山羊和绵羊嗜血支原体PCR检测方法的建立与应用

摘要

为建立羊嗜血支原体(M.ovis)病快速检测方法,本实验根据GenBank中登录的羊M.ovis 16S rRNA基因序列设计一对引物,以山羊和绵羊M.ovis基因组DNA为模板,建立M.ovis PCR检测方法,并进行特异性、敏感性及临床应用试验.结果显示,建立的M.ovis PCR检测方法扩增片段大小为508 bp,与GenBank中M.ovis参考株同源性达99％以上,该方法与猪嗜血支原体、牛嗜血支原体等病原体无交叉反应,最低检测40个拷贝的DNA;通过对延边地区60份山羊和绵羊血液样本的检测结果表明,建立的PCR检测方法具有特异、敏感、准确等优点,完全适用于M.ovis的检测.%In order to establish a rapid diagnostic method for Mycoplasma ovis,a pair of primers was designed based on the gene sequence of 16S rRNA of M.ovis (AF338268).A 508 bp DNA fragment was amplified from the genomic DNA extracted from blood samples of goats and sheep naturally infected with M.ovis in Yanbian,Jilin province.The results showed that the gene sequence shared more than 99％ homology with the gene sequences of M.ovis reference strains in GenBank.Moreover,this assay had no cross reactivity with related pathogens,including Mycoplasma suis and Mycoplasma wenyonii.The minimum detectable limit was about 40 copies.In addition,a total of 60 blood samples collected from goats and sheep in Yanbian area were detected and analyzed with this method,and the result indicated that the developed PCR diagnostic test in this study was specific,sensitive,accurate,and suitable for the diagnosis of M.ovis infection.