胞内劳森菌TaqMan荧光定量PCR检测方法的建立

Development of a TaqMan Quantitative Polymerase Chain Reaction Assay for Detecting Lawsonia intracellularis

为建立猪胞内劳森菌的TaqMan荧光定量PCR检测方法,针对胞内劳森菌的16SrDNA基因设计合成特异性的引物和TaqMan探针,经过优化各项反应条件,建立了TaqMan荧光定量PCR检测方法。该方法仅对胞内劳森菌靶基因有信号,其相关性方程为Ct=-3.318×log(conc)+38.840(R2=0.998),具有良好的线性关系,对标准质粒最低检出量为5.55copies·μL-1,组内样品的变异系数低于2%。表明该TaqMan荧光定量PCR方法的特异性强、敏感度高、重复性好,具有较好的实用性,能准确定量DNA样品中靶基因的拷贝数,适合于对临床样品中胞内劳森菌定性、定量检测和防治效果的评价。

Based on the 16 SrDNA,specific primers and probe were designed,and a TaqMan quantitative polymerase chain reaction assay(TaqMan-PCR)was developed to determinate Lawsonia intracellularis.The results showed that the correlation equation of this TaqMan-PCR method was Ct=-3.318×log(conc)+38.840,and had a good linear relativity(R2=0.998).The TaqManPCR prove to be a sensitive,specific,reproducible,and accurate method,and can detecting as little as 5.55copies·μL-1 standard plasmid with 16 SrDNA of L.intracellularis.The new TaqManPCR could be applied for qualitative and quantitative detection of L.intracellularis from clinical sample and the evaluation of treatment effect.