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海洋科学  2008 

实时定量RT-PCR方法检测太平洋牡蛎中的诺沃克样病毒

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Keywords: 牡蛎,诺沃克样病毒(Norwalk-like,virus,NLV),实时定量RT-PCR,Taqman,探针

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Abstract:

建立了Taqman实时定量RT-PCR方法检测诺沃克样病毒(Norwalk-likeVirus,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PrimerExpress2.0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(CrassostreagigasThunberg)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。

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