牛支原体TaqMan实时荧光定量PCR检测方法的建立

发布时间：2018-08-05 14:23

【摘要】：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。
[Abstract]:The aim of this study was to establish a real-time fluorescent quantitative PCR method for rapid detection of Mycoplasma bovis (Mycoplasma bovis). According to the OPPD/F gene sequence of Mycoplasma bovis contained in GenBank (accession number: AF130119), using Primer 5.0 software to design specific primers and TaqMan probes, using recombinant plasmid as absolute quantitative template, a real-time quantitative PCR detection method for Mycoplasma bovis was constructed. The results showed that the linear relationship of the method was good, the correlation coefficient of the standard curve was 0.994, and the amplification efficiency was 1.280. The sensitivity of fluorescence quantitative PCR was 10 times that of conventional PCR, the specificity was good, the detection of 9 related bacteria and viruses was negative, the reproducibility was good, and the Ct value of intra- and inter-group samples was less than 2. Compared with the conventional PCR method, the real-time fluorescence quantitative PCR method for detection of Mycoplasma bovis TaqMan has the advantages of rapidity, specificity, high sensitivity and good stability.
【作者单位】：内蒙古农业大学兽医学院;包头市农牧业局;内蒙古农业广播电视学校锡林郭勒盟分校;巴林右旗农牧业局;内蒙古大学哺乳动物生殖生物学与生物技术教育部重点实验室;
【基金】：国家科技支撑计划资助项目(2015BAI07B02-06)
【分类号】：S852.62

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