基于TaqMan探针的幽门螺杆菌实时荧光定量PCR检测方法的建立

Development of TaqMan-probe system based on real time florescent quantitative PCR for Helicobacter pylori detection

目的探讨基于TaqMan探针的幽门螺杆菌23SrDNA基因片段的实时荧光定量PCR检测的有效方法。方法根据幽门螺杆菌23SrDNA序列设计引物和探针,构建质粒标准品,建立实时荧光定量PCR体系并进行方法学评价,并对2012年5月至10月收集的100份临床样本进行检测同时应用免疫组化及银染方法进行比较分析。结果建立了检测23SrDNA的实时荧光定量PCR方法:107-102copies/ul范围内均有"S"型扩增曲线,最低检测浓度为102copies/ul,标准曲线相关系数为1。对临床其它消化道病原体不出现特异性的扩增曲线。对100份样本进行检测,实时荧光定量PCR能检出31份阳性,而免疫组化方法能检出25份阳性,银染方法能检出28份阳性。结论实时荧光定量PCR方法可成功检测幽门螺杆菌,且该方法具有灵敏度和特异性高及重复性好等优点。

Objective Develop a rapid and reliable method for Helicobacter pylori detection based on real time florescent quantitative PCR（FQ-PCR）.Methods The specific PCR primers and Taqman probe for Helicobacter pylori detection were designed based on its 23s rDNA sequence,the FQ-PCR reaction system was optimized and evaluated,then 100 samples were used for both FQ-PCR detection and immunohistochemistry detection.Results FQ-PCR reaction system was optimized and evaluated： FQ-PCR assay had a detection limit of102copies,and with a dynamic range of detection between 107 copies to 102copie,The correlation coefficient of standard curve was 1,no specific amplification curve for the other common pathogen in alimentary canal was found.There were 31 positive samples detected by FQ-PCR,while 25 positive samples detected by immunohistochemistry,28 positive samples detected by W-S silver staining.Conclusion Real time florescent quantitative PCR can detect Helicobacter pylori successfully with good sensitivity,specificity and reproducibility.