同时检测美洲型及欧洲型PRRSV的多重RT-PCR检测方法的建立及应用

Establishment and application of a multiplex RT-PCR assay for differential detection of the North American genotype and the European genotype PRRSV

为建立猪繁殖与呼吸综合征病毒(PRRSV)的快速鉴别检测方法,本研究针对PRRSV基因序列设计3对特异性引物,即第1对通用引物扩增美洲型和欧洲型病毒株ORF7基因(433 bp/398 bp)、第2对引物扩增美洲型经典病毒株和高致病性变异株(HP-PRRSV)Nsp2基因(338 bp/248 bp)、第3对引物扩增欧洲型病毒株ORF5基因(614 bp)。经过反应条件的优化,建立了能够同时检测并区分美洲型经典病毒株、HP-PRRSV株以及欧洲型病毒株的多重RT-PCR检测方法。该方法可以特异扩增3种病毒的基因,其重组质粒标准品的检出下限均为1.67×103拷贝;而与猪的其它重要病原均无交叉反应。应用该方法检测176份临床疑似病料样品,结果 PRRSV阳性45份,其中美洲型变异病毒株41份、经典病毒株4份,未检测到欧洲型病毒株。表明本研究建立的多重RT-PCR检测方法可以用于PRRSV的快速鉴别诊断和流行病学调查。

In this study, a multiplex RT-PCR was established for differential detection of the North American （NA） genotype and the European （EU） genotype porcine reproductive and respiratory syndrome virus （PRRSV）. The assay utilized three pairs of specific primers, of which one amplified 433 bp/398 bp ORF7 gene fragment of the NA genotype and the EU genotype, the other amplified 338 bp or 248 bp Nsp2 gene fragment of the classical strain or the highly pathogenic （HP-PRRSV） variant of the NA genotype, and the another amplified 614 bp ORF5 gene fragment of the EU genotype. It was able to specifically amplified from PRRSV, but not with other important porcine pathogens. It was highly sensitive with a detection limit of 1.67 x 103 copies of each initial template. The established assay were used to detect PRRSV in 176 clinical samples, of which 41 samples were positive for HP-PRRSV, 4 samples for classical PRRSV, while no sample for the EU genotype. The results indicated that this assay was suitable for rapid detection and epidemic surveillance of PRRSV.