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

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

doi:10.1515/jvetres-2016-0018

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.

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

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