Phat X. Dinh * , Trang T. T. Nguyen , Hiep L. X. Vu , & Thoai K. Tran

* Correspondence: Dinh Xuan Phat (email:

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This study aimed to simultaneously detect three important viruses reported to be involved in the reproductive problems of sows. A multiplex PCR (mPCR) test was developed to provide rapid diagnosis of porcine circovirus type 2 and 3 (PCV2, PCV3) and to illustrate parvovirus (PPV) prevalence in sow herds. Three pairs of specific primers were designed to target PCV2 Cap gene, PCV3 Cap gene and PPV NS1 gene, with predicted mPCR products of 702 bp, 267 bp and 380 bp, respectively. The detection limit of mPCR was 100 copies/reaction per target gene. The mPCR was run against a panel of 94 swine serum samples whose infection status had been pre-determined by commercial real-time PCR kits. Sequencing of mPCR products performed with clinical serum samples accurately confirmed the results. Overall, the results indicated that the mPCR functioned accurately and specifically and matched 100% with the single-target real-time PCRs. The mPCR was developed successfully and can be used in routine diagnosis of PCV2, PCV3 and PPV.

Keywords: mPCR, PCV2, PCV3, PPV, Swine

Article Details


Allan, G. M., McNeilly, E., Kennedy, S., Meehan, B., Moffett, D., Malone, F., Ellis, J., & Krakowka, S. (2000). PCV-2-associated PDNS in Northern Ireland in 1990. Porcine dermatitis and nephropathy syndrome. Veterinary Record 146(24), 711-712.

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. (1999). Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland. Veterinary Microbiology 66(2), 115-123.

Arruda, B., Pin˜eyro, P., Derscheida, R., Hause, B., Byers, E., Dion, K., Long, D., Sievers, D., Tangen, J., Williams, T., & Schwartz, K. (2019). PCV3-associated disease in the United States swine herd. Emerging Microbes & Infections 8(1), 684-698.

Calsamiglia, M., Fraile, L., Espinal, A., Cuxart, A., Seminati, C., Mart´ın, M., Mateu, E., Domingo, M., & Segal´es, J. (2007). Sow porcine circovirus type 2 (PCV2) status effect on litter mortality in postweaning multisystemic wasting syndrome (PMWS). Research in Veterinary Science 82(3), 299-304.

Cartwright, S. F., & Huck, R. A. (1967). Viruses isolated in association with herd infertility abortions and stillbirths in pigs. Veterinary Record 81(1967), 196-197.

Cartwright, S. F., Lucas, M., & Huck, R. A. (1969). A small hemagglutinating porcine DNA virus: I. Isolation and properties. The Journal of Comparative Pathology 79(3), 371-377.

Chen, G. H., Mai, K. J., Zhou, L., Wu, R. T., Tabg, X. Y., Wu, J. L., He, L. L., Lan, T., Xie, Q. M., Sun, Y., & Ma, J. Y. (2017). Detection and genome sequencing of porcine circovirus 3 in neonatal pigs with congenital tremors in South China. Transboundary and Emerging Diseases 64(6), 1650-1654.

Chen, H. Y., Li, X. K., Cui, B. A., Wei, Z. Y., Li, X. S., Wang, Y. B., Zhao, L., & Wang, X. Y. (2009). A TaqMan-based real-time polymerase chain reaction for the detection of porcine parvovirus. Journal of Virological Methods 156(1-2), 84-88.

Christianson, W. T. (1992). Stillbirths, mummies, abortions, and early embryonic death. Veterinary Clinics of North America: Food Animal Practice 8(3), 623-639.

Dinh, P. X., Nguyen, N. M., Nguyen, T. H., Tran, V. H., Tran, Q. D., Dang, K. H., Vo, D. T., Le, H. T., Nguyen, N. T. T., Nguyen, T. T., & Do, D. T. (2021). Porcine circovirus genotypes and their co-pathogens in pigs with respiratory disease in southern provinces of Vietnam. Archives of Virology 166(2), 403-411.

Ellis, J., Hassard, L., Clark, E., Harding, J., Allan, G., Willson, P., Strokappe, J., Martin, K., McNeilly, F., & Meehan, B. (1998). Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Canadian Veterinary Journal 39(1), 44-51.

Franzo, G., Legnardi, M., Hjulsager, C. K., Klaumann, F., Larsen, L. E., Segales, J., & Drigo, M. (2018). Full-genome sequencing of porcine circovirus 3 field strains from Denmark, Italy and Spain demonstrates a high within-Europe genetic heterogeneity. Transboundary and Emerging Diseases 65(3), 602-606.

Huynh, T. M. L., Le, V. P., Nguyen, V. G., & Trinh, D. T. (2015). Variety selection porcine circovirus type 2 (PCV2) for producing vaccines against piglets. Journal of Scientific Research and Development 13, 406-415.

Huynh, T. M., Nguyen, B. H., Nguyen, V. G., Dang, H. A., Mai, T. N., Tran, T. H., Ngo, M. H., Le, V. T., Vu, T. N., Ta, T. K., Vo, V. H., Kim, H. K., & Park, B. K. (2014). Phylogenetic and phylogeographic analyses of porcine circovirus type 2 among pig farms in Vietnam. Transboundary and Emerging Diseases 61(6), e25-34.

Johnson, R., & Collings, D. (1969). Experimental infection of piglets and pregnant gilt with a parvovirus. Veterinary Record 85(16), 446-447.

Kwon, T., Yoo, S. J., Park, C. K., & Lyoo, Y. S. (2017). Prevalence of novel porcine circovirus 3 in Korean pig populations. Veterinary Microbiology 207, 178-180.

Liu, J. K., Wei, C. H., Yang, X. Y., Dai, A. L., & Li, X. H. (2015). Simultaneous detection and differentiation of porcine circovirus type 2, type 2 porcine re-productive and respiratory syndrome virus, porcine parvovirus and pseudorabies virus in pigs with post-weaning multisystemic wasting syndrome (PMWS) by multiplex PCR. The journal Veterinarski arhiv 85(5), 511-521.

Milek, D., Wozniak, A., Podgorska, K., & Stadejek, T. (2020). Do porcine parvoviruses 1 through 7 (PPV1- PPV7) have an impact on porcine circovirus type 2 (PCV2) viremia in pigs? Veterinary Microbiology 242, 108613.

Nguyen, D. H. M., Luong, Q. T. X., Hoang, P. T., Do, D. T. T., Tran, T. K., & Dinh, P. X. (2018). Detecting toxin genes of Clostridium perfringens isolated from diarrhea piglets using multiplex PCR. The Journal of Agriculture and Development 17(6), 24-30.

Nguyen, V. G., Mai, T. N., Le, V. T., Vu, T. N., Vo, V. H., Ta, T. K. C., Vu, D. H., & Huynh, T. M. L. (2020). Presence of porcine parvovirus 1 (PPV1) in domestic pigs in Ha Noi and the neighborhood. Vietnam Journal of Agricultural Sciences 18, 495-503.

Oropeza-Moe, M., Oropeza Delgado, A. J., & Framstad, T. (2017). Porcine circovirus type 2 associated reproductive failure in a specific pathogen free (SPF) piglet producing herd in Norway: a case report. Porcine Health Management 3(1), 2-6.

Ouyang, T., Zhang, X., Liu, X., & Ren, L. (2019). Co-Infection of swine with porcine circovirus type 2 and other swine viruses. Viruses 11(2), 185.

Palinski, R., Pin˜eyro, P., Shang, P., Yuan, F., Guo, R., Fang, Y., Rui, G., Ying, F., Emily, B., & Ben, M. H. (2016). A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure. Journal of Virology 91(1), e01879-16.

Pham, H. Q., Nguyen, V. G., Nguyen, T. T. T., Pham, C. H., & Huynh, T. M. L. (2017). Study the circulation of new viruses (porcine circovirus 3 - PCV3) in pigs raised in some northern provinces of Vietnam. Vietnam Journal of Agricultural Science 15(11), 1520-1528.

Prinz, C., Stillfried, M., Neubert, L. K., & Denner, J. (2019). Detection of PCV3 in German wild boars. Virology Journal 16(1), 25.

Rosell, C., Segales, J., Ramos-Vara, J. A., Folch, J. M., Rodriguez-Arrioja, G. M., Duran, C. O., Balasch, M., Plana-Duran, J., & Domingo, M. (2000). Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome. Veterinary Record 146(2), 40-43.

Saraiva, G. L., Vidigal, P. M. P., Assao, V. S., Fajardo, M. L. M., Loreto, A. L. S., Fietto, J. R. L., Bressan, G. C., Lobato, Z. I. P., Almeida, M. R. D., & Silva- Ju´nior, A. (2019). Retrospective detection and genetic characterization of porcine circovirus 3 (PCV3) strains identified between 2006 and 2007 in Brazil. Viruses 11(3), 201.

Tisciier, I., Mields, W., Wolff, D., Vagt, M., & Griem, W. (1986). Studies on epidemiology and pathogenicity of porcine circovirus. Archives of Virology 91(3-4), 271-276.

West, K. H., Bystrom, J. M., Wojnarowicz, C., Shantz, N., Jacobson, M., & Allan, G. M. (1999). Myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus 2. Journal of Veterinary Diagnostic Investigation 11(6), 530-532.

Wozniak, A., Milek, D., Baska, P., & Stadejek, T. (2019). Does porcine circovirus type 3 (PCV3) interfere with porcine circovirus type 2 (PCV2) vaccine efficacy? Transboundary and Emerging Diseases 66(4), 1454-1461.

Xu, P. L., Zhang, Y., Zhao, Y., Zheng, H. H., Han, H. Y., Zhang, H. X., Chen, H. Y., Yang, M. F., & Zheng, L. L. (2018). Detection and phylogenetic analysis of porcine circovirus type 3 in central China. Transboundary and Emerging Diseases 65(5), 1163-1169.

Yang, K., Jiao, Z., Zhou, D., Guo, R., Duan, Z., Yuan, F., & Tian, Y. (2018). Detection of porcine circoviruses in clinical specimens using multiplex PCR in Hubei, central China. PeerJ Preprints 6, e27145v1.

Yang, K., Jiao, Z., Zhou, D., Guo, R., Duan, Z., & Tian, Y. (2019). Development of a multiplex PCR to detect and discriminate porcine circoviruses in clinical specimens. BMC Infectious Diseases 19, 778.

Zhao, K., Han, F., Zou, Y., Zhu, L., Li, C., Xu, Y., Zhang, C., Tan, F., Wang, J., Tao, S., He, X., Zhou, Z., & Tang, X. (2010). Rapid detection of porcine circovirus type 2 using a TaqMan-based real-time PCR. Virology Journal 7, 374.