In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10−5 ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.
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Allan G M, Ellis J A. 2000. Porcine circoviruses: a review. J Vet Diagn Invest, 12: 3–14.
Allan G M, McNeilly F, Kennedy S, et al. 1998. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J Vet Diagn Invest, 10: 3–10.
Bolin S R, Stoffregen W C, Nayar G P, et al. 2001. Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type 2 porcine circovirus. J Vet Diagn Invest, 13: 185–194.
Chen H T, Zhang J, Sun D H, et al. 2008. Rapid detection of porcine circovirus type 2 by loop-mediated isothermal amplification. J Virol Methods, 149: 264–268.
Chen L, Fan X, Wang Q, et al. 2010. A novel RT-LAMP Assay for Rapid and Simple detection of Classical Swine Fever Virus. Virol Sin, 25(1): 59–64
Chen H T, Zhang J, Yang S H, et al. 2009. Rapid detection of porcine parvovirus DNA by sensitive loop-mediated isothermal amplification. J Virol Methods, 158: 100–103.
Gao H, Lei Z, Jia J, et al. 2009. Application of loopmediated isothermal amplification for detection of Yersinia enterocolitica in pork meat. J Micro Methods, 77: 198–201.
Liu Y B, Sun Y H, Zhang L, et al. 2010. Isolation & genomic Sequence analysis of three PCV2 strains. Chin J Preven Vet Med, 8: 27–29. (in Chinese)
Meehan B M, McNeilly F, Todd D, et al. 1998. Characterization of novel circovirus DNA associated with wasting syndromes in pigs. J Gen Virol, 79: 2171–2179.
Mori Y, Nagamine K, Tomita N, et al. 2001. Detection of loopmediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun, 289: 150–154.
Notomi T, Okayama H, Masubuchi H, et al. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 28: e63.
Olvera A, Sibila M, Calsamiglia M, et al. 2004. Comparison of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs. J Virol Methods, 117: 75–80.
Parida M M. 2008. Rapid and real-time detection technologies for emerging viruses of biomedical importance. J Biosci, 33(4): 617–628.
Pham H M, Nakajima C, Ohashi K, et al. 2005. Loop-mediated isothermal amplification for rapid detection of Newcastle disease virus. J Clin Microbiol, 43: 1646–1650.
Poon L L M, Leung C S W, Chan K H, et al. 2005. Detection of human influenza a viruses by loop-mediated isothermal amplification. J Clin Microbiol, 43: 427–430.
Richt J A, Lager K M, Clouser D F, et al. 2004. Real-time reverse transcription-polymerase chain reaction assays for the detection and differentiation of North American swine influenza viruses. J Vet Diagn Invest, 16(5): 367–373.
Schorr E, Wentworth D, Hinshaw V S. 1994. Use of polymerase Real-time RT-PCR for swine influenza viruses polymerase chain reaction to detect swine influenza virus in nasal swab specimens. Am J Vet Res, 55: 952–956.
Segales J, Domingo M. 2002. Postweaning multisystemic wasting syndrome (PMWS) in pigs: a review. Vet Q, 24: 109–124.
Segales J, Allan, G M, Domingo M. 2005. Porcine circovirus diseases. Anim Health Res Rev, 6: 119–142.
Shang S B, Li Y F, Guo J, et al. 2008. Development and validation of a recombinant capsid protein-based ELISA for detection of antibody to porcine circovirus type 2. Res Vet Sci, 84(1): 150–157.
Tsugunori N, Okayama H, Harumi M, et al. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 28(12): E63–E63.
Walker I W, Konoby C A, Jewhurst V A, et al. 2000. Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2. J Vet Diagn Invest, 12: 400–405.
Zhou J Y, Chen Q X, Ye J X, et al. 2006. Serological investigation and genomic characterization of PCV2 isolates from different geographic regions of Zhejiang Province in China. Vet Res Communications, 30: 205–220.
Foundation item: Fifteenth National Science and Technology Support Program of China (2007BAD86B04-4)
These authors contributed equally.
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Liu, Yb., Zhang, L., Xue, Qh. et al. Development of a loop-mediated isothermal amplification assay for porcine circovirus type 2. Virol. Sin. 26, 214–220 (2011). https://doi.org/10.1007/s12250-011-3169-x
- Porcine circovirus type 2 (PCV2)
- Loop-mediated isothermal amplification (LAMP)
- Virus detection