猪繁殖与呼吸综合征病毒一步法多重RT-PCR检测方法的建立和应用

Development and application of one step multiplex RT-PCR method for the detection of porcine reproductive and respiratory syndrome virus

为提高实验室诊断猪繁殖与呼吸综合征病毒(PRRSV)的准确性,本研究分析了近几年国内流行的PRRSV毒株序列,针对主要流行的美洲型和欧洲型毒株设计合成特异性引物,建立PRRSV一步法多重RT-PCR检测方法,通过特异性、敏感性和重复性试验,与实时荧光RT-PCR对比检测田间样品,评价该方法的检测能力。结果显示,本研究筛选出了3对特异性引物,成功建立了PRRSV一步法多重RT-PCR检测方法,扩增目的片段大小分别为408 bp、548 bp、689 bp,至少扩增出任意一条特异性条带即可判为阳性;对口蹄疫病毒、猪瘟病毒、塞内卡病毒、非洲猪瘟病毒、猪伪狂犬病病毒、猪圆环病毒均无特异性条带扩增;检测美洲型经典毒株VR-2332、高致病性毒株GSWW15株和NADC30样毒株GSWW18的最低病毒量范围为25~50 TCID_(50)/mL;45份田间临床样品的比对检测结果显示,本研究建立的多重RT-PCR检测方法检出阳性率为33.3%,与实时荧光RT-PCR方法的检测结果符合率为95.6%。结果表明,本研究建立的PRRSV一步法多重RT-PCR检测方法特异性强、敏感性高、重复性好,可用于PRRSV流行病学的调查,具有良好的应用前景。

In order to improve the accuracy of laboratory diagnosis of porcine reproductive and respiratory syndrome virus(PRRSV),the sequences of PRRSV strains in China in recent years were analyzed in this study.Specific primers were designed and synthesized for the main American and European genotype strains prevalent in China,and a PRRSV one-step multiplex RT-PCR test method was established.The specificity,sensitivity,and repeatability tests were carried out to compare the detection ability of the method with real-time RT-PCR.The results showed that three pairs of specific primers were screened out in this study,and the one-step multiplex RT-PCR test method for PRRSV detection was successfully established for North American genotype and European genotype strains.The sizes of the target fragments were 408 bp,548 bp,and 689 bp,respectively.At least one specific band was identified as positive.There was no specific band amplification for foot-and-mouth disease virus(FMDV),classical swine fever virus(CSFV),senecavirus A(SVA),African swine fever virus(ASFV),porcine pseudorabies virus(PRV),and porcine circovirus(PCV).The minimum viral load for detecting North American genotype strains,including the classic strain,the highly pathogenic strain,and the NADC30-like strain,was 25—50 TCID_(50)/mL.The comparative results of detecting 45 field clinical samples showed that the positive rate of the multiplex RT-PCR method established in this study was 33.3%,and the coincidence rate with the detection results of the real-time RT-PCR method was 95.6%.The above results indicated that the one-step multiplex RT-PCR method established in this study has strong specificity,high sensitivity,and good repeatability,which can be used for the epidemiological investigation of PRRSV and has a good application prospect.