基于Taq Man探针的猪繁殖与呼吸综合征病毒荧光定量PCR检测方法的研究

Establishment of fluorescent quantitative PCR assay by Taq Man probe approach for PRRSV detection

通过条件优化,以定量的10倍系列稀释的质粒pMD-ORF7为标准品进行荧光定量PCR扩增并制作标准曲线建立了PRRSV的荧光定量RT-PCR检测方法。结果表明,该方法检测灵敏度可达1.0×100拷贝/μL;用该方法对6份不同的组织样品进行重复性检测,结果显示具有良好的重复性和重现性。对阳性组织病料的检测表明该方法与常规RT-PCR阳性符合率为100%,但检测灵敏度高出常规RT-PCR 100倍。

The PRRSV ORF7 genome was cloned into pMD-18 T vector and the recombinant plasmid pMD-ORF7 was constructed and used as standard positive template. Meanwhile,the plasmid pMD-ORF7 was identified by sequencing. The FQ-PCR assay was carried out by serial 10 fold dilutions of pMD-ORF7 cDNA and optimizing cycle parameters. A standard curve was achieved and the result showed the sensitivity of this method was 1.0×10^0copy/μL. Meanwhile,six different samples were detected by the FQ-PCR repeatedly. The result showed that the sensitivity of FQ-PCR was roughly 100 times greater than that of RT-PCR for detecting of PRRSV in positive samples.