实时定量RT-PCR方法检测太平洋牡蛎中的诺沃克样病毒

Detection of Norwalk-like virus in oysters(Crassostrea gigas) by real-time quantitative RT-PCR

建立了Taqman实时定量RT-PCR方法检测诺沃克样病毒(Norwalk-like Virus,NLV)的方法,并对中国的牡蛎样品进行了分析。选取GⅡ型NLV的RNA聚合酶区域,利用Primer Express 2.0软件设计引物和探针,建立了定量RT-PCR反应体系。将含有NLV扩增片段的质粒10倍梯度稀释,作为标准品进行反应以确定检测灵敏度并制备标准曲线。结果表明,质粒密度在6×106,6×105,6×104,6×103,6×102,6×101,6个拷贝之间,共7个数量级的范围内,定量RT-PCR反应都有"S"型扩增曲线,检测灵敏度为6个拷贝。制备的标准曲线中,病毒拷贝数(X)与Ct值的关系为Ct=-3.36lgX+37.11,相关系数R2=0.998。对中国37批太平洋牡蛎(Crassostrea gigas Thunberg)样品进行了定量检测,有6批样品为阳性,并根据标准曲线测定了NLV的含量。

A real-time quantitative RT-PCR assay was developed for detection of Norwalk-like Virus （NLV） from oysters. Primers and probes were designed based on the RNA polymerase region of the virus. The Taqman probe was labeled with fluorescent dye FAM on the 5＇ end and TAMRA on the 3 end. The real-time RT-PCR assay had a detection limit of 6 virus copies, with a dynamic range of detection between 6×10^6 to 6 virus copies. The standard curve was prepared based on the linear relationship between the amount of plasmid DNA （X） and cycle threshold （Ct）. For NLV virus, Ct=-3.36lg X＋ 37.11, with the correlation coefficient R^2 =0. 998. Thirty-seven oysters were tested and six were NLV positive and the virus was quantified. The results demonstrate that real-time RT-PCR method can be used as a rapid and highly sensitive detection and quantification method for NLV and it is amenable to high-throughout assay.