猪脑心肌炎病毒和猪繁殖与呼吸综合征病毒二重RT-PCR检测方法的建立

根据猪脑心肌炎病毒(EMCV)3D基因和猪繁殖与呼吸综合征病毒(PRRSV)N蛋白基因序列设计合成了两对特异性引物,经过PCR反应条件的优化,建立了EMCV和PRRSV二重RT-PCR方法,其PCR产物大小分别为286 bp和390 bp,并具有EMCV和PRRSV特征序列。该方法对EMCV和PRRSV的最低检测量分别为10×TC ID50和1×TC ID50;而对乙型脑炎病毒、猪瘟病毒、猪细小病毒、猪圆环病毒2型、猪伪狂犬病毒的PCR检测结果均为阴性;20份临床发病仔猪样品检测结果为PRRSV阳性者15份,EMCV阳性者3份,证明我国存在EMCV感染。该方法敏感、特异,可用于EMCV和PRRSV感染的检测。

Developmemt of a duplex RT-PCR assay for detection of encephalomyocarditis virus and porcine reproductive and respiratory syndrome virus

Based on the sequences of 3D of encephalomyocarditis virus(EMCV) and N gene of porcine reproductive and respiratory syndrome virus(PRRSV),two pairs of primers were designed and synthesized,and a duplex RT-PCR method was established for detection of the two viruses simultaneously.Two specific PCR products,286 bp for EMCV and 390 bp for PRRSV in length,were simultaneously amplified from the two viruses.The sensitivity and specificity tests showed that the assay could detect 10TCID50 of EMCV and 1TCID50 of PRRSV.There was no cross-reactions with those clinically relevant pathogens,ie.porcine parvovirus,porcine pseudorabies virus,porcine circovirus type 2,hog cholera virus and Japanese encephalitis virus.The detection results for 20 clinical samples with the duplex RT-PCR showed that 15 samples were positive for PRRSV and 3 for EMCV,which were identical to those with single RT-PCR.It suggested that EMCV exists in China and the duplex RT-PCR established in this study could be used as a detection method for coinfections with EMCV and PRRSV.