百日咳鲍特菌BP283基因的实时荧光定量PCR检测方法的建立

摘要

目的 探讨检测百日咳鲍特菌BP283基因片段的有效方法.方法 以百日咳鲍特菌BP283序列为目的基因,设计探针和引物,构建质粒标准品；建立FQ-PCR体系,优化反应条件,并进行方法学评价.结果 成功构建了重组质粒pCR2.1-BP283；建立了检测BP283基因片段的FQ-PCR方法:标准曲线相关系数为0.998,最低检测浓度为102 copies/μL,对临床其他常见呼吸道病原体不出现特异性扩增曲线,批内及批间变异系数均＜4％.结论 FQ-PCR方法可成功检测百日咳鲍特菌BP283基因,该方法具有敏感性好、特异性高及重复性好等优点.%Objective To evaluate the effective method for detection of Bordetella pertussis BP283 gene. Methods The primers and Taqman probe were designed according to the sequence BP283 of Bordetella pertussis, and the standards were constituted; then the real time fluorescent quantitative PCR(FQ-PCR) reaction system was optimized and evaluated. Results The recombinant vector (pCR2. 1-BP283) was cloned successfully; the FQ-PCR was well established; the corre-lation coefficient of standard curve was 0.998, the minimum detection limit concentration was 10 copies/μL , no specific amplification curve for the other common clinical pathogen in respiratory tract was found, both intra and inter assay coeffi-cients of variation were lower than 4% . Conclusion FQ-PCR can detect Bordetella pertussis BP283 gene successfully with good sensitivity, specificity and reproducibility.