人G1型轮状病毒TaqMan荧光定量检测方法的建立与应用

Establishment and application of TaqMan real-time fluorescence quantitative PCR for G1 serotype rotavirus detection

目的本研究旨在建立一种快速准确定量检测人G1型轮状病毒的TaqMan探针荧光实时PCR检测方法。方法以轮状病毒VP6基因为靶基因,分别设计2对引物及其相应的TaqMan探针,扩增目的片段,将目的片段克隆于PCDNA3.1+载体上,体外转录获得RNA,系列稀释后为标准品,建立TaqMan探针荧光定量实时PCR检测方法并对该方法进行验证。结果设计实验找到最优条件下的引物浓度(250nmol/L)、探针浓度(300nmol/L);通过对引起腹泻的人柯萨奇病毒、呼肠孤病毒等进行检测,结果均为阴性,表明该方法具有很好的特异性。该方法的最低检测量为5copies/μL。对该方法进行重复性实验,发现变异率均小于1%,重复性好。用已建立的方法对4份粪便临床样品进行3次重复试验,病毒RNA的检出率为100%。结论本实验初步建立了人G1型轮状病毒TaqMan探针荧光实时PCR检测方法,为G1型轮状病毒的诊断、检测和分子流行病学研究提供了一种新的方法。

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 300nmol/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 5copies/μL,and variation coefficient less than 1%.With the 3times repeated detection on 4clinical 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.