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Journal of Zhejiang University SCIENCE B 2007 Vol.8 No.3 P.162-169

http://doi.org/10.1631/jzus.2007.B0162


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


Author(s):  YANG Zong-zhao, HABIB Mudasser, SHUAI Jiang-bing, FANG Wei-huan

Affiliation(s):  Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China; more

Corresponding email(s):   whfang@zju.edu.cn

Key Words:  Porcine circovirus type 2 (PCV2), Real-time PCR, Sensitivity


YANG Zong-zhao, HABIB Mudasser, SHUAI Jiang-bing, FANG Wei-huan. Detection of PCV2 DNA by SYBR Green I-based quantitative PCR[J]. Journal of Zhejiang University Science B, 2007, 8(3): 162-169.

@article{title="Detection of PCV2 DNA by SYBR Green I-based quantitative PCR",
author="YANG Zong-zhao, HABIB Mudasser, SHUAI Jiang-bing, FANG Wei-huan",
journal="Journal of Zhejiang University Science B",
volume="8",
number="3",
pages="162-169",
year="2007",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.2007.B0162"
}

%0 Journal Article
%T Detection of PCV2 DNA by SYBR Green I-based quantitative PCR
%A YANG Zong-zhao
%A HABIB Mudasser
%A SHUAI Jiang-bing
%A FANG Wei-huan
%J Journal of Zhejiang University SCIENCE B
%V 8
%N 3
%P 162-169
%@ 1673-1581
%D 2007
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.2007.B0162

TY - JOUR
T1 - Detection of PCV2 DNA by SYBR Green I-based quantitative PCR
A1 - YANG Zong-zhao
A1 - HABIB Mudasser
A1 - SHUAI Jiang-bing
A1 - FANG Wei-huan
J0 - Journal of Zhejiang University Science B
VL - 8
IS - 3
SP - 162
EP - 169
%@ 1673-1581
Y1 - 2007
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.2007.B0162


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.

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

[1] 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.

[2] 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.

[3] 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.

[4] 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.

[5] 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.

[6] 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.

[7] 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.

[8] 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.

[9] 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.

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

[11] 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.

[12] 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.

[13] 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.

[14] 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.

[15] 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.

[16] 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.

[17] 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.

[18] 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.

[19] 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.

[20] 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.

[21] 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.

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

[23] 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.

[24] 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.

[25] 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.

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