猪生殖与呼吸综合征病毒的SYBR Green-Ⅰ荧光定量PCR检测

Establishment of SYBR Green-Ⅰ real-time fluorescent quantitative PCR for detection of porcine reproductive and respiratory syndrome virus

根据猪生殖与呼吸综合征病毒(PRRSV)GP5蛋白基因核酸序列设计引物,经BLAST比较发现,该引物既可以扩增较早发现的PRRSV毒株,也可以扩增近期发现的在Nsp2基因上有较大变异的PRRSV毒株,以含有该引物扩增序列的重组质粒作为标准品建立了检测PRRSV的SYBR Green-Ⅰ荧光定量PCR方法。结果表明,该方法标准曲线的相关系数大于0.99,敏感性可以达到1×101拷贝/μL,约为普通PCR的100倍;用该方法检测人工感染PRRSV的猪全血,结果阳性检出率为100%。敏感性、特异性和稳定性试验表明,该方法具有很高的敏感性、特异性和稳定性,可以用来快速检测PRRSV。

A pair of primers were designed based on the sequences of PRRSV GP5 protein gene. The amplicon was 502 bp,which was used to develop SYBR Green-Ⅰ fluorescent quantitative PCR for detecting PRRSV. The results showed that r values exceeded 0.99 in the standard curve of the developed PCR method. The detection limit of the PCR was 10 copies/μL, which was 100 fold sensitive than that of the conventional PCR. The 54 sera from the pigs infected-experimentally with PRRSV were examined by the PCR, and the positive detection rate was 100 %, This PCR assay was sensitive, specific and stable for rapid detection of PRRSV,with a positive detection rate of 100%.