猪肺炎支原体P97 TaqMan-BHQ荧光定量PCR检测方法的建立及应用

Development and application of TaqMan-BHQ real time PCR assay for detection of Mycoplasma hyopneumoniae P97

根据猪肺炎支原体(Mhp)P97基因的保守序列设计合成了引物和TaqMan-BHQ探针,建立了快速检测Mhp的TaqMan-BHQ荧光定量PCR方法。结果显示,该方法特异性良好,与其他病原不发生交叉反应;最低检测限可达10copies/μL,敏感性高;不同梯度定量模板的拷贝数的对数值与Ct值之间的线性关系表达式为Ct=-3.426×lg(conc)+43.343,有良好的线性关系,标准曲线的相关系数为0.997,扩增效率为95.85%,重复性良好。对江苏省某猪场的48份猪肺组织进行荧光定量PCR检测,结果有43份肺组织呈现阳性,阳性检出率为89.58%,比巢式PCR的敏感性高。表明,该检测方法能用于Mhp的快速检测,可对猪支原体肺炎的早期诊断和流行病学调查提供新的技术手段。

A pair of primers and TaqMan-BHQ probe were designed according to the Mycoplasma hyopneumoniae（Mhp） P97 sequence,and TaqMan-BHQ real time PCR assay was successfully established for detection of Mhp.In result,the developed method could specifically detect Mhp,and had no cross-reaction with pathogens of other swine diseases.There was an excellent linear correlation during the DNA concentration from 101 copies/μL to 107 copies/μL.The analytic sensitivity of the assay was 10 copies/ μL.The equation of the assay was Ct=-3.426×lg（conc）＋43.343,the correlation coefficient of the standard curve was 0.997,and amplification efficiency was 95.85% with good repeatability.The 48 pig lung tissues from pig farms in Jiangsu Province were detected,and the positive rates were 89.58%.The result showed that the TaqMan-BHQ real time PCR can be useful for rapid detection of Mhp in clinic detection and for epidemic investigation of mycoplasmal pneumonia of swine in field study.