牛支原体TaqMan实时荧光定量PCR检测方法的建立

The construction of the TaqMan real-time fluorescent quantitation PCR detection method for the Mycoplasma bovis

本研究旨在建立一种快速检测牛支原体(Mycoplasma bovis)的实时荧光定量PCR检测方法。根据GenBank中收录的牛支原体OPPD/F基因序列(登录号:AF130119),利用Primer 5.0软件设计特异性引物与TaqMan探针,以重组质粒作为绝对定量模板,构建检测牛支原体的TaqMan实时荧光定量PCR检测方法。结果显示该检测方法线性关系良好,标准曲线的相关系数R2=0.994,扩增效率E=1.280。荧光定量PCR法检测的敏感性是常规PCR的10倍;特异性良好,检测9种相关细菌和病毒均为阴性;重复性好,组内及组间样本检测Ct值均小于2%。牛支原体TaqMan实时荧光定量PCR检测方法较之于常规PCR方法有快速、特异性强、敏感性高、稳定性好的优点。

The study aimed at constructing a prompt TaqMan real-time fluorescent quantitation PCR method for detection of the Mycoplasma boris. According to the gene sequence of the Myco- plasma boris OPPD/F included by the Genbank（accession number： AF130119）,Primer 5.0 was used the to design specific primer and TaqMan probe conducted the recombinant plasmid as the template of absolute quantitation. The method showed good linear relationship, with the correla- tion coefficient R^2 =0. 994 and the amplification efficiency E=1. 28 of the related standard curve. The sensitivity of the TaqMan real-time fluorescent quantitation PCR method was 10 times higher than that of the regular PCR method. The TaqMan real-time fluorescent quantitation PCR method had good specificity without cross reaction to 9 kinds of bacteria and viruses. In addition, the detected Ct values of intra-group and interblock were all lower than 2% ,with good repetitiveness. The TaqMan real time fluorescent quantitation PCR detection method for the Mycoplasma boris had better specificity,sensitivity and repetitiveness than the regular PCR.