猪繁殖与呼吸道综合征病毒荧光定量PCR检测方法的研究

Establishment of Fluorescent Quantitative PCR Assay by TaqMan 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;利用该方法对3份不同的组织样品进行重复性检测,计算出平均循环阈值的标准差分别为0.14、0.14和0.44,变异系数分别为0.01%、0.01%和0.02%,说明该方法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的阳性符合率为100%,但其检测灵敏度较常规RT-PCR约高100倍。

The recombined plasmid pMD-ORF7 was constructed and used as standard positive template. And the FQ-PCR assay was constructed by quantitative concentration of serial 10 fold dilutions of pMD-ORF7 cDNA by optimizing circulation parameters.A standard curve was a- chieved. The result showed the sensitivity of this method was 1.0 ×10 ×^0copy/μl. To detect three different samples by FQ-PCR repeatedly, the av- erage circle threshold value was 0. 14, 0. 14 and 0.44, with the coefficient of variation of 0.01%, 0.01% and 0.02%, respectively. The FQ- PCR was proved to be with high sensitivity and repeatability. The detection of PRRSV in positive samples by both FQ-PCR and conventional RT- PCR was 100%, but the sensitivity of the former was 100 times higher than that of the latter.