Development and application of a TaqMan-MGB real-time RT-PCR assay for the detection of porcine epidemic diarrhoea virus strains in China

Yi-Xuan Hou; Chun Xie; Kang Wang; Yu-Ting Zhao; Yang-Yang Xie; Hong-Yan Shi; Jian-Fei Chen; Li Feng; Guang-Zhi Tong; Xiu-Guo Hua; Cong-Li Yuan; Yan-Jun Zhou; Zhi-Biao Yang

doi:10.1515/jvetres-2016-0018

.blurhash-client-img { display: none !important; }

Development and application of a TaqMan-MGB real-time RT-PCR assay for the detection of porcine epidemic diarrhoea virus strains in China

Journal of Veterinary Research

Volume 60 (2016): Issue 2 (June 2016)

By: Yi-Xuan Hou, Chun Xie, Kang Wang, Yu-Ting Zhao, Yang-Yang Xie, Hong-Yan Shi, Jian-Fei Chen, Li Feng, Guang-Zhi Tong, Xiu-Guo Hua, Cong-Li Yuan, Yan-Jun Zhou and Zhi-Biao Yang

Open Access

|May 2016

Abstract

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.

References

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, 209–219.10.1586/14737159.5.2.209
Search in Google Scholar
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, 33–40.10.1016/0167-5877(94)00429-M
Search in Google Scholar
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, 1471–1476.10.1007/s00705-010-0720-2708699020544234
Search in Google Scholar
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, 234–243.10.1128/JCM.02820-13391141524197882
Search in Google Scholar
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, 499–507.10.1007/s11262-012-0794-x708944222843324
Search in Google Scholar
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, 113–117.10.1007/s11262-012-0755-4708856722585338
Search in Google Scholar
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, 2417–2427.10.1128/JCM.41.6.2417-2427.200315648312791858
Search in Google Scholar
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. 4^th International Conference on Biomedical Engineering and Informatics (BMEI'11), 2011, Shanghai, pp. 1412–1415.10.1109/BMEI.2011.6098527
Search in Google Scholar
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, e00737–13.10.1128/mBio.00737-13381270824129257
Search in Google Scholar
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, 172–177.10.1016/j.jviromet.2007.06.021711965017697717
Search in Google Scholar
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, 1350–1353.10.3201/eid1803.120002
Search in Google Scholar
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, 1267–1273.10.1007/s00705-012-1592-4366812923389550
Search in Google Scholar
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, 181–185.10.1007/s11262-012-0735-822528639
Search in Google Scholar
14. Oldham J.: Pig Farming (Oct suppl), 1972, 72–73.
Search in Google Scholar
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.
Search in Google Scholar
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, 195–203.10.1186/1743-422X-9-195348793122967434
Search in Google Scholar
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, 2089–2092.10.3201/eid2012.131642425780525424875
Search in Google Scholar
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, 1533–1541.10.1007/s00705-013-1651-5708727923462888
Search in Google Scholar
19. Reed L.J., Muench H.: A simple method of estimating fifty per cent endpoints. Am J Epidemiol 1938, 27, 493–497.10.1093/oxfordjournals.aje.a118408
Search in Google Scholar
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, 229–235.10.1007/s11262-011-0570-3708943621286798
Search in Google Scholar
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, 44–49.10.1016/j.jviromet.2006.09.007711278917055070
Search in Google Scholar
22. Song D., Park B.: Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus genes 2012, 44, 167–175.10.1007/s11262-012-0713-1708918822270324
Search in Google Scholar
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, 161–163.10.3201/eid1801.111259338168322261231
Search in Google Scholar
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.
Search in Google Scholar
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.
Search in Google Scholar
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, 150–155.10.1016/j.jviromet.2014.06.00624928691
Search in Google Scholar
27. Zhou Y.J., Wu Y.L., Zhu J.P., Tong W., Yu H., Jiang Y.F., Tong G.Z.: Complete genome sequence of a virulent porcine epidemic diarrhea virus strain. J Virol 2012, 86, 13862.10.1128/JVI.02635-12350306123166262
Search in Google Scholar

Articles in this issue

DOI: https://doi.org/10.1515/jvetres-2016-0018 | Journal eISSN: 2450-8608

Journal RSS Feed

Language: English

Page range: 127 - 133

Submitted on: Sep 9, 2015

Accepted on: Apr 22, 2016

Published on: May 28, 2016

Published by: National Veterinary Research Institute in Pulawy

In partnership with: Paradigm Publishing Services

Publication frequency: 4 issues per year

Keywords:

porcine epidemic diarrhoea virus,

TaqMan-MGB real-time RT-PCR,

People’s Republic of China

Related subjects:

Life sciences,

Molecular biology,

Microbiology and virology,

Life sciences, other,

Medicine,

Veterinary medicine

© 2016 Yi-Xuan Hou, Chun Xie, Kang Wang, Yu-Ting Zhao, Yang-Yang Xie, Hong-Yan Shi, Jian-Fei Chen, Li Feng, Guang-Zhi Tong, Xiu-Guo Hua, Cong-Li Yuan, Yan-Jun Zhou, Zhi-Biao Yang, published by National Veterinary Research Institute in Pulawy
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Volume 60 (2016): Issue 2 (June 2016)