绵羊痘病毒和山羊痘病毒双重PCR检测方法的建立及应用

Differentiation of sheeppox virus and goatpox virus by duplex PCR

为建立鉴别绵羊痘病毒(SPPV)和山羊痘病毒(GTPV)的检测方法,本研究针对这2种病毒的基因组序列,分别设计2对特异性引物,通过对引物浓度、退火温度等的优化,建立了快速鉴别检测SPPV和GTPV的双重PCR方法。该方法分别扩增出SPPV长度为177 bp和GTPV长度为222 bp的目的片段。特异性试验结果显示,该方法对牛疙瘩皮肤病病毒、犬细小病毒、大肠杆菌O157、沙门氏菌、健康羊组织和牛组织均无扩增。敏感性试验显示,该方法最低可检测1.725×107copies/μL的SPPV和1.71×106copies/μL的GTPV。应用该方法对50份临床病料样品进行检测的结果与病毒分离鉴定结果一致,均检出5份感染GTPV的病料和2份感染SPPV的病料,表明该方法可以用于临床病料样品的检测。

To established differential detection method for sheeppox virus （SPPV） and goatpox virus （GTPV）, a duplex PCR was developed with two pairs of specific primers designed according to SPPV and GTPV sequences. Under optimized reaction conditions, two specific fragments of 177 bp （SPPV） and 222 bp （GTPV） were amplified by the duplex PCR with a detection limit of 1.725 x 107 copies for SPPV and 1.71 x 106 copies for GTPV. The specific tests indicated that no product was amplified from lumpy skin disease virus, canine parvovirus, E. coli O157, Salmonella. Tested on 50 clinical samples, of which 5 positive for GTPV and 2 positive for SPPV by either the duplex PCR or virus isolation.