猪繁殖与呼吸综合征3种毒株多重PCR检测方法的建立

Establishment of a multiplex PCR assay for differential detection of three strains of PRRSV

根据猪繁殖与呼吸综合征病毒(PRRSV)基因序列设计2对特异性引物,第1对通用引物扩增美洲型经典毒株和变异毒株Nsp2基因(466bp/376bp),第2对引物特异性扩增欧洲型毒株ORF7基因(580bp)。反应条件优化后,建立了能够同时检测美洲型经典毒株、美洲型变异毒株和欧洲型毒株的多重PCR检测方法。该方法可以特异性扩增3种毒株的基因,目的基因片段大小差异在90bp以上便于电泳观察,重组质粒标准品检测下限为1.93×103拷贝。应用该方法检测183份临床疑似病例,结果 PRRSV阳性35份,其中美洲型经典株2份,美洲型变异株31份,欧洲型毒株3份,美洲型变异株和欧洲型毒株混合感染1份。表明建立的多重PCR检测方法具有快速、准确、敏感等优点,对PRRSV 3种毒株的快速诊断有十分重要的意义。

Two pairs of specific primers were designed according to the porcine reproductive and respiratory syndrome virus gene sequence.One pair of the primers was used for the amplification of466 bp or 376 bp Nsp2gene fragment of the classical strain or the variant of the NA genotype,and the other pair of the primers was used for the amplification of 580 bp EU genotype.The method could specific amplification of three strains of PRRSV gene,the length of target gene fragment size differences were more than 90 bp,with a detection limit of 1.93×103 copies of each initial template.The established assay were used to detect PRRSV in 183 clinical samples,31 samples of the total were positive for variant PRRSV,2samples for classical PRRSV,3samples for the EU genotype,1sample for mixed infection of NA genotype and EU genotype.The Multiple PCR detection system is rapid,accurate,sensitive,for the rapid diagnosis of PRRSV three strains.