Development and application of a TaqMan-MGB real-time RT-PCR assay for the detection of porcine epidemic diarrhoea virus strains in China
2016
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
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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