猪繁殖与呼吸综合征病毒和牛病毒性腹泻病毒双重RT-PCR检测方法的建立

Establishment of duplex RT-PCR assay for detection of bovine viral diarrhea virus and porcine reproductive and respiratory syndrome virus

根据牛病毒性腹泻病毒(BVDV)5'端非编码区基因序列,设计合成了1对特异性引物,参考本实验室针对猪繁殖与呼吸综合征病毒(PRRSV)N蛋白设计的引物,经过PCR反应条件的优化,建立了BVDV和PRRSV双重RT-PCR的检测方法。对于PRRSV和BVDV的cDNA最低检测量分别为3.8×10-4 ng和7×10-4 ng,对于猪瘟病毒(CFSV)、脑心肌炎病毒(EMCV)和猪圆环病毒2型(PCV-2)的PCR扩增结果均为阴性;用该方法对江苏省不同地区采集的75份仔猪的肺脏、脾脏和淋巴结等病料进行了检测,结果PRRSV有55份阳性,BVDV有14份阳性,PRRSV和BVDV混合感染的有12份,与PRRSV和BVDV单一RT-PCR的检测结果符合率分别为89.3%和92%。证明建立的双重RT-PCR检测方法可用于临床样品中BVDV和PRRSV的检测。

According to the sequence of 5＇ untranslated region （ 5＇ UTR） of the bovine viral diarrhea virus （ BVDV ） genome, a pair of primers was designed and synthesized. Combined with the established primers of the N protein of porcine reproductive and respiratory syndrome virus （PRRSV） , a duplex RT-PCR was established for detection of the two viruses simultaneously based on the optimization of the reaction condition. The specific PCR products were 290 bp for BVDV and 374 bp for PRRSV in length. The detection limit of cDNA template in the RT-PCR was 3.8×10-4 ng for PRRSV and 7× 10-4 ng for BVDV. The high specificity of the method was demonstrated by detecting classical swine fever virus （ CSFV）, eneepbalomyocarditis virus （EMCV） and porcine circovirus 2 （ PCV-2 ）. Among 75 clinical samples, there were 55 samples with PRRSV positive, 14 samples with BVDV positive, and 12 samples with coinfection of BVDV and PRRSV. The coincidence rates were 89. 3% and 92% with the single RT-PCR for PRRSV and BVDV, respectively. It suggested that the duplex RT-PCR assay in this study could be applied for the detection of BVDV and PRRSV in clinical samples.