猪生殖与呼吸综合征病毒TaqMan荧光定量RT-PCR检测方法的建立

Development of real-time TaqMan-quantitative RT-PCR assay for detection of porcine reproductive and respiratory syndrome virus

根据GenBank登录的PRRSV保守基因序列设计合成了引物和探针,并对其进行了筛选;对荧光定量PCR的反应条件进行了优化,建立了TaqMan荧光定量RT-PCR检测方法。同时用建立的检测方法对组织病料进行了检测,并与常规RT-PCR做了对比。结果显示,所建立的TaqMan荧光定量RT-PCR方法灵敏度可达5.0×100拷贝/μL,比常规RT-PCR灵敏度高100倍。用该方法对东莞、增城、湛江等地的猪血清和多种组织样品进行了检测。结果,该方法与常规RT-PCR检测方法的阳性符合率为100%。用该方法对3份不同的组织样品进行了重复检测,结果表明,该方法具有良好的重复性,可满足当前PRRS的诊断需要。

The probes and primers were designed and synthesized according to the conserved gene ORF7 of PRRSV available in GenBank, and then reaction parameters were optimized to develop a real-time TaqMan-quantitative RT-PCR assay. The serum and other tissue samples from pigs on farms in Dongguan, Zengcheng, Zhanjiang and other areas of Guangdong Province were detected by using the established quantitative RT-PCR assay, and the results was compared with that of routine RT-PCR. The developed quantitative RT-PCR assay could detect 5.0 × 10^0 copy/μL of plasmid DNA and its sensitivity was 100 times higher than that of the routine RT-PCR, while the results of the quantitative RT-PCR were the same as that of the routine RT-PCR. Three samples were examined using the real-time RT-PCR repeatedly and the results indicated that the real-time quantitative RT-PCR was reproducible and could be used for the diagnosis of PRRSV infection.