绵羊肺炎支原体TaqMan荧光定量PCR检测方法的建立

Establishment of a TaqMan real-time fluorescent quantitative PCR assay for detection of Mycoplasma ovipneumoniae

为建立一种快速检测绵羊肺炎支原体(Mo)的检测方法,本研究根据GenBank登录的Mo Y-98株(KR021380.1)黏附素基因p113基因序列设计1对特异性引物和探针,建立了针对Mo的TaqMan荧光定量检测方法。结果显示,该方法可以特异性检测Mo,对丝状支原体山羊亚种、山羊支原体山羊肺炎亚种、莱氏无胆甾原体及羊传染性脓疱病毒(ORFV)等羊常见病原扩增结果均为阴性;该方法最低检测限为10拷贝/μL;组内和组间变异系数均小于2%;采用该方法对96份临床样品进行检测,结果 Mo的阳性率为67.7%(65/96),比常规PCR法及支原体分离鉴定法更加敏感。以上结果表明本研究建立的Mo TaqMan荧光定量PCR方法可以用于Mo的准确快速检测及羊支原体性肺炎的诊断和流行病学调查。

To develop a rapid method for detecting Mycoplasma ovipneumoniae （Mo）, the TaqMan real-time fluorescent quantitative PCR （RF-PCR） assay was established with a pair of specific primers and a TaqMan probe based on the p113 gene sequence of Mo Y-98 （KR021380.1） strain in GenBank. The results showed that the assay was specifically amplified target gene from Mo, and had no cross-amplification from Mycoplasma mycoides subsp, capri （Mmc）, Mycoplasma capricolumsubsp. capripenumoniae （Mccp）, Acholeplasmalaidlawii （AL） and Orf virus （ORFV）. The detection limit was 10 copies/μL of the Mo. The co-efficient of variations in intra- and inter-assays were both less than 2%. The method was applied to detect 96 clinical samples, and the result showed that the positive rate of 96 clinical samples was 67.7% （65/96）, which indicated that the assay was more sensitive than the conventional PCR and the isolation and identification for Mo. The above result indicated that the assay could be applied in rapid and accurate detection of Mo and diagnosis and epidemiologic investigation for Mycoplasma pneumonia of goats and sheep （MPGS）.