Skip to main content
SpringerLink
Log in

Detection of PCV2 DNA by SYBR Green I-based quantitative PCR

  • Published:
Journal of Zhejiang University SCIENCE B Aims and scope Submit manuscript

Abstract

We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 103 to 1011 copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was detected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Allan, G.M., McNeilly, F., Cassidy, J.P., Reilly, G.A., Adair, B., Ellis, W.A., McNulty, M.S., 1995. Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material. Vet. Microbiol., 44(1):49–64. [doi:10.1016/0378-1135(94)00136-K]

    Article  PubMed  CAS  Google Scholar 

  • Allan, G.M., McNeilly, F., Kennedy, S., Daft, B., Clarke, E.G., Ellis, J.A., Haines, D.M., Meehan, B.M., Adair, B.M., 1998. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J. Vet. Diagn. Invest., 10(1):3–10.

    PubMed  CAS  Google Scholar 

  • Allan, G.M., McNeilly, F., Meehan, B.M., Kennedy, S., Mackie, D.P., Ellis, J.A., Clark, E.G., Espuna, E., Saubi, N., Riera, P., Botner, A., Charreyre, C.E., 1999. Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland. Vet. Microbiol., 66(2):115–123. [doi:10.1016/S0378-1135(99)00004-8]

    Article  PubMed  CAS  Google Scholar 

  • Allan, G.M., McNeilly, F., Ellis, J., Krakowka, S., Meehan, B., McNair, I., Walker, I., Kennedy, S., 2000. Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication. Arch. Virol., 145(11):2421–2429. [doi:10.1007/s007050070031]

    Article  PubMed  CAS  Google Scholar 

  • Brunborg, I.M., Moldal, T., Jonassen, C.M., 2004. Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR. J. Virol. Methods, 122(2):171–178. [doi:10.1016/j.jviromet.2004.08.014]

    Article  PubMed  CAS  Google Scholar 

  • Chung, W.B., Chan, W.H., Chaung, H.C., Lien, Y., Wu, C.C., Huang, Y.L., 2005. Real-time PCR for quantitation of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in naturally-infected and challenged pigs. J. Virol. Methods, 124(1–2):11–19. [doi:10.1016/j.jviromet.2004.10.003]

    Article  PubMed  CAS  Google Scholar 

  • Fenaux, M., Opriessnig, T., Halbur, P.G., Elvinger, F., Meng, X.J., 2004. Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus in vivo. J. Virol., 78(24):13440–13446. [doi:10.1128/JVI.78.24.13440-13446.2004]

    Article  PubMed  CAS  Google Scholar 

  • Gilpin, D.F., McCullough, K., Meehan, B.M., McNeilly, F., McNair, I., Stevenson, L.S., Foster, J.C., Ellis, J.A., Krakowka, S., Adair, B.M., Allan, G.M., 2003. In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system. Vet. Immunol. Immunopathol., 94(3–4):149–161. [doi:10.1016/S0165-2427(03)00087-4]

    Article  PubMed  CAS  Google Scholar 

  • Hamel, A.L., Lin, L.L., Sachvie, C., Grudeski, E., Nayar, G.P., 2000. PCR detection and characterization of type-2 porcine circovirus. Can. J. Vet. Res., 64(1): 44–52.

    PubMed  CAS  Google Scholar 

  • Harding, J., 1997. Post-weaning multisystemic wasting syndrome (PMWS): preliminary epidemiology and clinical presentation. Proc. Am. Assoc. Swine Pract., 28:503.

    Google Scholar 

  • Huang, C., Hung, J.J., Wu, C.Y., Chien, M.S., 2004. Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses. Vet. Microbiol., 101(3):209–214. [doi:10.1016/j.vetmic.2004.04.007]

    Article  PubMed  CAS  Google Scholar 

  • Kennedy, S., Allan, G., McNeilly, F., Adair, B.M., Hughes, A., Spillane, P., 1998. Porcine circovirus infection in Northern Ireland. Vet. Rec., 142(18):495–496.

    PubMed  CAS  Google Scholar 

  • Kiatipattanasakul-Banlunara, W.T.R., Suzuki, K., Albarenque, S.M., Thanawongnuwech, R., Nakayama, H., Doi, K., 2002. Detection of porcine circovirus 2 (PCV-2) DNA by nested PCR from formalin-fixed tissues of post-weaning multisystemic wasting syndrome (PMWS) pigs in Thailand. J. Vet. Med. Sci., 64(5):449–452. [doi:10.1292/jvms.64.449]

    Article  PubMed  CAS  Google Scholar 

  • Kim, J., Chae, C., 2003. Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome. Can. J. Vet. Res., 67(2):133–137.

    PubMed  CAS  Google Scholar 

  • Ladekjær-Mikkelsen, A.S., Nielsen, J., Stadejek, T., Storgaard, T., Krakowka, S., Ellis, J., McNeilly, F., Allan, G., Botner, A., 2002. Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type 2 (PCV2). Vet. Microbiol., 89(2–3):97–114. [doi:10.1016/S0378-1135(02)00174-8]

    Article  PubMed  Google Scholar 

  • Larochelle, R., Antaya, M., Morin, M., Magar, R., 1999. Typing of porcine circovirus in clinical specimens by multiplex PCR. J. Virol. Methods, 80(1):69–75. [doi:10.1016/S0166-0934(99)00032-4]

    Article  PubMed  CAS  Google Scholar 

  • Liu, Q., Wang, L., Willson, P., Babiuk, L.A., 2000. Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome. J. Clin. Microbiol., 38(9):3474–3477.

    PubMed  CAS  Google Scholar 

  • Olvera, A., Sibila, M., Calsamiglia, M., Segales, J., Domingo, M., 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(1):75–80. [doi:10.1016/j.jviromet.2003.12.007]

    Article  PubMed  CAS  Google Scholar 

  • Ouardani, M., Wilson, L., Jette, R., Montpetit, C., Dea, S., 1999. Multiplex PCR for detection and typing of porcine circoviruses. J. Clin. Microbiol., 37(12):3917–3924.

    PubMed  CAS  Google Scholar 

  • Rovira, A., Balasch, M., Segales, J., Garcia, L., Plana-Duran, J., Rosell, C., Ellerbrok, H., Mankertz, A., Domingo, M., 2002. Experimental inoculation of conventional pigs conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2. J. Virol., 76(7):3232–3239. [doi:10.1128/JVI.76.7.3232-3239.2002]

    Article  PubMed  CAS  Google Scholar 

  • Sanchez, R.E.Jr, Nauwynck, H.J., McNeilly, F., Allan, G.M., Pensaert, M.B., 2001. Porcine circovirus 2 infection in swine foetuses inoculated at different stages of gestation. Vet. Microbiol., 83(2):169–176. [doi:10.1016/S0378-1135(01)00425-4]

    Article  PubMed  Google Scholar 

  • Segales, J., Domingo, M., 2002. Postweaning multisystemic wasting syndrome (PMWS) in pigs. A review. Vet. Q., 24(3):109–124.

    PubMed  CAS  Google Scholar 

  • Tischer, I., Gelderblom, H., Vettermann, W., Koch, M.A., 1982. A very small porcine virus with circular single-stranded DNA. Nature, 295(5844):64–66. [doi:10.1038/295064a0]

    Article  PubMed  CAS  Google Scholar 

  • Wellenberg, G.J., Stockhofe-Zurwieden, N., de Jong, M.F., Boersma, W.J., Elbers, A.R., 2004. Excessive porcine circovirus type 2 antibody titers may trigger the development of porcine dermatitis and nephropathy syndrome: a case-control study. Vet. Microbiol., 99(3–4):203–214. [doi:10.1016/j.vetmic.2004.01.001]

    Article  PubMed  CAS  Google Scholar 

  • Wen, L., Guo, X., Yang, H., 2005. Genotyping of porcine circovirus type 2 from a variety of clinical conditions in China. Vet. Microbiol., 110(1–2):141–146. [doi:10.1016/j.vetmic.2005.07.003]

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou, 310029, China

    Yang Zong-zhao, Habib Mudasser, Shuai Jiang-bing & Fang Wei-huan

  2. Hangzhou Husbandry and Veterinary Station, Hangzhou, 310020, China

    Yang Zong-zhao

Authors
  1. Yang Zong-zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Habib Mudasser
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shuai Jiang-bing
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fang Wei-huan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fang Wei-huan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, Zz., Habib, M., Shuai, Jb. et al. Detection of PCV2 DNA by SYBR Green I-based quantitative PCR. J. Zhejiang Univ. - Sci. B 8, 162–169 (2007). https://doi.org/10.1631/jzus.2007.B0162

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1631/jzus.2007.B0162

Key words

CLC number

Search

Navigation