Development and Evaluation of A Loop-Mediated Isothermal Ampliication (LAMP) Assay For Rapid Detection of Actinobacillus

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Pesq. Vet. Bras.

32(8):757-760, agosto 2012

Development and evaluation of a loop-mediated isothermal


ampliication (LAMP) assay for rapid detection of Actinobacillus
pleuropneumoniae based the dsbE-like gene1
Hongwei Ji3, Haitao-Li2, Ling Zhu3, Hui Zhang3, Yin Wang2, Zhicai Zuo2, Wanzhu Guo3
and Zhiwen Xu3*

ABSTRACT.- Ji H.W., Li H.T., Zhu L., Zhang H., Wang Y., Zuo Z.C., Guo W.Z. & Xu Z.W. 2012.
Development and evaluation of a loop-mediated isothermal ampliication (LAMP)
assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like
gene. Pesquisa Veterinária Brasileira 32(8):757-760. Key Laboratory of Animal Biotechno-
logy, Center of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural Uni-
versity, Ya’an, Sichuan, 625014, P.R. China. E-mail: [email protected]
This paper reports on the development and validation of a loop-mediated isothermal
ampliication assay (LAMP) for the rapid and speciic detection of Actinobacillus pleurop-
neumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the
dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae
reference/ield strains, 132 clinical isolates and 9 other pathogens. The results indicated
that positive reactions were conirmed for all A. pleuropneumoniae strains and specimens
by LAMP at 63°C for 60 min and no cross-reactivity were observed from other non-A.pleu-
ropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bor-
detella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine re-
productive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection
limit of the conventional PCR was 102 CFU per PCR test tube, while that of the LAMP was 5
copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover,
the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for labora-
tory diagnosis and pen-side detection due to ease of operation and the requirement of only
a regular water bath or heat block for the reaction.
INDEX TERMS: Actinobacillus pleuropneumoniae, loop-mediated isothermal ampliication (LAMP),
sensitivity.

INTRODUCTION present, 15 different serovars and two biotypes of A. pleu-


Actinobacillus pleuropneumoniae is a Gram-negative, en- ropneumoniae have been described based on nicotinamide
capsulated respiratory pathogen of porcine contagious adenosine dinucleotide (NAD) requirements (Rogers et al.
pleuropneumonia. Due to the aggressive nature of A. pleu- 1990). Serovar speciicity is predominately due to structu-
ropneumoniae, it is distributed worldwide and outbreaks ral differences in the capsular polysaccharides and for most
usually result in signiicant economic losses to the swine serovars also differences in lipopolysaccharides (Chatellier
industry (Sebunya & Saunders 1983). The disease is cha- et al. 1999). Recently, conventional cultivation of the bacte-
racterized by ibrinous pleuritis with hemorrhagic and ria from healthy carrier pigs has been improved by deve-
necrotic lesions in the lungs (Fenwick & Henry 1994). At lopment of A. pleuropneumoniae-selective media (Jacobsen
& Nielsen 1995, Sebunya & Saunders 1983). For the sero-
logical tests, such as immunomagnetic separation-based
1
Received on April 2, 2012. (IMS), tube agglutination test, agglutination in the presen-
Accepted for publication on April 27, 2012.
2
ce of 2-mercaptoethanol, complement ixation test, and en-
College of Veterinary Medicine of Sichuan Agricultural University, Ya’an,
Sichuan, 625014, P.R. China.
zyme-linked immunosorbent assay (ELISA) has been used
3
Key Laboratory of Animal Biotechnology Center of Sichuan Province, to detect the pathogen (Gagné et al. 1998, Gottschalk et al.
College of Veterinary Medicine of Sichuan Agricultural University, Ya’an, 1994, 1994b, Gottschalk et al. 1997, Catia & Itamar 2003).
Sichuan, 625014, P.R. China. *Corresponding author: [email protected] Detection of the bacterium by polymerase chain reaction

757
758 Hongwei Ji et al.

(PCR) ampliication has also been reported (Chiers et al. Table 1. Strains used test species-speciic of the LAMP for
2001, Gram & Ahrens 1998, Moral et al. 1999, Schaller et al. detection of Actinobacillus pleuropneumoniae
2001). Among these diagnostic methods, polymerase chain Strains result Reference/ Serotype Number of LAMP
reaction methods are the most rapid and able to detect a Field strain strains tested
small amount of bacterial chromosomes. Although the PCR A. pleuropneumoniae Reference 1 1 1/1
assay have been shown to be highly effective for A. pleu- Reference 2 1 1/1
ropneumoniae detection, the requirement of an expensive Reference 3 1 1/1
specialist thermal cycler limit their universal application, Reference 5a 3 3/3
Reference 5b 1 1/1
particularly in resource-limited or point-of-care settings
Reference 7 1 1/1
and clinical laboratories. Field 8 2 2/2
Loop-mediated isothermal ampliication (LAMP) is a Field 9 1 1/1
novel nucleic acid ampliication method (Nagamine et al. Field 12 1 1/1
2001, 2002, Notomi et al. 2000). The method is characteri- Haemophilus parasuis Field 1 0/1
Escherichia coli Reference 3 0/3
zed by employing a DNA polymerase with strand-displace- Pasteurella multocida Field 8 0/8
ment activity, along with two inner primers (FIP, BIP) and Bordetella bronchiseptica Field 4 0/4
two outer primers (F3, B3) to form auto-cycling immedia- Streptococcus suis Field 3 0/3
tes. The new assay is a novel technique which is quite sim- Salmonella enterica Reference 4 0/4
Staphylococcus Field 1 0/1
ple, requiring only a conventional water bath or heat block
PRRSV Field 6 0/6
for incubation under isothermal conditions. The sensitivity, Pseudorabies virus Field 5 0/5
speciicity, and applicability of the method have been re-
ported. Because of these advantages, it has been applied Table 2. Details of LAMP and PCR primers designed for
successfully to the detection of a lot of pathogen, such as detection of Actinobacillus pleuropneumoniae
Newcastle disease virus, Escherichia coli, PRRSV (Hong et Method Primer Sequence
al. 2004, Song et al. 2005, Chen et al. 2008).
LAMP F3 5’-CTGAAAGTTTCGTCAGCAC-3’
B3 5’-TCTGAAACTGAAAAGTATCCAC-3’
MATERIALS AND METHODS FIP (F1+F2) 5’-GCTCGAAATCCGGGTTATATCCT–
Bacterial strains. TTTT–ATTTAATTGATAAACACGGTGTG-3’
BIP(B1+B2) 5’-TCGTTTAAAAGCGTTACAAGAGGA–
All strains (China Institute of Veterinary Drugs Control) used
TTTT–AAACTGAAGACAAGCGGTA-3’
for this study were listed in Table 1. Actinobacillus pleuropneu- PCR F 5’-GATAAACCTTTTCCGGAATT-3’
moniae serotype 3 was used to develop a LAMP method and de- B 5’-TACCACACCGTGTTTATCAA-3’
termine the detection limit of the method. A. pleuropneumoniae
were grown overnight in tryptic soy broth (TSB) medium at 37°C primer, 0.2μM each of F3 and B3 primer, 1.4μM each deoxynucle-
shaking on a rotary shaker, supplemented with 10μg of NAD/ml oside triphosphate, 8 U of Bst DNA polymerase (New England Bio-
and 10% bovine serum. Bordetella bronchiseptica was grown on labs) using the manufacturer’s supplied 10 × buffer (containing
Bordet–Gengou agar supplemented with 10% sheep’s blood. All 2 mM of MgSO4, 0.8 M betaine) and 1 μl of extracted template
bacterium/virus were identiied by conventional PCR (or RT-PCR) DNA in a 0.2 ml Eppendorf tube. Before the reaction, to increase
and sequencing. the sensitivity of the LAMP, the reaction mixture (without the Bst
DNA polymerase) was heated at 96°C for 5 min and immediately
DNA/RNA extraction cooled on ice for 3 min. The ampliication reaction was performed
DNA from A. pleuropneumoniae and other species were prepa- at 63°C for 60 min and then terminated by heating at 95°C for 5
red as follows: bacterial cells of each strain from colonies on TSA min. Each test was repeated three times.
were resuspended in TE buffer [10mM Tris-HCl(pH 8.0), 1mM
EDTA] to achieve a concentration of approximately 106 CFUmL-1. Speciicity and sensitivity of LAMP for Actinobacillus pleurop-
All the strains were treated in a boiling water bath for 10 min and neumoniae
centrifuged for 10 min. The resulting supernatant was used as the To evaluate the species speciicity of the LAMP test, 35 strains
template for the LAMP. For PRRSV and Pseudorabies virus, DNA representing 9 species (Table 1), normally found in pigs, were
and RNA were extracted directly from virus with a Trizol reagent examined. Serial dilutions of 1, 5, 10, 102, 103, 104, and 105co-
(Invitrogen), according to the manufacturer’s instructions. Com- pies of DNA from A. pleuropneumoniae were used as template for
plementary DNA (cDNA) synthesis reaction was performed by the LAMP to detection limit of the method. The sensitivity of detec-
TranScript First-strand cDNA Synthesis SuperMix (Beijing Trans- tion was compared between LAMP and PCR (Chiers et al. 2001).
Gen Biotech Co., China) in accordance with the manufacturer’s
instructions. Evaluation of LAMP with clinical samples
For further evaluation of the LAMP assay, 132 samples of
Primer design and LAMP reaction tonsil tissues (n=132), all originating from different herds, were
A set of 6 primers for LAMP were designed, using the onli- obtained from pigs with an apparent infection of the respiratory
ne LAMP PRIMER DESIGN software (https://primerexplorer. tract. 35 tonsil samples (n=35) taken from 35 healthy pigs that
jp/e/),by targeting highly conserved and A .pleuropneumoniae was free of infection with A. pleuropneumoniae. Approximately to-
speciic sequences regions of dsbE-like gene (Chiers et al. 2001). tal of 0.5 g of each tonsils described above was reduced to small
Primer names and sequences for LAMP are shown in Table 2. The pieces with a scalpel and placed in sterile tubes added 5 mL of
LAMP reaction was carried out in a conventional water bath, the trypticase soy broth, 5 mL NAD and 500 mL sterilized fetal bovine
total 25-μL reaction mixture containing 2.0μM each of FIP and BIP serum and Bacterial growth was harvested after grown overnight

Pesq. Vet. Bras. 32(8):757-760, agosto 2012


A loop-mediated isothermal ampliication (LAMP) assay of Actinobacillus pleuropneumoniae based the dsbE-like gene 759

at 37°C. Take out 2 μl of each sample for LAMP and PCR4. The IMS 3, electrophoresis results revealed that it was possible to
bacterial isolation detection of A. pleuropneumoniae was perfor- detect 5 copies. However, as previously reported, the PCR
med according to the described previously (Gagne et al. 1998). assay could detect the DNA was 102 CFU per PCR test tube
(Chiers et al. 2001). Therefore, the LAMP method was more
RESULTS AND DISCUSSION sensitive than the PCR described previously. The detection
The analysis on agarose gel 2.5% indicated an ampliication sensitivity of LAMP was 20-fold higher than the PCR des-
successful by LAMP showed a ladder-like pattern appeared cribed previously
when using the Actinobacillus pleuropneumoniae as a tem- The feasibility of the LAMP assay for detecting A. pleu-
plate, whereas other bacterial was not ampliied (Fig.1). ropneumoniae in clinical material was assessed by using
The 9 A.pleuropneumoniae reference/ield strains could all healthy pigs and infected pigs. Among the 35 samples col-
get the positive reaction, indicated that these LAMP assay lected from healthy pig, there was no false positivity for the
could detect each of the A. pleuropneumoniae given in these three assays. For the sensitivity, the LAMP gave a total of 73
LAMP method (Fig.2). In order to rule out the possibility positive results and 19 samples were positive for LAMP but
of false positivity, all the positive products of LAMP were negative for the PCR method (Table 3). The positive results
sequenced and the results were as expected. The sensitivi- obtained by IMS bacterial isolation is 38, and these positive
ty of LAMP was demonstrated by using various A. pleurop- results also tested positive by both PCR and LAMP. Overall,
neumoniae DNA dilutions as templates. As shown in Figure the LAMP method demonstrated higher sensitivity than
that of PCR and IMS bacterial isolation.

Table 3. Comparison the sensitivity of the conventional PCR,


IMS bacterial isolationand LAMP for detection of Actinobacillus
pleuropneumoniae
Assay Positive samples (%)
Healthy material infected material
LAMP 0% (0/35) 53% (73/132)
PCR 0% (0/35) 41% (54/132)
IMS bacte-rial isolation 0% (0/35) 29% (38/132)

Porcine contagious pleuropneumonia is highly conta-


gious and may result in high herd mortality. Pigs from 2 to
6 months are particularly affected, even though all ages are
sensitive to the pathogen. Depending on the immune status
of animals, environmental conditions, infecting dose and
Fig.1. The speciicity of LAMP assay for detecting Actinobacillus strain virulence, pigs can develop a peracute, acute, suba-
pleuropneumoniae. Ampliication was detected by electro- cute or chronic form of the disease. These infected pigs can
phoretic analysis on a 2.5% agarose gel. Lane M, DNA marker transmit A. pleuropneumoniae to the other pigs of the herd.
DL2000, lane 1 A. pleuropneumoniae; lane 2-10: Haemophilus In this context, early detection of this bacterium is impor-
parasuis, Escherichia coli, Pasteurella multocida, Bordetella
tant for control and treatment of the disease.
Bronchiseptica, Streptococcus suis, Salmonella enterica, Staph
ephylococcus, PRRSV, Pseudorabies virus. Loop-mediated isothermal ampliication (LAMP) is a
novel ampliication method which was developed origi-
nally by Notomi (Notomi et al. 2000). This simple and rapid
method relies on strand-displacing DNA synthesis perfor-
med using the large fragment of Bst DNA polymerase under
isothermal conditions at 60-65°C within 60 min. A novel
system for the rapid and easy identiication of A. pleurop-
neumoniae was described. Another useful feature of LAMP
is that its products can be observed directly by naked eye,
because a white precipitate of magnesium pyrophosphate
forms in the reaction tube (Mori et al. 2001).
A novel system for the rapid and easy identiication of A.
pleuropneumoniae was described. Compared to PCR, use of
LAMP to detect the pathogen has many advantages. Sensiti-
vity, it requires only small amounts of target DNA to be pre-
sent in a sample for positive diagnosis, the detection limits
of the LAMP described in this study was 5 copies, but that
Fig.2. The LAMP assay for detecting 9 A. pleuropneumoniae refe- obtained by previously reported PCR was 102 CFU per PCR
rence/ield strains. Lane M, DNA marker DL2000, lanes 1-9: test tube. Moreover, in the ield trial, 53% and 41% sam-
Serotype 1, Serotype 2, Serotype 3, Serotype 5a, Serotype 5b, ples tested positive by LAMP and PCR, respectively. Time,
Serotype 7, Serotype 8, Serotype 9, Serotype 12, respectively. it will take 60 min for LAMP to detect the A. pleuropneu-

Pesq. Vet. Bras. 32(8):757-760, agosto 2012


760 Hongwei Ji et al.

moniae, whereas the PCR method typically requires 2–4h. F. 2001. Detection of Actinobacillus pleuropneumoniae in cultures from
Requirements for specialist equipment, the method can be nasal and tonsillar swabs of pigs by a PCR assay based on the nucleotide
sequence of a dsbE-like gene. Vet. Microbiol. 83:147-159.
performed under isothermal conditions in the temperature
Fenwick B.W. & Henry S. 1994. Porcine pleuropneumonia. J. Am. Vet. Med.
range of 60–65°C using a simple heating device such as a Assoc. 204:1334-1340.
water bath or heat block, but for PCR, a complicated ther-
Gagné A., Lacouture S., Broes A., D’Allaire S. & Gottschalk M. 1998. Deve-
mal cyclers was need. In conclusion, although PCR assays lopment of an immunomagnetic method for selective isolation of Acti-
possess many advantages, the LAMP assay showed supe- nobacillus pleuropneumoniae serotype 1 from tonsils. J. Clin. Microbiol.
rior sensitivity than the PCR for detection of A. pleuropneu- 36:251-254.
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tests in the ield with speciicity improved instruments was ation of a saline boiled extract, capsular polysaccharides and long-chain
not required and great sensitivity results can be received. lipopolysaccharides of Actinobacillus pleuropneumoniae serotype 1 as
antigens for the serodiagnosis of swine pleuropneumonia. Vet. Micro-
biol. 42:91-104.
s s s s s
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o o o o p diagnosis of swine pleuropneumonia due to Actinobacillus pleuropneu-
5 c 4 c 3 c 2 c co cop cop moniae serotypes 7 and 4 using long-chain lipopolysaccharides. Can. J.
M 10 10 10 10 10 5 1 Vet. Res. 61:62-65.
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Fig.3. The detection limits of LAMP for the detection of A. pleu- niae and its use in a PCR assay for rapid identiication. J. Clin. Microbiol.
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showed in the picture, represented the concentrations of DNA Mori Y., Nagamine K., Tomita N. & Notomi T. 2001. Detection of loop-me-
from APP as a template for LAMP. diated isothermal ampliication reaction by turbidity derived from mag-
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CONCLUSION
Nagamine K., Kuzuhara Y. & Notomi T. 2002. Isolation of single-stranded
In conclusion, a novel system for the rapid and easy iden- DNA from loop-mediated isothermal ampliication products. Biochem.
tiication of Actinobacillus pleuropneumoniae was descri- Biophys. Res. Commun. 290:1195-1198.
bed which was rapid, highly sensitive, simple, and inex- Nagamine K., Watanabe K., Ohtsuka K. & Hase T. 2001. Loop-mediated
pensive. This method can potentially be used for tests in isothermal ampliication reaction using a nondenatured template. Clin.
the ield. Chem. 47:1742-1743.
Notomi T., Okayama H., Masubuchi H. & Yonekawa T. 2000. Loop-mediated
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Pesq. Vet. Bras. 32(8):757-760, agosto 2012

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