ORFV和Mo双重PCR检测方法的建立及应用

摘要

羊传染性脓疱病毒(Orf virus,ORFV)是羊传染性脓疱病(Contagious ecthyma,CE)的病原,绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)是羊支原体性肺炎(Mycoplasma pneumonia of goats and sheep,MPGS)的主要病原,近年来此两种病原混合感染日益增多,目前采用单一PCR方法检测这两种病原,临床上迫切需要能同时快速检测这两种病原的方法.为了建立能够直接从样品中同时检测ORFV核酸和Mo核酸的双重PCR方法,本研究根据ORFV的主要膜蛋白B2L基因(major envelope protein B2L gene)和Mo的膜蛋白P80基因(membrane protein P80 gene)序列,设计合成了2对特异性引物.结果显示,该方法可以同时扩增出ORFV的402 bp和Mo的700 bp特异性片段,而对其他常见病原的DNA模板均无扩增,显示出良好的特异性,该方法对ORFV和Mo的最低检出量分别为1.1×103 copies/μL和3.47× 103 copies/μL,比单一PCR敏感10倍.对来自福建省部分羊场的883份病山羊(Capra hircus)临床样品进行检测,双重PCR检出ORFV阳性样品28份(阳性率为33.7％),Mo阳性样品33份(阳性率为39.8％),同时感染ORFV和Mo的阳性样品9份(阳性率为10.8％),检测结果与单一ORFV和Mo PCR结果一致.本研究所建立的双重PCR方法可用于ORFV和Mo的临床检测,为CE和MPGS的临床快速鉴别诊断和流行病学调查提供了有效的方法.%Orfvirus (ORFV) is the pathogen of Contagious ecthyma (CE).Mycoplasma ovipneumoniae (Mo) is the main pathogen of Mycoplasma pneumonia of goats and sheep (MPGS).In recent years,the mixed infection of ORFV and Mo is increasing.Currently,the main detection method is single PCR,it is imperative to develop a method for rapid and simultaneous detection of ORFV and Mo in clinics.Thus,two pairs of specific primers were designed according to the major envelope protein B2L gene of ORFV and membrane protein P80 gene of Mo.The results showed that the assay could amplify 402 bp for ORFV and 700 bp for Mo simultaneously,but other common pathogen's DNA could not amplify,indicating good specificity.The detection limits of the method was 1.1 × 103 copies/μL for ORFV and 3.47 × 103 copies/μL for Mo,respectively,and these limits were 10 times more sensitive than the single PCR assay.The duplex PCR and single PCR were simultaneously performed to detect 83 clinical samples from Fujian,and the results showed that the positive rate of ORFV and Mo were 33.7％ and 39.8％,respectively,co-infection rate of ORFV and Mo was 10.8％.The date indicated that the duplex PCR established in this study can be used for clinical rapid detection of ORFV and Mo and epidemiological investigation of CE and MPGS.