绵羊肺炎支原体实时荧光定量PCR检测方法的建立

Establishment of Real-time Fluorescent Quantitative PCR Assay for Detection of Mycoplasma ovipneumoniae

为研究绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)侵害山羊的组织嗜性、筛选其最佳培养基及建立快速检测方法,针对Mo16SrRNA基因设计特异性引物FQs/FQa,以Mo Y98株16SrRNA基因重组质粒pMD19-MoFQ为模板,通过优化反应体系构建标准曲线,建立可定量检测Mo 16SrRNA基因的FQ-PCR方法,并对所建方法进行重复性、特异性、敏感性及应用性检测。结果表明,所建Mo 16SrRNA基因检测FQ-PCR方法具有较好的重复性、特异性、临床应用性,Mo核酸拷贝最低检出量可至10μL-1。该方法不仅可作为Mo临床检测方法,且为后续Mo研究工作提供技术支持。

To stu screen the best c dy the situation of organ of goat which infected Mycoplasma ovipneumoniae （Mo）, ulture medium and establish detecting method, a real-time fluorescent quantitative PCR assay for detection of Mo was established by optimizing reaction condition and constructing standard curve after specific primers designed and recombinant plasmid pMD18-MoFQ of positive tem- plate prepared. The results showed the assay had good repeatability and specificity. It could detect low to 10 copies/μL of template. The establishment of FQ-PCR not only supplied a detecting assay of Mo, but also provided technical support for the future research.