为建立同时检测非洲猪瘟病毒(ASFV)、猪瘟病毒(CSFV)和高致病性猪繁殖与呼吸道综合征病毒(HP-PRRSV)的方法,本研究根据ASFV CP530R基因、CSFV 5'UTR基因和HP-PRRSV NSP2基因,分别设计了3对特异性引物和TaqMan水解探针,建立了同时检测这3种病毒的多重荧光定量RT-PCR方法,并对其反应条件进行优化.结果表明,该方法仅对ASFV、CSFV和HP-PRRSV呈现特异性扩增,不与伪狂犬病毒、猪细小病毒和猪圆环病毒2型的DNA以及猪流行腹泻病毒和猪流感病毒的cDNA发生交叉反应;该方法对ASFV、CSFV和HP-PRRSV的最低检出量分别为61拷贝/μL、11拷贝/μL和41拷贝/μL;组内和组间重复性试验的Ct值变异系数均小于2.5％,具有良好的重现性.用该方法对276份临床样品进行ASFV、CSFV和HP-PRRSV的检测,所有样品的ASFV检测结果均为阴性,CSFV检测单阳性样品6份,HP-PRRSV检测单阳性样品22份,CSFV和HP-PRRSV检测双阳性样品4份.本研究建立的方法为临床样品中ASFV、CSFV和HP-PRRSV的同时检测提供了一种快速、敏感和特异的技术手段.%To develop an assay for simultaneous differential detection of African swine fever virus(ASFV),classical swine fever virus (CSFV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV),a multiplex real-time RT-PCR method was established with 3 pairs of specific primers targeting the sequence of ASFV CP530R gene,CSFV 5'UTR and HP-PRRSV NSP2 gene,along with 3 kinds of TaqMan probes labeled with different,distinguishable reporter dye for each virus.After optimizing the reaction conditions,the multiplex real-time RT-PCR assay was able to specifically detect ASFV,CSFV and HP-PRRSV simultaneously with the detection limit of 61 copies/μL for ASFV,1 lcopies/μL for CSFV and 41 copies/μL for HP-PRRSV,respectively,but no genomic DNA or cDNA from other virus were amplified such as pseudorabies virus.Moreover,the variation coefficient of intra-and inter-assays were both less than 2.5％.Furthermore,276 samples tested by the multiplexed assay,all samples were negative for ASFV,whilst 6 were positive for CSFV,22 positive for HP-PRRSV and 4 positive for both CSFV and HP-PRRSV.The method established in this study could provide a rapid,sensitive and specific technique for simultaneous detection of ASFV,CSFV and HP-PRRSV in clinical samples.
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