大鼠微小病毒和细小病毒双重荧光定量PCR检测方法建立

摘要

目的 建立快速、准确地同时检测和鉴别大鼠细小病毒(RPV)和大鼠微小病毒(RMV)的双重TaqMan实时荧光定量PCR方法.方法 根据GenBank中已公布的RMV NTU1株全基因组序列(JX627317.1),在1 705～1 808 nt处设计引物和TaqMan探针,根据RPV NTU1毒株的全基因组序列(JX827169),在863-967nt处设计引物和TaqMan探针.以构建的质粒参考物为模板建立荧光定量PCR检测方法,并对该鉴别检测方法的灵敏度、稳定性和特异性进行评价;用建立的荧光定量方法对50份临床样品进行检测,并用酶联免疫吸附试验(ELISA)的商品试剂盒进行验证.结果 建立的RMV和RPV的鉴别荧光定量PCR方法标准曲线的线性关系良好,R2值可达0.99,最低检测限均为10拷贝数/μL;应用鉴别荧光定量PCR方法和ELISA检测试剂盒,对100份大鼠肝脏和50份血清样品病料进行检测,结果均为阴性.结论 建立的RMV和RPV的TaqMan实时荧光定量PCR方法具有特异、灵敏的特点,适用于RMV和RPV的临床监测.%Objective To establish TaqMan real-time fluorescent quantitative PCR method which can detect rat minute virus (RMV) and rat parvovirus (RPV) quickly and accurately in clinic.Method According to the whole genome sequence of strain RMV NTU1 (Accession No.JX627317.1 in Genbank) the primers and TaqMan probe were designed from the 1705～1808 nt.And according to the whole genome sequence of strain RPV NTU1 (JX827169) the primers and TaqMan probe were designed from the 863-967nt.The stability,specificity,and sensitivity of the method were evaluated through realtime quantitative PCR method,which is based on the standardized plasmid constructed.50 clinical samples were detected using this fluorescence quantitative method,which validated with the traditional ELISA method.Result The real-time quantitative PCR for detecting RMV and RPV showed a perfect linear relationship of standard curve,and R2 value reached 0.99 with a high specificity.The sensitivity of the real-time PCR was 10 copies/μL at minimum.Due to dual specificity of primer and probe,TaqMan quantitative PCR is extremely accurate.One hundred liver samples and 50 serum samples were negative via ELISA and real time quantitative PCR respectively.Conclusion The TaqMan probe-based real-time PCR method is established with good specificity and sensitivity,which can make a powerful technical support for RMV and RPV investigation and detection.