1. The Affiliated Hospital of Kunming University of Science and Technology, Kunming 650034, China ; 2. Kunming University of Science and technology, faculty of life science and technology,Yunnan Provincial research center of molecular medicine,Kunming 650034, China
Abstract：The aim of this study is to develop a method of TaqMan real-time PCR for quantitative detection on G1 serotype human rotavirus. Based on the VP6 gene, a set of primer and TaqMan probe were designed. The VP6 gene fragment was amplified and cloned into the PCDNA3.1+ vector. RNA was transcribed in vitro and serial diluted to establish the external standards. With the optimization for PCR parameters, the optimized primer concentration was determined as 250 nM and probe concentration as 300 nmol/L. The human coxsackievirus and reovirus couldn’t be detected which proved the high specificity of this method. The low limitation of this method was detected to be less than 5 copies/μL, and variation coefficient less than 1%. With the 3 times repeated detection on 4 clinical samples, this method was verified to have the good performance. Finally, a TaqMan real-time fluorescence quantitative PCR for human rotavirus detection was established, which may provide a new approach for the RV diagnosis, food safety inspection and epidemiological investigation.
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2016, Vol. 32 
