccine candidate and diagnostics against other burdensome viral diseases such as
PCV2, PRRS, or Avian influenza.
Key words Baculovirus expression vector system (BEVS), Virus-like particles (VLP), Vaccine, FMDV,
PCV 2
1 Introduction
Since the first expression of human beta interferon in insect cells by

infection with a recombinant Autographa californica multiple
nuclear polyhedrosis virus (AcMNPV), the baculovirus expression
vector system (BEVS) has become a very popular platform for the
expression of recombinant proteins [1, 2]. In addition to high
expression levels, the insect cell successfully allows posttranslational
modifications such as glycosylation, oligomerisation, disulfide bond
351
352 Hemanta Kumar Maity et al.
formation, acylation, and phosphorylation [3, 4] so that recombi-

nant proteins are produced in a near authentic state. AcMNPV is a
rod shaped double-stranded DNA virus, predominantly infecting
insect larvae in the order Lepidoptera such as Moth and Butterfly.
The non-essential polh gene which expresses the polyhedrin protein
(which normally protects the virion particle from environmental
exposure) is replaced by a foreign gene of interest under polh
promoter control for the expression of recombinant proteins in
the very late phase of the virus replication cycle [5]. Other
non-essential genes such as p10, cathepsin and chitinase may be
replaced for the expression of multiple recombinant proteins in a
similar way [6, 7]. AcMNPV produces two types of virus particle,
budded virus (BV) and occlusion derived (polyhedra based) bacu-
lovirus (ODV), the former of which continue to replicate when
genes for the latter phenotype are deleted or replaced, making a
helper independent expression system. The combination of high-
level expression and the ability to express multiple recombinant
proteins has made the system a natural choice for the production
of VLPs for a number of enveloped and non-enveloped viruses
[8, 9]. VLPs produced in insect cells have shown specific antibody
responses in vaccinated animals that are protective with an efficacy
similar to commercially available inactivated vaccines suggesting
that a commercial vaccine supply may be viable [10, 11].
VLPs assemble spontaneously following the expression of the
viral structural proteins associated with virus capsid assembly. They
are structurally similar to authentic virus but devoid of any genomic
material, providing a safe choice compared to the risk associated
with the handling live virus. In addition, they offer a solution for
vaccine development in cases where emergent strains do not grow
well in tissue culture and the possibility of further engineering, for
example to improve stability or yield [12]. Notably, the three-
dimensional conformational epitopes on the outer surface of VLPs
are antigenically indistinguishable from those on authentic virus and
induce strong humoral and cell mediated immunity that cannot be
matched by the individual structural proteins alone [13–15].
Many experimental vaccine prototypes have adopted VLPs as their
candidate immunogen including FMDV, HFMD, poliovirus [16–19],
EV71 [20], PCV 2 [21], Duck hepatitis A virus [22], and Porcine
Reproductive and Respiratory Syndrome Virus (PRRSV) [23].
However, a concern associated with VLP production in the BEVS
system is that the final vaccine preparation contains baculovirus parti-
cles released by the infected cells. These may have to be inactivated to
comply with legislative requirements on the release of genetically
modified organisms which can be an issue in vaccine production
even if the risk from the baculoviruses themselves is minimal [7]. In
this chapter we provide a detailed protocol for purification of VLPs of
an FMDV O serotype from baculovirus infected cells. The method for
the purified VLPs described here can be used similarly for other VLP
vaccine and diagnostic candidates [24].
Production of Virus-like Particles Using the Baculovirus Expression System. . . 353
2 Materials
1. Insect cell: Sf9 cells (Spodoptera frugiperda), Tnao38 cell line

(Ascalapha odorata).
2. Medium: Insect cell lines were cultured in Insect-XPRESS
protein-free cell medium (Lonza, Switzerland).
3. Fetal calf serum (FCS).
4. T25, T75, T175 vented tissue culture flasks (Greiner
Bio-One).
5. 125 ml, 250 ml, 500 ml, 1 l vented conical flasks (Corning).
6. Lipofectin Transfection Reagent (Fisher).
7. Benzonase® Nuclease (Sigma).
8. TAE buffer (50) (Geneflow).
9. TBS (10) Tris buffer saline (Geneflow).
10. SuperBlock™ (TBS) Blocking Buffer (Thermo Scientific).
11. Complete protease inhibitor cocktail (Roche).
12. Vivaspin column, 2 ml, MWCO 100,000 kDa (GE healthcare).
13. HRP-conjugated anti-rabbit antibody (Agilent
Technology, USA).
14. Carbon coated formvar 200 square mesh copper grids (Agar
Scientific).
15. Beckman Tube, Thinwall, Ultra-Clear™, 38.5 ml,
25 89 mm (Beckman).
16. Beckman Tube, Thinwall, Ultra-Clear™, 14 ml, 14 95 mm
(Beckman).
3 Methods
3.1 Cell Culture Insect cell lines Spodoptera frugiperda (Sf9) were developed from
and Media the ovarian tissue of the Fall Armyworm [25] and Ascalapha odor-
Requirement ata (Ao38), originally thought to derive from eggs of the black
witch moth [26], were later shown to be from Trichoplusia ni and
3.1.1 Bacterial Strain renamed Tnao38 [27], were used for virus stock production and
and Insect Cell Lines empty capsid protein expression, respectively.
3.1.2 Culture Conditions Insect cell lines Sf9 and Tnao38 were cultured in Insect-XPRESS
and Stock Preparation protein-free cell medium (Lonza, Switzerland) supplemented with
2% FCS. The Sf9 cells were cultured as monolayers at 27 C for
virus stock preparation, and Tnao38 cells were cultured as suspen-
sion cultures in a 27 C shaker incubator at 100 rpm. The Tnao38
cells were used for expression and purification. For preservation of
baculovirus stocks, infected insect cells were harvested and centri-

fuged at 3000 g for 10 min and the supernatant containing
baculovirus was stored at 4 C in the dark.
3.2 Generation Recombinant plasmid vectors with the target gene encoding one or
of Recombinant more viral structural proteins were constructed as described [11]
Baculovirus and co-transfected into Sf9 cells with suitable baculovirus DNA
Expressing Gene (such as flashBAC™ GOLD (FBG, Mirus Bio) or ProEasy
of Interest (AB Vector LLC, San Diego, USA)) to produce a recombinant
baculovirus via homologous recombination as described
[11]. Briefly, 1 ml of Sf9 cells were seeded into a 6-well tissue
culture plate at a concentration of 0.9 106 cells/well and incu-
bated at room temperature for 30 min to settle. The recombinant
plasmid (100 ng/μl) 5 μl was mixed with 2 μl baculovirus DNA
(100 ng/μl) and 5 μl nuclease free water (NFW) in a sterile Eppen-
dorf tube and, separately, a lipofection mixture (12 μl) was prepared
by mixing 8 μl lipofectin reagent and 4 μl sterile water. Both the
lipofectin (12 μl) and plasmid DNA-baculovirus contents (12 μl)
were mixed together and incubated at room temperature for
30 min. Following incubation, the DNA-Lipo mixture (24 μl)
was mixed with 1 ml serum free media by gentle pipetting. The
seeded Sf9 cells were washed with serum free media and the 1 ml of
DNA-Lipo mix was layered gently onto the Sf9 monolayer and
incubated at 27 C for 24 h. The next day, 1 ml of complete growth
media containing 2% fetal calf serum (FCS) was added per well and
incubation continued at 27 C for 4–5 days. After five days, the
media were harvested and centrifuged at 2000 x g for 10 min. The
supernatant was collected and labeled as the P0 passage virus.
Similarly, 0.05 ml of P0 virus was used to infect additional Sf9
cells and kept for 4–5 days for the generation of the P1 virus
stock and the same process repeated for P2 and P3 virus passages.
The final virus titer was ~2.0 108 pfu/ml with a volume depen-
dent on the volume of the passage and the intended use. A flow-
chart of the process is shown in Fig. 1.
3.3 Detection Obtaining an accurate virus titer is essential for determining the
of Recombinant mutiplicity of infection (MOI) necessary for optimum protein pro-
Baculoviral Titer by duction and the titer of recombinants expressing the P1 region of O
Plaque Assay Bangkok FMDV was measured by plaque assay. Sf9 cells seeded in a
6-well dish at a concentration 0.5 106/ml were incubated at
3.3.1 Baculoviral Titer by 27 C for 30 min to adhere and after attachment the cell growth
Plaque Assay media was removed and the monolayer washed with serum free
media (Insect-XPRESS™ protein-free cell medium; Lonza, Swit-
zerland). A series of tenfold serial dilutions of the virus to be
titrated was prepared by adding 100 μl of viral sample into 900 μl
of growth media to a final dilution of 106. One milliliter of each
viral dilution was added into the respective wells and incubated for
1 h at room temperature to allow virus adsorption. The supernatant
Fig. 1 Flowchart for preparation of recombinant baculovirus
was then carefully removed and the monolayer overlaid with plaque
media (0.75% low melting point agarose in 50% serum free media)
at a final temperature of approximately 33 C. The plaque media
was allowed to solidify and 1 ml of complete growth media was
overlaid on top of the agarose and incubation continued at 27 C
for 5–6 days. After incubation, plates were stained with 1 ml of
neutral red solution (Sigma-Aldrich) for 1 h at room temperature
and then drained by inverting the plates. Plaque numbers of 3–30
per well were considered ideal for counting and an end point titer
was evaluated by the formula below. Alternatively, a 50% cytopathic
effect score was used and the TCID 50 determined by the Reed and
Muench method [28].
Virus titerðpfu=mlÞ ¼ average plaque count dilution factor
10:
3.3.2 Titration by Sf9 An alternative and quicker method of detection of viral titer was by
ET Cell use of the Sf9 ET cell line [29]. Sf9 ET cells were seeded into
96-well plates at a concentration 0.9 106/ml, 100 μl/well, in
Insect-XPRESS™ medium supplemented with 2% FCS and
allowed to attach for 30 min at 27 C. The serially diluted baculo-
virus stock from 101 to 108 was incubated with the cells in
respective wells. In the negative control, only insect cell media
was added. The plate was incubated at 27 C for 48–72 h until
green fluorescent foci had appeared when viewed under fluores-

cence microscopy. The foci were counted and the virus titre deter-
mined as before.
3.4 Purification 100 ml cultures of Tnao38 suspension cell cultures

of Empty Capsids from (at 1 106 cells/ml) were infected with recombinant baculovirus
Insect Cell (titer ~108/ml) at an MOI of ~3 and incubated in a 500 ml cell
culture conical flask for 3 days with shaking. The viablity of the cells
at the end of this period was typcially 50% when measured by trypan
blue exclusion. Following incubation, the cell suspension was pel-
leted, 2000 g for 10 min at 10 C. Pelleted cells were lysed in a 1/
20th volume of lysis buffer containing 50 mM HEPES (pH 8.0)
supplemented with 400 mM NaCl, 1% NP40, benzonase 3 μl and
one tablet of complete protease inhibitor cocktail (Roche). Lysates
were incubated in ice and vortexed intermittently for approximately
3 h. After treatment, the cell lysate was centrifuged at 15,000 g
for 30 min at 10 C. The clarified lysis supernatant was layered
above a 30% sucrose cushion (in lysis buffer) and any VLPs present
were sedimented through the cushion by ultracentrifugation at
100,000 g for 2 h at 4 C. The pellet was allowed to resuspend
overnight at 4 C and then loaded on top of a 15–45% sucrose
velocity gradient and ultracentrifugation repeated at 100,000 g
for 16 h at 4 C. The sucrose gradient samples were fractionated
into eight equal fractions from the top, preserved at 4 C, and all
fractions assessed by ELISA, Western blot analysis, or TEM as
described below.
3.5 Analysis Expressed proteins were analyzed by sodium dodecyl sulfate poly-
of Empty Capsids acrylamide gel electrophoresis (SDS PAGE) on precast 4–12% Nu
PAGE gels in 1 MES buffer (Invitrogen). All protein samples
were prepared by adjusting to 1 loading buffer (Novex) and
heated at 98 C for 10 min before loading on the gel. The protein
samples were loaded into the wells and the gels were electrophor-
esed at a constant voltage of 200 V for 30 min. The separation of
the protein samples was compared with a prestained protein ladder
(Geneflow). After completion of electrophoresis, the gels were
removed from the gel cast and subjected to either Coomassie
staining or transfer to PVDF membrane for Western blot analysis.
3.5.1 Analysis Protein bands were visualized by staining the electrophoresed gels
of Expression of Capsid with Coomassie brilliant blue stain (40% methanol v/v, 10% glacial
Protein by Coomassie acetic acid, and 0.5% Coomassie brilliant blue) at room temperature
Staining with constant rocking for 1 h. The stain was removed from the gel
by incubating overnight with destaining solution (40% methanol
v/v, 10% glacial acetic acid) at room temperature with continuous
rocking.
3.5.2 Analysis Proteins were transferred electrophoretically to polyvinylidene

of Expression of Capsid difluoride (PVDF) membranes (Immobilon® Membranes, Merck,
Protein by Western Blot Germany) to detect protein by immunoblot. The PVDF mem-
branes were pre-wetted with methanol and subsequently with
Western blot transfer buffer. Electrophoresis was performed in
semidry format at 90 V for 80 min in transfer buffer with 20%
methanol. The complete transfer of the proteins was confirmed by
the transfer of the prestained ladder into the PVDF membrane.
Membranes were blocked with SuperBlock™ Blocking Buffer
(Fisher Scientific) and incubated with primary antibody rabbit
anti-VP0 (available in the host laboratory) at 1:400 dilution fol-
lowed by washing three times and incubation with polyclonal goat
HRP-conjugated anti-rabbit antibody (Agilent Technology, USA).
The protein bands were developed by addition of ECL Western
blot Detection Reagent (GE Healthcare Life Sciences) for 2 min
incubation and imaged by a Syngene G-Box.
3.5.3 Analysis of Capsids The assembly of FMDV capsid proteins into virus-like particles
Protein Assembly by (VLP) were analyzed by transmission electron microscopy
Transmission Electron (TEM). The purified capsid protein containing fraction was con-
Microscopy (TEM) centrated by spin dialysis prior to absorption to the grids. Firstly,
100 μl of the peak western blot positive gradient factions were
diluted threefold in TBS to reduce the sucrose concentration and
re-concentrated tenfold using a microspin column (500 μl Vivaspin
column, MWCO 100,000, GE Healthcare) dialysis by tabletop
centrifugation at 4 C. The concentrated final capsid sample was
absorbed to the grid (carbon-coated formvar 200 square mesh
copper grids, Agar Scientific) by floating it, glossy side down, on a
10 μl droplet of the concentrated sample for 10 min at room
temperature. Followed two washes with distilled water each of
2 min duration, the grids were stained by two 30 s incubations on
a droplet of 2% uranyl acetate. Excess stain was removed by blotting
and the grid examined on a Joel transmission electron microscope
(JEOL USA, Inc.) operating at 200 kV. The empty FMDV capsid
particles expressed in insect cells were visualized with an approxi-
mate diameter of 30–35 nm (Fig. 2).
3.5.4 Detection The antigenicity of the visualized capsid particles was analyzed by
of Antigenicity by immunogold staining. For labeling the particle grids were prepared
Immunogold Staining as described up to the wash stage. The grids were then blocked by
floating on drops of SuperBlock™ TBS blocking buffer for 10 min
and then incubated with a 10 μl drop of 1:10 diluted VP0 poly-
clonal rabbit antibody for 10 min. Following washing the grid was
then incubated with 10 μl of diluted anti-rabbit Ig (1:100) conju-
gated with 10 nm gold. Finally, the grids were washed and stained
as before. The nanogold particle immunologically attached with
the empty capsid particle as seen in Fig. 3.
Fig. 2 The empty capsid sample of FMDV O Bangkok expressed in insect cell
(A038), prepared from 15–45% peak sucrose gradient visualized under
transmission electron microscopy by negative staining with 2% uranyl acetate
Fig. 3 The antigenicity of the assembled capsid particle analyzed by immunogold

staining with the anti-VP0 polyclonal antisera raised in rabbit, followed by
incubation with anti-rabbit IgG conjugated with 10 nm gold. The specific
attachment of the gold particle to the capsid structure is indicated
3.6 Detection of Yield ELISA assay components were provided by the FMD World Refer-
of Empty Capsid by ence Laboratory at The Pirbright Laboratory, UK. Briefly, polysty-
ELISA rene plates were coated in triplicate with 50 μl of capture antibody
O1BFS polyclonal rabbit sera at 1:100 dilution (in sodium bicar-
bonate buffer, pH 9.6) and incubated overnight at 4 C. The plate
was washed three times with PBST (1 PBS with 0.1% Tween-20)
and blocked with SuperBlock™ PBS blocking buffer (Thermo
Scientific) for 1 h at 37 C. After blocking 50 μl of purified antigen

was diluted 1:2 in the original well and then further diluted in a
twofold serial dilution and incubated for 1 h at 37 C. After
washing, the plates were probed with 50 μl per well of O1 Manisa
polyclonal guinea pig sera diluted (1:100) in PBS and incubated for
1 h at 37 C. After washing three times, 50 μl of 1:1000 diluted
HRP-conjugated anti-guinea pig antibody (Abcam, UK) was added
per well and incubated 1 h at 37 C. The plates were finally washed
three times with PBST and 50 μl of TMB substrate (Thermo
Scientific) was added to each well and incubated for 20 min in the
dark. Then 50 μl 0.03 M sulfuric acid was added per well as stop
solution and the absorbance was measured at 420 nm in a 96-well
plate reader (Tecan). The data was analyzed by GraphPad Prism8
software.
3.7 Use of Empty The empty capsid can be also used to detect serum antibody by
Capsid to Detect ELISA. The seroconversion of different serum sample was detected
Serum Antibody by by ELISA by collecting sera samples, pre-bleed and final bleed from
ELISA immunized animals and assay can be performed by ELISA as
described in column 6 except that the probing layer was formed
of the test sera, not the control serum provided by the kit and the
detecting layer was an anti-mouse HRP conjugate.
4 Notes
1. To prepare virus stocks in Sf9 cells, the cells need to be seeded

at low confluency, no more than 50%, checked every day fol-
lowing virus addtion for cytopathic effect (CPE). Virus stocks
can be harvested when more than 50% cells showed CPE, by
gently tapping in the flask to dislodge the monolayer and then
clarification by centrifugation as described.
2. Purification of protein was performed by infecting Tnao38 cell
line with recombinant baculoviruses in suspension culture by
incubation at 27 C in shaker incubator. The progress of the
infection be checked every day by routine microscopy and
trypan blue viability. Generally, cells will look intact on the
first day post infection but will become slightly granular after
48 or 72 h. Cell counting is the best measure to check the
density of the cells and infection is normally done as a density of
approximately ~1.0 106 ml. Cell counts may rise for 1 more
day but will then plateau and decline such that a viability of
<50% is apparent after 72–96 h. This is the best time to harvest
the cells for VLP purification as cell debris is not excessive and
the clarified culture supernatent is clear. Virus titration in Sf9
cells with each recombinant baculovirus is needed to ensure
adequate titres for expression experiments, as is preliminary
epression analysis to verify recombinant protein expression

with an expected pattern. It is not practical to check expression
during a large scale infection study.
3. Despite the generally robust natue of virus capsids purification
of empty capsids (VLPs) from insect cell is best performed as
quickly as possible, with all necessary manisplation steps per-
formed at ambient temperature and all storage steps kept below
10 C to minimise protein degradation. Highly purified VLPs
are less prone to degradation and working stocks can be stored
at 4 C with longterm storage at 20 C in 5% glycerol.
4. The purification of empty capsids from the baculovirus vector is
very useful to remove the background in TEM. A method that
uses sucrose gradient fractionation naturally achieves this as the
middle fractions (sucrose density 30–35%) contain empty cap-
sid, whereas the lower fractions (sucrose density 40–45%) con-
tains mostly baculovirus. Simple concetration of the VLPs, for
example by size exclusion chromatography may not achieve the
same removal of baculovirus background.
Acknowledgments
This review has been promoted and supported by Felix Trust and
The Department of Biotechnology, Government of India.
References
1. Smith GE, Summers MD, Fraser MJ (1983) 7. Van Oers MM, Pijlman GP, Vlak JM (2015)
Production of human beta interferon in insect Thirty years of baculovirus-insect cell protein
cells infected with a baculovirus expression vec- expression: from dark horse to mainstream
tor. Mol Cell Biol 3:2156–2165 technology. J Gen Virol 96(Pt 1):6–23
2. Chambers AC, Aksular M, Graves LP et al 8. Rohrmann G (2019) The baculovirus replica-
(2018) Overview of the baculovirus expression tion cycle: effects on cells and insects. Baculo-
system. Curr Protoc Protein Sci 91:541–546 virus Molecular Biology [Internet]. 4th
3. Liu F, Wu X, Li L et al (2013) Use of baculo- edition. National Center for Biotechnology
virus expression system for generation of virus- Information (US); 2019
like particles: successes and challenges. Protein 9. Li Y, Shen S, Hu L et al (2018) The functional
Expr Purif 90:104–116 oligomeric state of tegument protein GP41 is
4. Qu Z, Li M, Guo Y et al (2020) Expression, essential for baculovirus budded virion and
purification, and characterisation of recombi- occlusion-derived virion assembly. J Virol
nant ferritin in insect cells using the baculovirus 92(12):e02083-17
expression system. Biotechnol Lett 42:57–65 10. Mohana Subramanian B, Madhanmohan M,
5. Gwak WS, Lee SN, Choi JB et al (2019) Bacu- Sriraman R et al (2012) Development of foot-
lovirus entire ORF1629 is not essential for viral and-mouth disease virus (FMDV) serotype O
replication. PLoS One 14(8):e0221594 virus-like-particles (VLPs) vaccine and evalua-
6. López-Vidal J, Gómez-Sebastián S, Sánchez- tion of its potency. Antivir Res 96:288–295
Ramos I et al (2013) Characterization of a 11. Porta C, Xu X, Loureiro S et al (2013) Efficient
Trichoplusia ni hexamerin-derived promoter production of foot-and-mouth disease virus
in the AcMNPV baculovirus vector. J Biotech- empty capsids in insect cells following down
nol 165:201–208 regulation of 3C protease activity. J Virol
Methods 187:406–412
12. Le DT, Müller KM (2021) In vitro assembly of 22. Wang A, Gu L, Wu S et al (2018) Duck hepati-
virus-like particles and their applications tis A virus structural proteins expressed in
13. Noad R, Roy P (2003) Virus-like particles as insect cells self-assemble into virus-like particles
immunogens. Trends Microbiol 11:438–444 with strong immunogenicity in ducklings. Vet
14. Syomin BV, Ilyin YV (2019) Virus-like parti- Microbiol 215:23–28
cles as an instrument of vaccine production. 23. Garcı́a Durán M, Costa S, Sarraseca J et al
Mol Biol 53:323–333 (2016) Generation of porcine reproductive
15. Qian C, Liu X, Xu Q et al (2020) Recent and respiratory syndrome (PRRS) virus-like-
progress on the versatility of virus-like particles. particles (VLPs) with different protein compo-
Vaccine 8(1):139 sition. J Virol Methods 236:77–86
16. Gong M, Zhu H, Zhou J et al (2014) Cryo- 24. Fabre LL, Arrı́as PN, Masson T et al (2019)
electron microscopy study of insect cell- Baculovirus-derived vectors for immunization
expressed enterovirus 71 and coxsackievirus and therapeutic applications. In: Emerging and
A16 virus-like particles provides a structural reemerging viral pathogens: volume 2: applied
basis for vaccine development. J Virol 88(11): virology approaches related to human, animal
6444–6452 and environmental pathogens. Academic, pp
197–224
17. Xiao Y, Chen HY, Wang Y et al (2016) Large-
scale production of foot-and-mouth disease 25. Vaughn JL, Goodwin RH, Tompkins GJ,
virus (serotype Asia1) VLP vaccine in Escher- McCawley P (1977) The establishment of two
ichia coli and protection potency evaluation in cell lines from the insect spodoptera frugi-
cattle. BMC Biotechnol 16:56 perda. In Vitro 13:213–217
18. Adeyemi OO, Nicol C, Stonehouse NJ et al 26. Hashimoto Y, Zhang S, Blissard GW (2010)
(2017) Increasing type 1 poliovirus capsid sta- Ao38, a new cell line from eggs of the black
bility by thermal selection. J Virol 91: witch moth, Ascalapha odorata (Lepidoptera:
e01586-16 Noctuidae), is permissive for AcMNPV infec-
tion and produces high levels of recombinant
19. Zhang W, Dai W, Zhang C et al (2018) A virus- proteins. BMC Biotechnol 10:50
like particle-based tetravalent vaccine for hand,
foot, and mouth disease elicits broad and bal- 27. Hashimoto Y, Zhang S, Zhang S et al (2012)
anced protective immunity article. Emerg Correction: BTI-Tnao38, a new cell line
Microbes Infect 7:1–12 derived from Trichoplusia ni, is permissive for
AcMNPV infection and produces high levels of
20. Wang X, Ku Z, Zhang X et al (2017) Structure, recombinant proteins. BMC Biotechnol 12:12
immunogenicity, and protective mechanism of
an engineered enterovirus 71-like particle vac- 28. Reed LJ, Muench H (1938) A simple method
cine mimicking 80S empty capsid. J Virol 92: of estimating fifty per cent endpoints. Am J
e01330-17 Epidemiol 27:493–497
21. Li Y, Yi X, Zhuang Y et al (2016) Regulation of 29. Hopkins RF, Esposito D (2009) A rapid
porcine circovirus type 2-like particles method for titrating baculovirus stocks using
expressed in baculovirus expression system. the Sf-9 Easy Titer cell line. BioTechniques 47:
Bioresour Bioprocess 3:37 785–788
Chapter 26
Good Laboratory Practices and Biosafety Containments

in a Virology Laboratory
Yashpal Singh Malik, Anuradha Sharma, Niraj Kumar Singh,
B. T. Naveen Kumar, and Naveen Kumar
Abstract
Infectious disease outbreaks keep challenging human and veterinary health worldwide since decades.
Disease outbreaks such as smallpox, influenza, polio, SARS, Ebola, foot-and-mouth disease, African
swine fever, and the most recent and devastating COVID-19, all point to the need for a more proactive
approach to developing diagnostics and treatment methods for these deadly diseases. Because the patho-
genic agents that cause these diseases are highly transmissible, careful containment of these agents within
the laboratories is necessary, with little or no exposure to working personnel. Different regulatory autho-
rities across the world provide guidelines and procedures to ensure that research and diagnostic laboratories
operate safely. This chapter delves into the many events that occur as a result of lab-mediated disease spread,
as well as the need for, importance of, and guidelines for good lab practices and biosafety.
Key words Biosafety, Lab-acquired infections, Containment, Good laboratory practices, Virology,
Risk groups, Biosafety levels
1 Introduction
Clinical, biomedical, and research laboratories employ a vast num-

ber of laboratory personnel worldwide, and biosafety is a key con-
cern in these environments. Laboratory personnel are exposed to a
wide range of pathogenic microorganisms and are always at risk of
contracting infection if basic biosafety measures such as personal
protective equipment, engineering setup, standard operating pro-
cedures, and administrative controls fail or are not in place [1].
Viruses, bacteria, fungi, and parasites are the most common causa-
tive agents of lab-acquired infections (LAIs), which are not only
dangerous to the laboratory personnel but also pose a substantial
risk of community outbreaks if they go unnoticed. Such disease
outbreaks also result in social and economic losses all across the
world.
363
364 Yashpal Singh Malik et al.
The infectious diseases have a complex epidemiology, and only

a few diseases such as Corona virus disease 19 (COVID 19) and
severe acute respiratory syndrome (SARS) share clinical symptoms
[1]. Because identifying and distinguishing causative agents only
based on clinical signs is difficult, laboratory methods/technologies
have become a key aspect of disease diagnosis and subsequent
clinical treatment. Good Laboratory Practices (GLPs) aid in the
maintenance of international quality standards in laboratory opera-
tions by allowing control over laboratory protocols, resulting in
precision, accuracy, and reproducibility of results. Furthermore,
GLPs help to ensure the safety of laboratory personnel involved
in infectious disease diagnosis and research [2]. Non-clinical
research with environmental safety and quality drug development
for infectious diseases around the world is also boosted by GLPs.
2 History of Lab-Escaped Pathogens
Individuals working in laboratories have long been aware that they

can become infected by the pathogenic agent they are working
with, either directly or indirectly, making their profession an occu-
pational hazard. Dr. HT Ricketts, for example, died in 1910 after
contracting Typhus infection while studying the disease with a less
specific containment setup. In 2001, a researcher at the United
States contracted anthrax after an antimicrobial disinfectant was
switched incorrectly in a public health laboratory [3]. Some reports
and surveys of LAIs around the world have been in the past [4–7]
and the largest survey of LAIs was conducted by Pike [5], who
reported 159 pathogenic agents caused 4079 LAIs, with ten major
infectious agents accounting for more than half of the cases.
The following are the major incidents of viral escapes from the
lab in the past.
2.1 Smallpox Virus The World Health Organization’s (WHO) campaign to eradicate
in 1966–1978 smallpox identified the virology labs as a potential source of infec-
tion and epidemic. Between 1963 and 1978, only 4 cases of small-
pox infection were documented in smallpox endemic areas in the
United Kingdom, yet 80 cases and three deaths were reported from
three different lab escapes of smallpox virus during the same period.
This notion was backed by the fact that smallpox lab escapes
reported in three different years, 1966, 1972, and 1978, all
were from the areas where smallpox virus was being handled for
research purpose at that time. These infections were extremely
similar, and the individuals who first became infected in 1966 and
1978 worked at the same location where Variola minor research was
being conducted. Following the identification of these lab escapes,
different investigations were conducted, and stringent guidelines
for handling pathogens, personnel training, and sufficient rooms/
Good Laboratory Practices and Biosafety Containments in a Virology Laboratory 365
cabinets ventilation were established. These guidelines served as the

foundation for biosafety protocols at all levels [8, 9].
2.2 Human Influenza The H1N1 influenza virus was found circulating in humans in
H1N1 in 1977 1977. It started in China and the Soviet Union and spread
throughout the world. This pandemic was also known as Russian
flu, and it primarily affected people under the age of 21 years. For
more than three decades, scientists debated whether the pandemic
was caused by lab release or by some other factor. The 1977 H1N1
influenza virus strain was found closely related to the H1N1 virus
strain circulating in 1949–1950, based on extensive genomic and
serological tests. The isolation of H1N1 RNA from Siberian Lake
meltwater, which is frequently visited by migratory birds and could
have frozen since long times, suggested a reason other than lab
escape in a 2006 report [10]. However, in 2008, natural stasis was
ruled out, and it was found that the H1N1 isolation from meltwa-
ter was caused by contamination in the lab, where the H1N1
(1977) strain was being used as a positive control [11]. The advent
of flu was thus clearly proposed to be an escape from a viral sample
lot frozen in a virology lab since 1950, and the reason for the virus’s
thawing out was the consequence of the US vaccine campaign
against H1N1 influenza in 1976 [4].
2.3 Venezuelan VEE is a hemorrhagic fever-causing viral epizootic disease that first
Equine Encephalitis appeared in South Armenia in 1969–1971. Veterinarians utilized
(VEE) in 1995 the inactivated whole VEE virus as a vaccine against this lethal
disease at that time, and inactivating VEE is notoriously difficult.
The viral strain found in 1970 outbreak was similar to the one used
for immunization in the 1930s, implying the incomplete inactiva-
tion of VEE virus and that the virus was able to escape [4, 12].
2.4 SARS (Severe The SARS outbreak in 2002–2003 resulted 8000 infections and
Acute Respiratory 774 deaths in 29 countries; however, the epidemic was contained in
Syndrome) Virus 2003 due to implementation of strict containment and quarantine
in 2002–2003 procedures [13, 14]. In terms of transmission, SARS is a super
spreader, and even a single LAI can cause the disease to spread
[15]. After 2003, no natural outbreaks of SARS were recorded;
nonetheless, six lab-escaped outbreaks (Singapore 1, Taiwan 1,
China 4) were reported from the labs handling SARS virus for
vaccine research [16, 17]. As a result, labs conducting SARS-CoV
research and retaining specimens of SARS patients pose the greatest
risk of virus exposure and transmission.
In addition to these incidents, lab escapes of the Ebola virus,
and foot-and-mouth disease virus were also recorded in the early
2000s [4]. All of these events of viral lab escapes emphasize the
significance of following stringent GLPs and biosafety guidelines.
3 Good Laboratory Practices
Good Laboratory Practices are the set of rules established to ensure

the integrity and quality of laboratory studies conducted in support
of government-sponsored research or marketing policies of pro-
ducts. GLPs were initially adopted in 1972 in Denmark and
New Zealand and then, in 1979 in the USA [19, 20]. Later, the
Organization of Economic Cooperation and Development
(OECD) released GLP guidelines to help with various toxicological
studies and the production of high-quality data for environmental
and human risk assessment. Institute, personnel, experiment facil-
ity, quality assurance system, experiment system, experiment item,
standard operating procedures (SOPs), experiment recording, and
reporting of findings are all covered under the OECD GLP guide-
lines [19]. According to the OECD, GLP is “a quality system
concerned with the organizational process and the conditions
under which non-clinical health and environmental safety studies
are planned, performed, monitored, recorded, archived and
reported.” Because non-validated data generated by various labora-
tories is not acceptable in various countries throughout the world,
the OECD member countries took the initiative to create the
national monitoring authorities to ensure that GLP is followed in
their respective countries. GLP-compliant labs follow a set of stan-
dard protocols while conducting the experiment/studies/tests,
and analyses. This helps in the planning, execution, monitoring,
reporting, documenting, and validation of findings in order to
deliver the data/products that are compliant with international
standards. Because GLPs allow interchange of information, these
practices result in the removal of technical and international trade
barriers as well as a reduction in input/operational expenses.
Henceforth, GLPs make it easier for international cooperation to
work toward a common goal of protecting human health and the
environment [19]. The International Council for Harmonization
of Technical Requirements for Pharmaceuticals for Human Use
(ICH), an organization that oversees pharmaceutical quality, effi-
cacy, and safety has proclaimed GLP to be a prerequisite for inter-
national pharmaceutical registration [20].
The GLP regulations include pre-established plan and standard
operating procedures. These set the rules for good practices on the
test site and help the technical or research personnel (s) in conduct-
ing the study according to a pre-designed and approved plan. The
GLPs are based on the following fundamental principles:
1. Resources: organization, personnel, facilities, and equipment.
2. Characterization: test items and test systems.
3. Rules: study plans (or protocols) and written procedures.
4. Results: raw data, final report, and archives.
5. Quality Assurance: determine the quality of product/report

generated from the non-clinical research independently
[21, 22].
The GLP principles were developed by an OECD expert panel
in 1978, and adopted by member countries in 1981. These princi-
ples were further amended in 1997 (Annex to the Council Decision
on the Mutual Acceptance of Data in the Assessment of Chemicals;
amended Annex II of the 1981 Council Decision) [2]. Table 1
summarizes the various GLP principles.
4 Biosafety Requirements While Working with Infectious Agents
Biosafety guidelines are developed to ensure the safety of indivi-

duals working in research and clinical labs by implementing various
measures such as establishing primary and secondary barriers
between personnel and biological hazards, some of which are sup-
ported by recent advances in engineering and material science.
Strict regulations with appropriate protective equipment, practices,
and facilities are required when handling pathogenic agents in order
to prevent contracting infection by laboratory personnel. Accord-
ing to the World Health Organization (WHO), each country
should identify and categorize the infectious agents found inside
its borders into distinct risk groups, and imply the regulations
accordingly. Authorities from various countries, including the
USA, Canada, Europe, and Australia, have published biosafety
guidelines, and infectious agents grouping is mostly the same
(Standards Australia [25]; American Biological Safety Association
[26]; Public Health Agency of Canada [27]; Directive 2000/54/
EC of European parliament [28]).
In India, all biosafety regulatory framework activities are gov-
erned by the Rules for the Manufacture, Use, Import, Export, and
Storage of Hazardous Microorganisms/Genetically Engineered
Organisms or Cells are advised by the Ministry of Environment,
Forest and Climate Change [29], Government of India, under the
Environment (Protection) Act, 1986 [30]. The institutional bio-
safety committee, which was constituted with the Department of
Biotechnology’s approval, plays an important role in the implemen-
tation of biosafety principles, rules, regulations, and guidelines.
This committee provides the biosafety regulatory framework to
the institution/scientists/industry for handling dangerous micro-
organisms, genetically engineered (GE) organisms, and products
derived from GE organisms [31]. Table 2 shows that competent
authorities assigned to implement the biosafety regulations under
Rules 1989. The Genetic Engineering Appraisal Committee
(GEAC), Review Committee on Genetic Manipulation (RCGM),
and Institutional Biosafety Committee (IBSC) have approval and
Table 1
A list of GLPs principles
S. no. Principles Details

1. Test facility, organization, l Test facility should ensure safety while performing experiments.
and personnel l Each experiment should have test and references; responsibility
of each individual should be defined and staff should be trained
properly.
l Study plans and sites should be approved by the director and
project investigator (PI), and SOPs should be followed strictly,

along with cooperation/communication between officials and
working staff.
2. Quality assurance program l The experiment should be conducted as per GLP guidelines.
l Quality assurance personnel should prepare the records of
experiment including all details, and communicate to
management, PI, and the director.
l Quality assurance personnel should prepare the final reports
detailing all the inspections and meetings that have taken place.
3. Facilities l The best location to satisfy the study’s requirements with the
least amount of disturbance should be selected and facility
should be constructed in accordance with GLP guidelines.
l Proper facility for collection, storage, handling, disposal, and
transportation should be arranged while adhering to GLPs.

4. Apparatus, material, and l Proper maintenance, calibration on regular basis, and
reagents corresponding records should be followed.
l SOPs should be maintained and followed for preparing
reagents/chemicals, proper arrangement and records with

detailed expiry date, safety and storage instructions.
5. Test systems l Appropriate equipment with capacity and quality required for
the study should be used and maintained as per industry
standards.
l Before starting the experiment, procured animals/plants should
go through biosecurity, quarantine, and acclimatization

procedures and all records pertaining to their source, strain,
dosage, cleaning, health, and so on should be maintained.
6. Test and reference items l Cross-contamination of test and reference products should be
strictly avoided and tracking facility for the samples should be
available.
l Proper records for all the test and reference products should be
maintained.
7. Standard operating l SOPs should be written in accordance with the study objectives
procedures (SOPs) and approved by the test facility management committee.
l SOPs should be designed in such a way that a technical person
can perform the experiment with little or no guidance.

8. Performance of the study l The study should be performed strictly as per approved SOPs,
and records and experimental details should be maintained.
9. Reporting of study results l PI should maintain the raw data and report the findings to the
study director.
l After statistical analysis of data, a report should be prepared, and
(continued)
Table 1
(continued)
S. no. Principles Details

any changes in report should be approved by the study
director/director and the PI.
10. Storage and retention of l Records should be properly documented, organized in a timely
records and materials manner, stored and made available to competent authorities as
and when needed with restricted access.
Table 2
Various competent authorities assigned for implementation of biosafety regulations
Competent authorities Mandate and functions

Recombinant DNA Advisory RDAC comes under Department of Biotechnology (DBT). Its role
Committee (RDAC) is to review the advances in biotechnology at national and
international levels and recommend appropriate and suitable
safety regulations for India.
Institutional Biosafety Committee IBSC regulates research activities related to hazardous and
(IBSC) genetically engineered microorganisms in institute according to
the guidelines/manuals of RCGM.
Review Committee on Genetic RCGM comes under DBT. It functions to monitor the safety-
Manipulation (RCGM) associated aspects in on-going activities and research projects
encompassing hazardous microorganisms/genetically
engineered organisms. The RCGM has important functions of
preparing manuals of guidelines postulating procedure for
supervisory process concerned to activities linking to GE
organisms in research activity and its industrial applications for
safety of personal, laboratory, and environmental.
Genetic Engineering Appraisal The GEAC comes under the MOEF & CC. It is accountable for
Committee (GEAC) approval of activities related with bulk scale use of recombinants
and hazardous microorganisms in research and industrial
production. It is also concerned in preparation of proposals
related to release of GE organisms and products obtained from
GE organisms.
State Biotechnology Coordination SBCC related with the control measures and safety in the various
Committee (SBCC) installations/institutions in given state involves in handling
hazardous microorganisms/genetically engineered organisms.
District Level Committee (DLC) DLC related with the control measures and safety in the various
installations/institutions in given district involves in handling
hazardous microorganisms/genetically engineered organisms.
regulatory functions, whereas the Recombinant DNA Advisory

Committee (RDAC) has advisory functions. At the district and
state levels, the District Level Committee (DLC) and the State
Biotechnology Coordination Committee (SBCC) are in charge of
monitoring operations involving genetically modified organisms

(GMOs), respectively [31].
5 Risk Groups, Biosafety Levels, and Physical Containment Levels
Pathogenic agents are classified into four risk groups (RG) based on
a variety of factors such as pathogenicity, ease and mode of trans-
mission, host range, and local control and prevention measures.
Pathogenic agents of risk group 1 (RG1) pose a low risk to indivi-
duals and communities, and comprise microorganisms that are
already present in the environment and are unlikely to infect healthy
hosts.
RG2 agents pose a moderate risk, and while they can cause
disease, they are unlikely or difficult to transmit. The infection
caused by RG2 agents can be prevented or treated. RG3 agents
are of high risk for individuals and can cause serious infections to
laboratory workers. These agents, while posing a limited risk to the
community, can pose a major threat if the pathogen escapes into the
environment; nonetheless, effective preventive and treatment mea-
sures are available for RG3 agents. Furthermore, RG4 pathogenic
agents pose a high risk to individuals and communities, and because
they are easily transmissible, they can cause serious infections
among laboratory workers. For these agents, there are usually no
effective preventive or treatment options [32]. Figure 1 shows
some examples of microorganisms belonging to various risk
groups.
Biosafety levels (BSL) and physical containment levels (PCL)
are established for each risk group to ensure worker safety and
prevent the escape of pathogenic microorganisms. RG, BSL, and
PCL are interconnected to each other (Fig. 2).
For each biosafety laboratory, a competent supervisory com-
mittee is assigned, and a range of primary barriers, such as gloves,
protective equipment, biosafety cabinets, and other containment
equipment, are installed between workers and infectious patho-
gens. The Lab Incharge is primarily responsible for the lab’s safe
operation and biosafety guidelines, and his judgment and recom-
mendations are critical for assessing risks and handling a specific
agent. Recommended BSL offers an environment in which an
infectious agent can be handled/modified without the risk of it
escaping. As per the U.S. Centers for Disease Control and Preven-
tion (CDC), BSLs are classified into four categories: BSL-1, BSL-2,
BSL-3, and BSL-4; characteristics, practices, safety equipment (pri-
mary barriers), and facilities (secondary barriers) are summarized in
Table 3, along with the allowed risk group and physical contain-
ment levels [4, 33].
Four distinct types of animal biosafety levels (ABSLs) are nec-
essary for handling of vertebrate animals exposed to agents that
Fig. 1 Examples of biological agents from different risk groups
may infect humans, just like four types of BSL laboratories

(required for microorganism handling). These ABSLs, which
include ABSL-1, ABSL-2, ABSL-3, and ABSL-4, provide practices,
equipment, and facilities that are comparable to the laboratory
BSLs. The ABSL-1 facility is appropriate for working with well-
characterized agents that are unlikely to cause adverse effects in
humans and pose a low risk to the environment and personnel.
ABSL-2 is suitable for working with experimental animals infected
with human disease-causing agents and posing a modest hazard to
the environment and personnel. ABSL-3 facility is suitable for
working with experimental animals infected with aboriginal or
exotic agents that can be transmitted through aerosols. Hazardous
agents can induce serious or potentially deadly disease. Animals
inoculated with a hazardous or exotic organism that can transmit
via aerosol and poses a serious risk of death must be housed in an
ABSL-4 facility. Attendants or animal care personnel essentially
should have extensive and systematic training in handling of haz-
ardous, infectious agents and inoculated animals [34, 35].
6 Working Safely in a Virology Laboratory
Different types of investigations are carried out in a basic virology

research laboratory, including virus isolation, cell culture, DNA and
RNA isolation, transfection, molecular biology and recombinant
Fig. 2 Biosafety levels (BSL), physical containment levels (PCL), and risk groups (RG) of a particular infectious
agent are interrelated
DNA techniques for genetic modifications, and so on. Some of the

low to moderate risk viruses such as Adenovirus, Parvovirus, and
Canine Distemper virus are utilized in research projects and gradu-
ate student dissertations, and require a Biosafety Cabinet-1 or
Biosafety Cabinet-2 to handle them. Despite the fact that these
viruses pose a low to moderate risk to healthy individuals with a low
possibility of transmission, accidental contact with virus and viral
genetic material may occur while performing experiments. Such
mishaps could put laboratory workers’ health at danger, and thus
they must be avoided. A typical BSL-2 design is shown in Fig. 3.
While working in a virology laboratory, routine biosafety
guidelines such as limited access to working areas, immediate and
proper disinfection of work surfaces upon spills, no mouth pipet-
ting, solid and liquid waste decontamination, proper disposal of
waste and sharps, no eatables, smoking, cosmetics in laboratory
areas, no food storage in laboratory refrigerators, washing hands
after performing experiments, removing gloves and leaving labora-
tory, wearing appropriate protective equipments, minimization of
aerosol generation while working with viable material, following
proper guidelines while transferring or transporting the research
Table 3
Summary of biosafety levels for handling infectious biological agents (as per CDC guidelines)
RG
and
Description Lab practices Primary barriers Secondary barriers PCL
BSL- For handling agents or l Standard l No special l Work surface that RG-
1 toxins not known to microbiological equipment can be easily 1
cause disease to practices required disinfected PCL-
healthy individuals l Work can be l Gloves, lab coat, l Open sink to wash 1
conducted on etc. could be used hands
open working as and when
bench needed
BSL- For handling agents l Restricted access l Class I or II l Open sink for RG-
2 which pose a while work is in biosafety cabinets washing hands 2
moderate health risk progress (BSCs) and eyes PCL-
upon ingestion, l Guidelines for l Appropriate PPE l Autoclave to 2
injury, or exposure sharps disposal, is required decontaminate the
to mucous waste l Face shield and eye laboratory waste
membrane decontamination protection gear
and limited required
biohazard signs
BSL- For handling agents l All BSL-2 practices l All BSL-2 primary l Open and hands- RG-
3 which can cause l Decontamination barriers free sink near exit 3
lethal diseases, and of waste before l Respiratory l Self-closing, PCL-
are transmitted via disposal and protection is double door 3
aerosols, could be clothing before necessary access, sealed
prevented or treated laundry windows
l Controlled access l Direction flow of
l Significant training air from

of working non-laboratory
individual areas to laboratory
areas
l No recirculation of
exhaust air
BSL- For handling agents l All BSL-3 practices l All work to be l All BSL-3 RG-
4 which can cause life- l Change of clothing performed in secondary barriers 4
threatening disease before entering BSC III or in l Separated/isolated PCL-
conditions via l Shower on exit BSC I or II while unit with 4
aerosol transmitted l Each and every wearing full body dedicated supply,
lab escapes, no material needs to protective gears exhaust, vacuum
prevention or be with positive air and
treatment therapy decontaminated pressure decontamination
available before exit personnel suit systems
l Immunizations of
lab staff for the

microbes they are
working on
(recommended)
Fig. 3 Biosafety Level-II laboratory plan for a basic virology lab
items from one place to other, regular maintenance of records, and

reporting of spills should be followed. The biological samples
should only be handled by trained personnel. To avoid cross-
contamination or unintentional thawing, which could lead to
lab-acquired infections, storage of biological samples/viruses
should be properly recorded and updated on a regular basis. Proper
biohazard labels should be placed on storage units having potential
pathogens and access should be limited to only those employees
who are aware of and trained to handle these pathogens.
Even if all of these biosafety guidelines are followed, there is still
a danger of mishaps or research interruptions, thus risk manage-
ment strategies should be implemented immediately. Spills, expo-
sures, power loss, fire, and other disruptions should be anticipated
in advance, and an action plan of “what to do when” should be kept
ready. Each floor should have its own fire safety devices and emer-
gency exits. In the event of an accidental contact or inhalation of
virus, first aid should be done immediately, followed by obtaining
medical assistance. Every laboratory member should be informed
of the risks posed by viruses being handled in the lab, as well as the
minimal protection/response measures.
There is a long history of lab-acquired viral infections, some of
which have been discussed previously. Sometimes infection
contracted from lab by an individual may not result in secondary

infections, but if not reported promptly, it can cause anxiety among
the entire research staff, chaos, wastage of time and money in
tracing the possible contacts. For example, a scientist infected
with the Sabia virus did not report the initial viral exposure and
was only diagnosed after 5 days of illness, delaying the implemen-
tation of effective control measures. A total of 142 people were
identified as potential contacts, with the bulk of them being labo-
ratory workers. Although no secondary infections were identified,
the fear and anxiety of contacts, the 6-days surveillance program,
and cost of investigation by various agencies strongly suggest
aggressive and compulsory reporting of mishaps/exposures in lab,
immediate use of available medical facilities (identified in advance),
and identification of surveillance or quarantine methods for such
mishaps [36].
All the information on experiments, spills, and other minor to
major incidents should be communicated to the lab supervisor. In
most cases, poor communication is one of the main causes of
laboratory mishaps. The lab incharge and biosafety officer should
ensure that all staff and students receive mandatory training upon
joining the facility, and that inspections are conducted on a regular
basis to ensure that GLPs and biosafety guidelines are being fol-
lowed effectively.
7 Conclusion
Because pathogens are emerging and re-emerging, research prac-

tices for prevention and treatment of these pathogens are in high
demand, raising the risk of lab-acquired infections even with a
minor negligence. Therefore, all laboratories should strictly adhere
to GLPs and biosafety guidelines provided by their respective
country’s governmental authority. With the recent SARS-CoV2
pandemic, even the common person has realized the importance
of proper disinfection and containment practices. One can assure
safe working research practices with minimal risk of accidental
infection spread by following guidelines, being aware of potential
dangers, and utilizing our best judgment in the event of unantici-
pated mishaps.
References
1. Mourya DT, Yadav PD, Majumdar TD, Chau- 2. Mohanty SK, Satapathy A, Naidu MM,
han DS, Katoch VM (2014) Establishment of Mukhopadhyay S, Sharma S, Barton LM et al
biosafety level-3 (BSL-3) laboratory: impor- (2020) Severe acute respiratory syndrome
tant criteria to consider while designing, con- coronavirus-2 (SARS-CoV-2) and coronavirus
structing, commissioning & operating the disease 19 (COVID-19)—anatomic pathology
facility in Indian setting. Indian J Med Res perspective on current knowledge. Diagn
140(2):171–183 Pathol 15(1):1–17
3. OECD (1998) Principles on good laboratory 17. WHO Global Alert and Response (GAR)
practice. OECD. https://www.oecd.org/ (2004) China reports additional SARS cases—
chemicalsafety/testing/good-laborator y- update 23 April 2004. http://www.who.int/
practiceglp.htm csr/don/2004_04_23/en/index.html
4. Burnett LC, Lunn G, Coico R (2009) Bio- 18. WHO Global Alert and Response (GAR)
safety: guidelines for working with pathogenic (2003) Severe acute respiratory syndrome
and infectious microorganisms. Curr Protoc (SARS) in Taiwan, China. http://www.who.
Microbiol 13(1):1A.1 int/csr/don/2003_12_17/en/. Accessed
5. Furmanski M (2014) Laboratory escapes and 17 Dec 2003
“Self-fulfilling prophecy” epidemics. Center 19. Baldeshwiler AM (2003) History of FDA good
for Arms Control and Nonproliferation laboratory practices. The Quality Assurance
6. Pike RM (1976) Laboratory-associated infec- Journal: The Quality Assurance Journal for
tions: summary and analysis of 3921 cases. Pharmaceutical, Health and Environmental
Health Lab Sci 13(2):105–114 Professionals 7(3):157–161
7. Weinstein RA, Singh K (2009) Laboratory- 20. Alatgi AC, Chougule SB (2015) Good labora-
acquired infections. Clin Infect Dis 49(1): tory practice. J Evol Med Dent Sci 4(103):
142–147 16901–16906
8. Wurtz N, Papa A, Hukic M, Di Caro A, 21. Turnheim D (1994) The OECD policy for the
Leparc-Goffart I, Leroy E et al (2016) Survey implementation of the principles of good labo-
of laboratory-acquired infections around the ratory practice. Annali-Istituto Superiore Di
world in biosafety level 3 and 4 laboratories. Sanita 30:395–395
Eur J Clin Microbiol Infect Dis 35(8): 22. Cavero I (2009) Exploratory safety pharmacol-
1247–1258 ogy: a new safety paradigm to de-risk drug
9. Fenner F, Henderson DA, Arita I, Jezek Z, candidates prior to selection for regulatory sci-
Ladnyi ID (1988) Smallpox and its eradication, ence investigations. Expert Opin Drug Saf
vol 6. World Health Organization, Geneva. 8(6):627–647
http://apps.who.int/iris/handle/10665/ 23. Akyar I (2011) GLP: good laboratory practice.
39485 Modern Appr Quality Control:37–56
10. Shooter RA (1980) Report of the investigation 24. Nahler G (2009) Good laboratory practice
into the cause of the 1978 Birmingham small- (GLP). In: Dictionary of pharmaceutical medi-
pox occurrence. HMSO, London. https:// cine. Springer, Vienna, p 82
www.nlm.nih.gov/nichsr/esmallpox/report_ 25. Standards Australia (2010) Safety in lab part 3:
1978_london.pdf. Accessed Nov 2021 microbiological safety and containment. Stan-
11. Zhang G, Shoham D, Gilichinsky D, dards Australia. AS/NZS, Sydney, pp
Davydov S, Castello JD, Rogers SO (2006) 22433–22010
Evidence of influenza A virus RNA in Siberian 26. American Biological Safety Association. Risk
Lake ice. J Virol 80(24):12229–12235 group database. https://my.absa.org/
12. Worobey M (2008) Phylogenetic evidence Riskgroups. Accessed Aug 2021
against evolutionary stasis and natural abiotic 27. Public Health Agency of Canada. Pathogen
reservoirs of influenza A virus. J Virol 82(7): safety data sheets and risk assessments.
3769–3774 https://www.canada.ca/en/public-health/
13. Weaver SC, Pfeffer M, Marriott K, Kang W, services/laboratory-biosafety-biosecurity/
Kinney RM (1999) Genetic evidence for the pathogen-safety-data-sheets-risk-assessment.
origins of Venezuelan equine encephalitis virus html. Accessed Aug 2021
subtype IAB outbreaks. Am J Tropic Med 28. Directive 2000/54/EC of the European par-
Hygiene 60(3):441–448 liament. https://eur-lex.europa.eu/eli/dir/
14. Abraham T (2007) Twenty-first century 2000/54/oj. Accessed Aug 2021
plague: the story of SARS. JHU Press 29. MoEF & CC (1989) Ministry of Environment
15. Lim PL, Kurup A, Gopalakrishna G, Chan KP, and Forests, Notification, New Delhi, the 5th
Wong CW, Ng LC et al (2004) Laboratory- December, 1989. https://www.indiacode.nic.
acquired severe acute respiratory syndrome. N in/bitstream/123456789/4316/1/ep_act_
Engl J Med 350(17):1740–1745 1986.pdf. Accessed Nov 2021
16. Shen Z, Ning F, Zhou W, He X, Lin C, Chin 30. EPA (1986) The Environment (Protection)
DP et al (2004) Superspreading sars events, Act, 1989 (Act No. 29 of 1986). https://
Beijing, 2003. Emerg Infect Dis 10(2): w w w. i n d i a c o d e . n i c . i n / b i t s t r e a m /
256–260 123456789/4316/1/ep_act_1986.pdf.
Accessed Nov 2021
31. Department of Biotechnology (ed) (2020) 34. World Health Organization (2004) Laboratory
Handbook for institutional biosafety commit- biosafety manual, 3rd edn. WHO. https://
tees (IBSCs), 3rd edn. Department of Biotech- w w w. w h o . i n t / p u b l i c a t i o n s / i / i t e m /
nology. https://ibkp.dbtindia.gov.in/ 9241546506
Content/FlashPDF/IBSC%20Handbook.pdf 35. Sewell DL (1995) Laboratory-associated infec-
32. Kaufer AM, Theis T, Lau KA, Gray JL, Raw- tions and biosafety. Clin Microbiol Rev 8(3):
linson WD (2020) Laboratory biosafety mea- 389–405
sures involving SARS-CoV-2 and the 36. Kortepeter MG, Martin JW, Rusnak JM, Cie-
classification as a Risk Group 3 biological slak TJ, Warfield KL, Anderson EL, Ranadive
agent. Pathology 52(7):790–795 MV (2008) Managing potential laboratory
33. Department of Biotechnology (2017) Regula- exposure to Ebola virus by using a patient bio-
tions and guidelines for recombinant DNA containment care unit. Emerg Infect Dis 14
research and biocontainment. Department of (6):881–887
Biotechnology. https://rcb.res.in/upload/Bio
safety_Guidelines.pdf
INDEX
A D
Absorbent pad ............................................. 198, 202, 203 Diagnosis ................................... 9, 14, 15, 22–26, 29, 39,
Acrylamide gel electrophoresis (AGE)......................... 128 41, 50, 64, 67–88, 92, 93, 127–135, 137, 138,
Alkaline lysis method .......................................95, 98, 103 140, 144, 146, 153, 156, 161, 171, 172, 187,
Annealing temperature ........................73, 74, 80, 83, 84, 195–203, 205–212, 231–237, 239, 240, 248,
143, 233, 248 251–253, 256, 257, 259, 260, 270–273,
Antibodies ....................................... 23, 33, 42, 113, 116, 275–287, 291, 298, 303–304, 306–308, 327,
138, 160, 162, 164–166, 171–185, 187–190, 332, 334–339, 341–348, 351–360, 364,
196–201, 203, 215–218, 220–228, 245, 246, BNF–134
265, 266, 268, 270–273, 287, 297–305, 308, Droplet digital polymerase chain reaction
309, 313, 314, 319–326, 336–347, 352, 353, (ddPCR) .................................. 206–208, 210–212
357–359 Droplet generation...................................... 207, 208, 210
Aptamers.......................................................159–166, 177 Dye-CsC1 gradient method ........................................... 96
AuNP-Conjugation....................................................... 144
E
B
Economics ................................................ 1–3, 15, 16, 18,
BHK-21 cells .......................................................... 33, 309 22, 86, 137, 151, 195, 240, 247, 252, 253, 335,
Biosafety .......................................................219, 363–375 363, 366
Biosafety levels...................................................... 370–372 EID50 or median egg infectious dose ............................ 34
Biosecurity ........................................... 4–7, 9, 15–17, 368 Electrochemical .......................................... 146, 166, 177,
Biosensors .......................................... 140, 143, 144, 146, 181, 182, 184, 188–191
160, 162–166, 172, 173, 176–178, Electron microscopy (EM) ..................................... 25, 29,
181–191, 248 40, 41, 44, 276, 286, 357, 358
Electrophoresis ..................................................58, 72, 94,
C 95, 99, 104, 105, 116, 121–123, 127, 129, 130,
135, 232, 233, 235, 237, 245, 246, 282, 286,
Cell cultures.......................................................32, 35, 44,
51, 62, 67, 139, 172, 196, 216, 218, 219, 309, 313, 315, 317, 324, 326, 327, 356, 357
251–260, 267, 270, 276, 286, 287, 309, End-point titer ........................................ 34–39, 308, 355
Enzyme linked immunosorbent assay (ELISA) .......9, 40,
353–354, 356, 371
Cell lines ...................................... 39, 162, 252–260, 268, 42–44, 164, 179, 196, 197, 215, 246, 265, 268,
341, 353, 355, 359 271, 272, 286, 297–310, 337, 343–347, 356,
358, 359
Chemiluminescence method ........................................ 321
Cloning ...............................................112, 113, 116–120, Epidemics ........................................ 3, 4, 6, 9, 14, 71, 92,
142, 270, 276, 278, 279 151, 156, 211, 231, 232, 240, 247, 258, 330,
342, 345, 347, 364, 365
Clustered regularly interspaced short palindromic repeats
(CRISPR).................................111, 112, 151–156 Epidemiology .................................... 7, 14, 18, 171, 287,
Colony PCR .................................................................. 120 330, 334, 342, 364
Column chromatography .........................................95–97
F
Competent cells............................................................. 119
Conjugates.................................................. 116, 123, 124, Fluid phase immunoassay ............................................. 337
197–199, 201–203, 297, 301, 305, 306, 359 Fluorescent detection ................................................... 248
Containments .................................................15, 363–375 Fluorescent microsphere assay............................. 291–295
Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-2043-4,
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer
Nature 2022
379
PROTOCOLS FOR THE DIAGNOSIS OF PIG VIRAL DISEASES
380 Index
Fluorophores ........................................ 41, 166, 215–218, 318–323, 325, 327, 330, 332–340, 353–360,
221–224, 227, 228, 246, 320, 321 364, 375
Focus-forming assays (FFA) ........................................... 33 Molecular assays .............................................................. 22
Foot and mouth disease (FMD) ....................5, 9, 22, 27, Monoclonal antibody (mAb) ............................. 171, 173,
33, 64, 68, 151–153, 195, 240, 248, 256, 257, 201, 265–273, 341
259, 260, 298, 303–304, 306–308, 346, Multiplex PCR (M-PCR) ............................68–81, 85–88
347, 358
N
G
Nitro-cellulose membrane (NCM) ............ 116, 318, 319
Genetic engineering ............................109, 110, 367, 369 Non-structural proteins ....................................... 304, 305
Genome quantification ................................................... 40 Northern blotting ........................................283–284, 313
Gold nanoparticles (AuNP)....................... 140, 143–146, Nucleic acid hybridization (NAH)..............276, 282–287
163, 165, 182, 188, 197, 199, 202 Nucleic acid sequence-based amplification
Good laboratory practices (GLPs) ..............364–368, 375 (NASBA)......................... 151–156, 232, 240, 338
NUCLISENS ................................................................ 154
H
O
Haemadsorption assay .................................................... 39
HAT selection................................................................ 269 OIE ............................................................... 4, 5, 7, 9, 15,
Hemagglutination (HA) assay........................................ 40 162, 163, 253, 310
His-tag antibody ........................................................... 116 One-health............................................................ 331, 332
Hybridomas ................................ 171–173, 266–272, 341 Optical density ............................................ 125, 308, 309
Hypoxanthine-guanine phosphoribosyl transferase
(HGPRT).................................................. 269, 270 P
Peptide nucleic acid (PNA) ....... 137–146, 160, 164–165
I
Permeabilizers ............................................. 220, 221, 226
ID50 or median infectious dose...................................... 33 Piezoelectric ........................................177, 184, 186–188
Imidazole ..................................................... 114, 116, 122 Plaque assay .......................................32, 33, 44, 354–356
Immobilization........................................... 178–181, 184, Plasmid isolation .....................................................95–105
190, 191, 203, 246, 300 Plasmids ................................................ 91–106, 111, 113,
Immunofluorescence .........................215–228, 256, 258, 115, 118–121, 125, 232, 268, 278, 354
265, 271, 276, 291, BNF–228 PNA-clamping PCR............................................. 142–143
Infectivity assays ...........................................31–35, 37, 39 Point-of-care (POC) ............................................. 93, 137,
138, 144, 166, 173, 241, 248
L Poisson distribution ............................................... 33, 205
Polyacrylamide gel electrophoresis (PAGEs) .... 116, 123,
Lab acquired infections (LAIs)............................ 363–365
Lateral flow assay (LFA) .................................9, 196–198, 126–130, 132, 134, 315, 316, 323–324, 356
200–203, 245, 272, 273 Polymerase chain reaction (PCR) ....................23, 32, 40,
LD50 or median lethal dose............................................ 33 41, 44, 65, 67–88, 99, 111, 116–120, 138–140,
142, 143, 152, 153, 162, 164, 196, 205–212,
Ligase detection reaction .............................................. 292
Loop mediated isothermal amplification (LAMP).....152, 232, 234, 240, 241, 244–246, 248, 276, 278,
153, 232, 235, 240 279, 292–294, 338, 339, 341–348
Polymerase spiral reaction (PSR) ................231–235, 237
M Polyvinylidene difluoride (PVDF) membrane............. 314
Primer dimers ................................... 72, 79, 80, 241, 246
Methods............................................. 4, 9, 13, 14, 18, 23, Probe labeling ...................................................... 279–280
31–33, 36–45, 50–53, 57, 58, 62, 63, 67, 80, 88, Protein purification .............................................. 116, 122
92, 93, 95–105, 112, 113, 115–126, 128–135, Pseudopeptide ............................................................... 160
138–146, 151–156, 160–166, 175, 177,
180–182, 189–191, 196, 199–202, 206, Q
210–212, 218–225, 228, 232, 234–235, 240,
241, 244, 246, 248, 252, 260, 267, 268, Quantal assay or end-point dilution assay ..................... 33
Quantitative real-time PCR (qPCR)............................. 40,
270–272, 276, 277, 279, 280, 285–287,
291–295, 298, 299, 303, 306–310, 314–316, 41, 43, 44, 138, 206–208, 210–212, 240
PROTOCOLS FOR THE DIAGNOSIS OF PIG VIRAL DISEASES
Index 381
R Spot hybridization................................................ 284–285
Sub-culture .................................................................... 252
Recombinant DNA technology ...........95, 110, 174, 180 Surface plasmon resonance (SPR)...................... 144, 146,
Recombinase polymerase amplification (RPA)...........232, 162, 177, 185, 186, 188
240–248, BNF–248 Swine in the Laboratory ................................................. 22
Risk groups ...................................................367, 370–371 Systematic evolution of ligands by exponential enrichment
RNA-PAGE .........................................128–131, 133, 135 (SELEX).................................................... 160, 162
S T
Sample pad ..........................................197–199, 202, 203 TCID50 or median tissue culture infectious dose ......... 34
Sensitivities .............................................7, 40, 67, 73, 78, Thymidine kinase (TK)........................................ 269, 270
80, 81, 85, 88, 126, 134, 152, 153, 156, 162,
163, 166, 178, 180–183, 190, 191, 197, 198, V
200, 203, 211, 216, 241, 244, 245, 247, 248,
257, 265, 269, 270, 285–287, 292, 297, 298, Viral diagnosis ................................ 23, 49, 216, 276, 291
300, 310, 320, 325, 334, 338, 339 Virology ........................22, 44, 162, 216, 314, 323–324,
Silver nitrate ......................................................... 128, 135 363–375
Silver staining ......................................128, 134, 135, 323 Virus detection ....................................156, 161, 196, 216
Single cell dilution method .......................................... 270 Virus isolation .............................................. 25, 161, 196,
Sodium dodecyl sulphate-polyacrylamide gel 251–260, 276, 287, 291, 298, 334, 342–344, 371
electrophoresis (SDS-PAGE)......... 271, 309, 313, Virus quantification............................... 31–33, 38, 40–44
315–322, 324
W
Southern blotting.........................................282–284, 313
Specificities .............................................7, 67, 73, 74, 78, Western blot ...................................... 116, 123, 124, 126,
80, 81, 88, 145, 154, 156, 159–164, 166, 171, 271, 313, 320, 322–325, 327, 356, 357, 360
172, 177, 180, 181, 184, 203, 210, 216, 223, Western blotting...........................................215, 313–327
227, 228, 234, 235, 237, 244, 247, 265, 266,
268, 270, 271, 281, 285, 297, 299, 301, 302,
320, 325, 334

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Rajib Deb · Ajay Kumar Yadav

Swaraj Rajkhowa · Yashpal Singh Malik

For further volumes:

Rajib Deb, Ajay Kumar Yadav and Swaraj Rajkhowa

Yashpal Singh Malik

Swaraj Rajkhowa Yashpal Singh Malik

ISSN 1949-2448 ISSN 1949-2456 (electronic)

Guwahati, Assam, India Rajib Deb

1 Requirements and Preparedness for Attending a Viral Disease

11 Aptamers as Diagnostic Markers for Viral Infections

25 Production of Virus-like Particles Using the Baculovirus Expression

SUSHILA MAAN • Department of Animal Biotechnology, College of Veterinary Sciences, Lala

Requirements and Preparedness for Attending a Viral

Key words Epidemic, Biosecurity, Epidemiology, Economics, Health

emerged as industry for its huge commercial potential. Globally,

S. No. Name of the disease Causative agent

Some of these viral diseases can be effectively controlled while

S. No. Name of the disease Country

1. Level of biosecurity practices: Biosecurity is the implementation

(back-yard) to medium farms with poor biosecurity practices

5. Consumer’s behaviors: Many consumers still prefer buying pork

2 Infectious Disease Outbreak and Its Spread

As discussed, the globalization, intensive swine production,

3 Epidemiological “Know-How”: An Important Pillar for Development

To substantiate the risk of any exotic disease(s) or status of endemic

Fig. 1 An overview of the steps involved in designing of a surveillance system

diagnostic tests for unbiased interpretation of the surveillance data

4 Investigation of Viral Disease Outbreak in Swine

It depends upon the clear-cut objective, availability of facilities,

peoples such as veterinary workers at village, pig farmers, local/

biological and allied sample collection accessories, pig restraint

(b) Appropriate recommendations: The suitable recommenda-

9. Once a major epidemic is ongoing, the following observations

5 Outbreak Investigation Steps for Infectious Diseases

An outbreak investigation is a systematic procedure which is used to

5.1 Requirements 1. Laboratory Diagnostic capabilities: The rapid and accurate

5.2 Descriptive Who?

Zoonoses in human population, associated risk factors, such as

Example: In a farm free from any infectious disease, a single

6 Implementation of Viral Disease Control Program at Various Levels

Level Control measures

On herd level l Implementation of strict biosecurity measures

Level Control measures

level due to its notifiable status

Occurrence of diseases leads to a huge economic loss in terms of

4. Vehicles used during farm visit should be properly disinfected

The unprecedented increase in the frequency of infectious disease

pathogenic organisms, thus ensures better farm health manage-

Collection of Samples, Their Preservation

Porcine species is closely related to humans in the mean of an

Pig is an important livestock species, which plays a crucial role in the

laboratory such as real time polymerase chain reaction (PCR),

3 Suitable Criteria for Collection and Transport and Preservation of Samples

The following criteria are suitable for proper selection of specimen

contamination during necropsy, it is best to collect a routine set of

bacteriologic and parasitologic examinations. Samples should be

to the laboratory within a reasonable time, serum should be

4 Collection and Transport and Preservation of Samples

Sample selected for laboratory analyses depend, of course, on the

Therefore, Log10 TCID50 ¼ ð1Þ ð1Þ½4 0:5 ¼ 4:50

Log10 50%end‐point dilution ¼ ½4 þ 0:5 1

17. Centrifuge at 4 C for 30 min at 10,000 rpm, wash with