猪繁殖与呼吸综合征病毒SYBR GreenⅠ荧光定量PCR方法的建立与应用

Establishment and application of SYBR GreenⅠreal-time PCR assay for the detection of porcine reproductive and respiratory syndrome virus

本研究根据猪繁殖与呼吸综合征病毒(PRRSV)ORF7基因序列,设计出一对引物,建立了检测该病毒的SYBR GreenⅠ染料荧光定量PCR方法。此法特异性高,与猪流行性腹泻(PEDV)、猪瘟病毒(CSFV)、塞内卡谷病毒(SVV)、脑心肌炎病毒(EMCV)、伪狂犬病病毒(PRV)和猪圆环病毒2型(PCV2)均无交叉反应;采用不同滴度的PRRSV测定,当Ct≤32且有明显的指数扩增曲线,判定为PRRSV阳性,Ct>32或者无Ct值则判定为PRRSV阴性。该方法检测下限为1×10^0.5TCID50病毒,高于常规RT-PCR国家标准(GB/T18090-2008),批内和批间变异系数均小于1%。PRRSV人工感染仔猪15份肺脏组织和60份血清样品检测结果均为阳性,SPF仔猪8份肺脏组织和25份血清检测结果均为阴性。采集245份临床仔猪肺脏组织病料检测,PRRSV阳性率为67.35%,明显高于普通RT-PCR检测阳性率(56.33%)。上述结果表明,本方法可用于PRRSV临床病料检测。

In this study,a pair of primers was designed based on the porcine reproductive and respiratory syndrome virus(PRRSV) ORF7 gene sequence,and a quantitative PCR method for SYBR Green I dye was established.The method is highly specific,and porcine epidemic diarrhea(PEDV),swine fever virus(CSFV),Seneca virus(SVV),encephalomyocarditis virus(EMCV),pseudorabies virus(PRV) and porcine circovirus 2 type(PCV2) had no cross-reaction;using different titer PRRSV measurement,when Ct≤32 and obvious exponential amplification curve,it was judged as PRRSV positive,Ct≥32 or no CT value was judged as PRRSV negative.The detection limit of this method is 1×10^0.5 TCID50 virus,which is higher than the national standard of conventional RT-PCR(GB/T18090-2008),and the intra-and inter-assay coefficient of variation is less than 1%.The 15 lung samples and 60 serum samples of PRRSV artificially infected piglets were positive.8 lung samples and 25 serum samples of SPF piglets were negative.The lung tissue samples in 245 clinical piglets were detected, and the positive rate of PRRSV was 67.35 %, which was significantly higher than that of ordinary RT-PCR(56.33%).The above-mentioned results showed that this method can be used for PRRSV clinical sample detection.