猪鼻支原体TaqMan MGB探针荧光定量PCR方法的建立

Development of TaqMan MGB probe real-time PCR for detection of Mycoplasma hyorhinis

为建立快速检测猪鼻支原体(M.hyorhinis)的荧光定量PCR方法,本研究根据GenBank中登录的p37基因序列设计并合成引物及MGB探针,构建含有p37基因的重组质粒,以其为标准品绘制标准曲线,并检测该方法的特异性、敏感性和重复性。结果显示该方法具有良好的特异性,与猪肺炎支原体、猪絮状支原体、猪滑液支原体、鸡毒支原体、副猪嗜血杆菌、猪胸膜肺炎放线杆菌、猪圆环病毒、猪瘟病毒及猪流感病毒无交叉反应,对M.hyorhinis的检测敏感性为10拷贝/μL,并且稳定性好,变异系数小于2%。利用建立的荧光定量PCR方法对55份临床样品进行检测,阳性检出率为87.3%(48/55),而分离培养方法和普通PCR的阳性检出率分别为41.8%(23/55)和29.1%(16/55)。该结果表明,建立的方法特异性强、敏感性高、稳定性好,可以用于M.hyorhinis临床样品的检测,对M.hyorhinis的快速和定量检测具有重要意义。

To establish a rapid assay for the detection of Mycoplasma hyorhinis, the TaqMan MGB real-time PCR was developed with a pair of specific primers and the MGB probe targeting the p37 gene. This detection results showed that the assay was specific for amplification of M.hyohinis with a detection limit of 10 copies, but no amplification for other related bacteria or virus, such as M.hyopneumoniae, M.flocculare, M.hyosynoviae, M.gallisepticum, H.parasuis, A.pleuropneumoniae, porcine circovirus type 2, swine fever virus and swine influenza virus. The coefficient of variation was less than 2%. In addition, fifty-five clinical specimens were detected by the real-time PCR assay and the results demonstrated the positive rate was 87.3% （48/55）, but the positive rates by bacteria isolation and conventional PCR were 41.8% （23/55） and 29.1% （16/55）, respectively. These data suggested that the real-time PCR assay targeting M.hyohinis p37 gene was of rapid, sensitive, specific, and quantifiable in M.hyohinis detection.