高致病性猪繁殖与呼吸综合征病毒strand-specific荧光定量RT-PCR检测方法的建立

Development of strand-specific real-time RT-PCR for distinguishing HP-PRRSV vRNA and cRNA

为了解高致病性猪繁殖与呼吸综合征病毒(PRRSV)的复制和转录机制,准确定量其在复制过程中基因组不同类型RNA(v RNA和c RNA)的含量,本研究根据PRRSV基因组Nsp12基因设计含有链特异性标签的特异性引物,建立了一种针对病毒基因组不同链RNA的strand-specific荧光定量RT-PCR方法。结果表明,以重组质粒标准品构建的标准曲线在101拷贝/μL^107拷贝/μL范围内具有良好的线性关系,对v RNA和c RNA的检测下限为7.40拷贝/μL和6.52拷贝/μL,能够特异性的区分病毒复制过程中产生的v RNA链和c RNA链,具有很高的特异性、灵敏性和重复性。HP-PRRSV感染MARC-145细胞后不同时间段检测v RNA和c RNA含量显示,病毒不同链RNA含量在感染过程中持续增加,v RNA和c RNA含量在感染后24 h约为7.0×107拷贝/μL和7.2×106拷贝/μL。本研究建立的方法可以用于区分和定量PRRSV在复制过程中产生的不同链RNA,为研究病毒的复制和致病机理提供了一种有效的检测手段。

To differentially detect and quantify the types of viral RNA （i.e.,vRNA and cRNA） in highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） infected cells,a strand-specific real-time RT-PCR assay was established to distinguish the viral RNAs of HP-PRRSV based on the specific primers tagged Nsp12 gene.The results showed that the assay had a linear relation was in the range from l01 copies/μL to 107 copies/μL.The real-time RT-PCR ensured the specificity for quantifying the two types of RNA and features high specificity,sensitivity and repeatability.In addition,the vRNA and cRNA levels were detected in MARC-145 cells infected with HP-PRRSV at different time points post-infection （PI）.The data showed that the copy numbers of the two RNAs gradually increased throughout the infection,and the vRNA increased to 7.0× 107 copies/μL and cRNA was to 7.2 ×l06 copies/μL at 24 hours PI.These data demonstrated that strand-sprecific real-time RT-PCR could be used to quantify the different types of RNA in PRRSV infection and could be an effective method for the investigation of replicantion and pathogenesis of HP-PRRSV.