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

文献类型： 外文期刊

第一作者： Hou, Yi-Xuan

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

作者机构：

关键词： porcine epidemic diarrhoea virus;TaqMan-MGB real-time RT-PCR;People's Republic of China

期刊名称：JOURNAL OF VETERINARY RESEARCH （ 影响因子：1.744； 五年影响因子：1.838 ）

ISSN：

年卷期：

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收录情况： SCI

摘要： 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.

分类号： S85

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