2022 Book ProtocolsForTheDiagnosisOfPigV
2022 Book ProtocolsForTheDiagnosisOfPigV
2022 Book ProtocolsForTheDiagnosisOfPigV
Protocols for
the Diagnosis of
Pig Viral Diseases
SPRINGER PROTOCOLS HANDBOOKS
Edited by
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
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.
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
vii
viii Contents
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
Sciences University (GADVASU), Ludhiana, Punjab, India
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
Sciences University (GADVASU), Ludhiana, Punjab, India
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
Sciences University (GADVASU), Ludhiana, Punjab, India
B. V. SUNIL KUMAR • College of Animal Biotechnology, Guru Angad Dev Veterinary and
Animal Sciences University (GADVASU), Ludhiana, Punjab, India
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
Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, Haryana,
India
SINÉAD LYONS • School of Biological Sciences, University of Reading, Reading, UK
Contributors xv
NIRAJ KUMAR SINGH • College of Animal Biotechnology, Guru Angad Dev Veterinary and
Animal Sciences University (GADVASU), Ludhiana, Punjab, India
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
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.
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.
Table 1
Important viral diseases of swine [6, 7]
Table 2
Notable outbreaks of swine viral disease in Asian and other countries [7]
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
Requirements and Preparedness for Attending a Viral Disease Outbreak. . . 9
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
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.
14 Dipak Deka et al.
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?
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.
7 Prevention and Control Measures for the Viral Disease Outbreaks in Swine
Population
8 Conclusion
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
Requirements and Preparedness for Attending a Viral Disease Outbreak. . . 19
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
20 Dipak Deka et al.
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
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.
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_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
21
22 Ashok Kumar et al.
2 Introduction
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
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)
Collection of Samples, Their Preservation and Transportation 27
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
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
5 Conclusion
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
30 Ashok Kumar et al.
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
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
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_3,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
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
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
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:
Methods for Quantification of Viruses 35
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 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
3.1 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
Table 1
Tissue culture infectivity data for determination of 50% end-point by Reed–Muench method
Table 2
Tissue culture infectivity data for determination of 50% end-point by Spearman–Karber method
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).
38 Mukesh Bhatt et al.
Table 3
Tissue culture infectivity data for determination of 50 % end-point by Ramakrishnan method (based
on Fig. 4)
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:
RBCs
Viral proteins
Virus
Indicator cells/PBMCs
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].
Table 4
Common enzymes and substrate combinations used in ELISA
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
Methods for Quantification of Viruses 43
7 Conclusion
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
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
Methods for Quantification of Viruses 47
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
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.
1 Introduction
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_4,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
49
50 Nihar Nalini Mohanty et al.
3 Lysis Buffer
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.
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.
58 Nihar Nalini Mohanty et al.
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.
Protocols for Isolation of Genetic Materials from RNA Viruses 59
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
microcentrifuge tube.
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.
60 Nihar Nalini Mohanty et al.
6 Affinity Matrices
10. The pellet was washed with 75% ethanol and re-suspended in
20 μL TE by heating at 60 C for a few minutes.
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.
Protocols for Isolation of Genetic Materials from RNA Viruses 65
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.
66 Nihar Nalini Mohanty et al.
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
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_5,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
67
68 Manjisha Choudhury et al.
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
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.
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
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
Table 5
Three examples of polymerases used in multiplex PCRs
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
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.
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.
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
Multiplex PCR for Diagnosis of Porcine Diseases 85
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
Multiplex PCR for Diagnosis of Porcine Diseases 87
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
88 Manjisha Choudhury et al.
9 Conclusion
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
Multiplex PCR for Diagnosis of Porcine Diseases 89
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
90 Manjisha Choudhury et al.
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
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.
1 Introduction
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_6,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
91
92 Vinod Kumar Singh et al.
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.
5. Water bath
6. Power supply and accessories for electrophoresis
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
96 Vinod Kumar Singh et al.
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
Protocols for Isolation of Plasmid DNA 97
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.
98 Vinod Kumar Singh et al.
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.
Protocols for Isolation of Plasmid DNA 99
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.
100 Vinod Kumar Singh et al.
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.
Protocols for Isolation of Plasmid DNA 101
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.
3.3.4 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.
104 Vinod Kumar Singh et al.
4 Notes
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
Protocols for Isolation of Plasmid DNA 107
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
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.
1 Introduction
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_7,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
109
110 Rajib Deb et al.
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
Recombinant Antigen-Based Diagnostic Assays of Pig Viral Diseases 113
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
Recombinant Antigen-Based Diagnostic Assays of Pig Viral Diseases 115
2 Materials
3 Methods
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.
Recombinant Antigen-Based Diagnostic Assays of Pig Viral Diseases 117
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
microcentrifuge tube.
118 Rajib Deb et al.
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
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
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.
120 Rajib Deb et al.
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
Recombinant Antigen-Based Diagnostic Assays of Pig Viral Diseases 121
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.
122 Rajib Deb et al.
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.
Recombinant Antigen-Based Diagnostic Assays of Pig Viral Diseases 123
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
124 Rajib Deb et al.
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.
3.6.2 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.
126 Rajib Deb et al.
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
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
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_8,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
127
128 Naveen Kumar et al.
2 RNA-PAGE Principle
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.
130 Naveen Kumar et al.
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
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:
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
RNA-PAGE-Based Diagnosis of Viral Diseases 133
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.
134 Naveen Kumar et al.
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
RNA-PAGE-Based Diagnosis of Viral Diseases 135
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
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.
1 Introduction
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_9,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
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].
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.
3.1 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
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
140 Vinay G. Joshi et al.
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]
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
Peptide Nucleic Acid (PNA): A Diagnostic Molecule for Infectious Diseases 141
3.4 Procedure The standard protocol for the Kaiser test [8].
for the Kaiser Test
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
142 Vinay G. Joshi et al.
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
Peptide Nucleic Acid (PNA): A Diagnostic Molecule for Infectious Diseases 143
Denaturation
PNA Extension
Primer
clamping Annealing
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
144 Vinay G. Joshi et al.
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
Peptide Nucleic Acid (PNA): A Diagnostic Molecule for Infectious Diseases 145
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.
146 Vinay G. Joshi et al.
Fig. 4 Principal of colorimetric assay based on agglomerative nature of AuNPs with PNA in presence and
absence of complementary RNA sequence
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.
148 Vinay G. Joshi et al.
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
Peptide Nucleic Acid (PNA): A Diagnostic Molecule for Infectious Diseases 149
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
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
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_10,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
151
152 Ajay Kumar Singh et al.
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
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,
Nucleic Acid Sequence-Based Amplification (NASBA) Methods and CRISPR/Cas13. . . 157
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
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
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_11,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
159
160 Victoria C. Khangembam and Dimpal Thakuria
2 Selection of Aptamers
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
162 Victoria C. Khangembam and Dimpal Thakuria
5 Peptide Aptamers
7 Advantages of Aptamers
8 Future Prospects
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
168 Victoria C. Khangembam and Dimpal Thakuria
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
Aptamers as Diagnostic Markers for Viral Infections of Veterinary Importance 169
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
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.
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
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_12,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
171
172 Nirmita Dutta et al.
Fig. 2 Hybridoma technology for monoclonal antibody production (HGPRT Hypoxanthine-guanine phosphor-
ibosyltransferase enzyme, HAT medium Hypoxanthine-aminopterin-thymidine medium)
Fig. 3 Recombinant DNA technology for chimeric recombinant antibody (rAb) production
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)
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].
Antibody-Based Sensors for Pathogen Detection 177
4 Antibody Immobilization
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,
Antibody-Based Sensors for Pathogen Detection 179
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.
4.4 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
Antibody-Based Sensors for Pathogen Detection 181
5 Biosensor Types
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
184 Nirmita Dutta et al.
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
186 Nirmita Dutta et al.
6 Biosensor Fabrication
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.
6.4 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.
Fig. 14 Schematic for the graphene film-based immunosensor for rotavirus detection
190 Nirmita Dutta et al.
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
192 Nirmita Dutta et al.
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
Antibody-Based Sensors for Pathogen Detection 193
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
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_13,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
195
196 Aditya Prasad Sahoo and Rajib Deb
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.
198 Aditya Prasad Sahoo and Rajib Deb
3 Materials
4 Methods
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.
Tube No. 1 2 3 4 5
IgG conc. 1 μg 5 μg 10 μg 20 μg 50 μg
Lateral Flow Assay for Diagnosis of Pig Viral Diseases 201
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).
202 Aditya Prasad Sahoo and Rajib Deb
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
References
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–com- ing 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
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
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_14,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
205
206 Yoya Vashi and Sachin Kumar
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])
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
208 Yoya Vashi and Sachin Kumar
Table 1
Reaction setup for DNA amplification in ddPCR
Table 2
Reaction setup for RNA amplification in one step RT-ddPCR
Table 3
PCR cycling conditions
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
5 Conclusion
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
Droplet Digital PCR-Based Diagnosis for Porcine Viral Diseases 213
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
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.
1 Introduction
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_15,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
215
216 Deepika Bisht et al.
Table 1
Commonly used fluorophores: excitation and emission wavelengths
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.
2 Materials
3 Method
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).
220 Deepika Bisht et al.
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.
Table 2
Commonly used permeabilizers, their working concentrations and preferred uses
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
222 Deepika Bisht et al.
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
3.9.3 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)
224 Deepika Bisht et al.
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
Table 3
Fixatives: Major advantages and disadvantages
5 Conclusion
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
Protocols for Immunofluorescence Techniques 229
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
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
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_16,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
231
232 Vikas Gupta et al.
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.
Table 1
Example of primers set for polymerase spiral reaction
Table 2
Initial reaction set up for polymerase spiral reaction
Table 3
Colorimetric dyes used for detection amplification on visual basis
4 Notes
References
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
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.
1 Introduction
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_17,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
239
240 Yoya Vashi and Sachin Kumar
Table 1
Economic and zoonotic viral pathogens of swine. (Reproduced from [1]
2 RPA Mechanism
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]
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])
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),
244 Yoya Vashi and Sachin Kumar
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].
3.4 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
Recombinase Polymerase Amplification-Based Diagnostics of Porcine Viral Diseases 245
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
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
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
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
Recombinase Polymerase Amplification-Based Diagnostics of Porcine Viral Diseases 249
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)
250 Yoya Vashi and Sachin Kumar
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
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
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_18,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
251
252 Vishal Rai et al.
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
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
254 Vishal Rai et al.
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
256 Vishal Rai et al.
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].
Cell Culture System for Porcine Virus Isolation and Propagation 257
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
258 Vishal Rai et al.
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].
Cell Culture System for Porcine Virus Isolation and Propagation 259
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].
4.8 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].
260 Vishal Rai et al.
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
Cell Culture System for Porcine Virus Isolation and Propagation 261
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
262 Vishal Rai et al.
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
Cell Culture System for Porcine Virus Isolation and Propagation 263
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
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.
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_19,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
265
266 Kaushal Kishor Rajak et al.
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
268 Kaushal Kishor Rajak et al.
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).
An Overview of Mouse Monoclonal Antibody Production 269
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
270 Kaushal Kishor Rajak et al.
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
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.
An Overview of Mouse Monoclonal Antibody Production 271
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
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
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
274 Kaushal Kishor Rajak et al.
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
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
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_20,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
275
276 B. V. Sunil Kumar et al.
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
3 Sample Preparation
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
278 B. V. Sunil Kumar et al.
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
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
280 B. V. Sunil Kumar et al.
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].
5.4 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].
282 B. V. Sunil Kumar et al.
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
Nucleic Acid Hybridization Techniques for Viral Disease Diagnosis... 283
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)
284 B. V. Sunil Kumar et al.
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.
6.3.1 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
Nucleic Acid Hybridization Techniques for Viral Disease Diagnosis... 285
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.
9 Conclusion
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
288 B. V. Sunil Kumar et al.
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
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.
1 Introduction
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_21,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
291
292 A. Raja
2 Principles of LDR
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
5 Methods
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
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
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_22,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
297
298 Ramya Kalaivanan and Sankar Palanisamy
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
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
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.
2 Materials
NaCl 80 g
KCl 2g
Na2HPO4 14.4 g
KH2PO4 2.45 g
Triple distilled water to 1000 ml
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
ELISA as a Diagnostic Weapon 305
l Blocking buffer.
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).
ELISA as a Diagnostic Weapon 307
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.
308 Ramya Kalaivanan and Sankar Palanisamy
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
ELISA as a Diagnostic Weapon 309
4 Notes
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
ELISA as a Diagnostic Weapon 311
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
312 Ramya Kalaivanan and Sankar Palanisamy
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.
1 Introduction
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_23,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
313
314 Mukesh Bhatt et al.
2 Sample Preparation
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
4 SDS-PAGE
Table 1
Suggested acrylamide concentration in resolving gel for separation of proteins based on molecular
weight
Table 2
Composition of stacking and resolving gel for SDS-PAGE
5 Native PAGE
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.
318 Mukesh Bhatt et al.
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
SDS-PAGE and Western Blotting: Basic Principles and Protocol 319
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
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].
320 Mukesh Bhatt et al.
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
SDS-PAGE and Western Blotting: Basic Principles and Protocol 321
Fig. 2 Detection of target protein. (a) Chemiluminescence method and (b) Fluorescent 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.
322 Mukesh Bhatt et al.
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.
SDS-PAGE and Western Blotting: Basic Principles and Protocol 323
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.
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)
326 Mukesh Bhatt et al.
Sr.
no. Problem Possible causes Interventions needed
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)
SDS-PAGE and Western Blotting: Basic Principles and Protocol 327
Sr.
no. Problem Possible causes Interventions needed
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
328 Mukesh Bhatt et al.
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
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
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_24,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
329
330 Prabhakar Maurya et al.
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
Table 3
Classification of various immunoassays and their characteristics (Adapted from [27])
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.
4.2 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.
Immune Assays as Diagnostic for Pig Viral Diseases 339
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.
342 Prabhakar Maurya et al.
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.
7.7 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.
7.9 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.
7.11 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.
344 Prabhakar Maurya et al.
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.
7.14 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.
7.23 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.
8 Conclusion
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
Immune Assays as Diagnostic for Pig Viral Diseases 349
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
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
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_25,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
351
352 Hemanta Kumar Maity et al.
2 Materials
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
354 Hemanta Kumar Maity et al.
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
Production of Virus-like Particles Using the Baculovirus Expression System. . . 355
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
356 Hemanta Kumar Maity et al.
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.
Production of Virus-like Particles Using the Baculovirus Expression System. . . 357
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.
358 Hemanta Kumar Maity et al.
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
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
Production of Virus-like Particles Using the Baculovirus Expression System. . . 359
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
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
Production of Virus-like Particles Using the Baculovirus Expression System. . . 361
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
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
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_26,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
363
364 Yashpal Singh Malik et al.
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
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.
366 Yashpal Singh Malik et al.
Table 1
A list of GLPs principles
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
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
(continued)
Good Laboratory Practices and Biosafety Containments in a Virology Laboratory 369
Table 1
(continued)
Table 2
Various competent authorities assigned for implementation of biosafety regulations
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
Good Laboratory Practices and Biosafety Containments in a Virology Laboratory 371
Fig. 2 Biosafety levels (BSL), physical containment levels (PCL), and risk groups (RG) of a particular infectious
agent are interrelated
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
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
7 Conclusion
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
376 Yashpal Singh Malik et al.
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
Good Laboratory Practices and Biosafety Containments in a Virology Laboratory 377
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
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,
© 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