Development and Evaluation of A Loop-Mediated Isothermal Ampliication (LAMP) Assay For Rapid Detection of Actinobacillus
Development and Evaluation of A Loop-Mediated Isothermal Ampliication (LAMP) Assay For Rapid Detection of Actinobacillus
Development and Evaluation of A Loop-Mediated Isothermal Ampliication (LAMP) Assay For Rapid Detection of Actinobacillus
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.
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
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.
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.
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moniae and the LAMP assays can potentially be used for Gottschalk M., Altman E., Charland N., De L.F. & Dubreuil J.D. 1994. Evalu-
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
p ie pie p ie p ie ie ies y Gottschalk M., Altman E., Charland N., De L.F. & Dubreuil J.D. 1997. Sero-
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.
Gottschalk M., De L.F., Radacovici S. & Dubreuil J.D. 1994b. Evaluation of
long chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneu-
moniae serotype 5 for the serodiagnosis of swine pleuropneumonia. Vet.
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Gram T. & Ahrens P. 1998. Improved diagnostic PCR assay for Actinoba-
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Hong T.C., Mai Q.L., Cuong D.V., Parida M., Minekawa H., Notomi T., Hasebe
F. & Morita K. 2004. Development and evaluation of a novel loop-media-
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Jacobsen M.J. & Nielsen J.P. 1995. Development and evaluation of a selec-
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Moral C.H., Soriano A.C., Salazar M.S. & Marcos J.Y. 1999. Molecular cloning
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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-
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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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