副猪嗜血杆菌荧光定量PCR检测方法的建立

Development of Fluorescent Quantitative PCR for Detection of Haempohlius parasuis

为了建立基于SYBR Green Ⅰ的快速检测副猪嗜血杆菌(HPS)的实时荧光定量PCR方法,参照GenBank公布的HPS的infB基因序列(DQ:781933.1),设计特异性引物;提取HPS基因组,进行PCR扩增,回收目的片段;构建阳性标准模板,建立标准曲线;验证其敏感性、重复性和特异性。结果显示,本试验得到了阳性标准质粒,并建立了标准曲线,线性关系在102copies/μL～108copies/μL检测范围内良好;该方法敏感性达到10copies/μL,比常规PCR高100倍,重复性试验变异系数均小于2.5%,同时对HPS具有良好的特异性。结果表明,本试验建立的副猪嗜血杆菌SYBR GreenⅠ荧光定量PCR检测方法可用于临床对HPS的快速诊断和定量检测。

To establish a fluorescent quantitative PCR based on SYBR Green Ⅰ for rapid detection of Haempohlius parasuis,a pair of primers were designed specificly for infB gene of HPS according to the published nucleotide sequence（GenBank accession №：781933.1）.The target fragment was amplified using routine PCR and retrieved from extracted DNA of HPS.Positive recombinant plasmid was built and used as a positive quantitative template to establish a standard curve.Then the sensibility,repeatability and specificity of the assay were tested.The results showed that this assay obtained positive recombinant plasmid and established a standard curve.The linear relation was fine and the wide dynamic range was from 102 to 108copies/μL.The detection limit of this assay attained 10 copies/μL,which was 100 times higher than that of routine PCR.The CV of the intra repeatability test was not more than 2.5%,indicating that the developed fluorescence quantitive PCR has stability.Meanwhile the fluorescenee quantitative PCR has specificity able to detect HPS.Finally,the assay demonstrated that the development of SYBR GreenⅠfluorescent quantitative PCR for detection of Haempohlius parasuis could be used for the rapid diagnosis and quantitative detection of HPS in the clinic.