1 Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China
2 Division of Swine Infectious Diseases, Shanghai Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Shanghai 200241, People’s Republic of China
3 Division of Swine Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150001, People’s Republic of China
Introduction: A real-time RT-PCR method for identification and quantification of porcine epidemic diarrhoea virus (PEDV) strains in China was developed.
Material and Methods: Based on the conserved sequence of the PEDV nucleocapsid (N) gene, a primer pair and probe were designed to establish a TaqMan-MGB real-time RT-PCR assay for quantitative detection of the virus. The sequence was cloned into the pMD18-T vector and a series of diluted recombinant plasmids were used to generate a standard curve with an R2 value of 0.999.
Results: The developed quantitative PCR assay detected viral titres as low as 0.1 TCID50 with high specificity and no cross-reaction with other porcine viruses (PoRV, TGEV, PRRSV, or CSFV). The intra-batch and inter-batch coefficients of variation were both less than 1%, which indicated good reproducibility. Thirty clinical diarrhoea samples obtained from pigs in Shanghai and Fujian were analysed using this quantitative PCR assay. Out of these samples, 93.3% were found to be PEDV positive.
Conclusion: This approach is suitable for clinical sample identification and pathogenesis studies.
1. Aryam M., Shergill I.S., Williamson M., Gommersall L., Arya N., Patel H.: Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn 2005, 5, 209–219.
2. Carvajal I.L., Rafael D., Pedro R., Pedro G.: Seroprevalence of porcine epidemic diarrhea virus infection among different types of breeding swine farms in Spain. Prev Vet Med 1995, 23, 33–40.
3. Chen J., Wang C., Shi H., Qiu H., Liu S., Chen X., Zhang Z., Feng L.: Molecular epidemiology of porcine epidemic diarrhea virus in China. Arch Virol 2010, 155, 1471–1476.
4. Chen Q., Li G., Stasko J., Thomas J.T., Stensland W.R., Pillatzki A.E., Gauger P.C., Schwartz K.J., Madson D., Yoon K.J.: Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak in US swine. J Clin Microbiol 2013, 52, 234–243.
5. Chen X., Yang J., Yu F., Ge J., Lin T., Song T.: Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) samples from field cases in Fujian, China. Virus Genes 2012, 45, 499–507.
6. Fan J.H., Zuo Y.Z., Li J.H., Pei L.H.: Heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in China. Virus Genes 2012, 45, 113–117.
7. Gardner S.N., Kuczmarski T.A., Vitalis E.A., Slezak T.R.: Limitations of Taq-Man PCR for detecting divergent viral pathogens illustrated by hepatitis A, B, C, and E viruses and human immunodeficiency virus. J Clin Microbiol 2003, 41, 2417–2427.
8. Gu J.P., Yue X.W., Li C.Y., Xing R., Yuan C.L., Cui L., Hua X.G., Yang Z.B. Development and primary application of the SYBR Green I RT-qPCR assay for the detection of the transmissible gastroenteritis virus (TGEV) S gene. 4th International Conference on Biomedical Engineering and Informatics (BMEI'11), 2011, Shanghai, pp. 1412–1415.
9. Huang Y.W., Dickerman A.W., Pineyro P., Li L., Fang L., Kiehne R., Opriessnig T., Meng X.J.: Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States. mBio 2013, 4, e00737–13.
10. Kim S.H., Kim I.J., Pyo H.M., Tark D.S., Song J.Y., Hyn B.H.: Multiplex real-time RT-PCR for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus. J Virol Methods 2007, 146, 172–177.
11. Li W., Li H., Liu Y., Pan Y., Deng F., Song Y., Tang X., He Q.: New variants of porcine epidemic diarrhea virus, China, 2011. Emerg Infect Dis 2012, 18, 1350–1353.
12. Li Z., Chen F., Yuan Y., Zeng X., Wei Z., Zhu L., Sun B., Xie Q., Cao Y., Xue C., Ma J., Bee Y.: Sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (PEDV) strains in China. Arch Virol 2013, 158, 1267–1273.
13. Li Z.L., Zhu L., Ma J.Y., Zhou Q.F., Song Y.H., Sun B.L., Chen R.A., Xie Q.M., Bee Y.Z.: Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in south China. Virus genes 2012, 45, 181–185.
15. Ouyang S.Y., Yang D., Ouyang H.S., Ma H.W.: Real-time fluorescent quantitative PCR technique and its application. Chem Life Sci 2004, 26, 74–76.
16. Pan X.T., Li W., Zhou Q.F., Wang D.D., Bi Y.Z., Chen F., Song Y.H.: Isolation and characterization of a variant porcine epidemic diarrhea virus in China. Virol J 2012, 9, 195–203.
17. Park S., Kim S., Song D., Park B.: Novel porcine epidemic diarrhea virus variant with large genomic deletion, South Korea. Emerg Infect Dis 2014, 20, 2089–2092.
18. Park S.J., Song D.S., Park B.K.: Molecular epidemiology and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field isolates in Korea. Arch Virol 2013, 158, 1533–1541.
19. Reed L.J., Muench H.: A simple method of estimating fifty per cent endpoints. Am J Epidemiol 1938, 27, 493–497.
20. Ren X., Li P.: Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus. Virus Genes 2011, 42, 229–235.
21. Salin C., Thaweesak C., Mai L Q., Sudarat D., Arunee C., Alongkorn A., Olfert L., Thaweesak S., Apiradee T.B., Yong P.R. H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes. J Virol Methods 2007, 139, 44–49.
22. Song D., Park B.: Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus genes 2012, 44, 167–175.
23. Sun R.Q, Cai R.J., Chen Y.Q., Liang P.S., Chen D.K., Song C.X.: Outbreak of porcine epidemic diarrhea in suckling piglets, China. Emerg Infect Dis 2012, 18, 161–163.
24. Zhang K., He Q.G.: Development and application of multiple RT-PCR assay for PEDV, TGEV and PoRV. Chin. J Anim Vet Sci 2010, 41, 1001–1005.
25. Zhang S.F., Jia Y., Wang M.X., Ni Y.X., He K.W., Chen F.Y.: Establishment of nested RT-PCR diagnostic method for porcine epidemic diarrhea virus. Virol Sinica 2004, 19, 174–176.
26. Zhao P.D., Bai J., Tang T.S., Li Y.F., Tan C. Shi X.: Development of a multiplex TaqMan probe-based real-time PCR for discrimination of variant and classical porcine epidemic diarrhea virus. J Virol Methods 2014, 206, 150–155.