梅毒螺旋体荧光定量PCR检测方法的建立和初步临床应用

Development and clinclal application of a novel fulorescent quantitative PCR method for treponema detection

目的建立一种利用MGB-Taqman探针的快速、灵敏、特异、准确定量检测梅毒螺旋体的方法。方法选择梅毒螺旋体的基因组的保守区域,设计合成引物和MGB-Taqman探针,构建质粒标准品pMD18-T-TP,优化定量PCR反应体系,并进行方法学评价。结果1)成功构建了重组质粒pMD18-T-TP。2)建立了利用MGB-Taqman探针的荧光定量PCR方法,其线性范围:101～1010copies/μl;灵敏度:10copies/μl;重复性:批内CV为2.02%,批间CV为2.83%,日间CV为4.23%;特异性:100%。3)初步临床应用证明:该方法比血清学检测方法灵敏度更高,假阳性率低。结论利用MGB-Taqman探针定量检测梅毒螺旋体的方法灵敏度高,特异性高,操作简便,适合临床检验诊断的要求。

Objective To establish a novel real-time fluorescence PCR method to detect treponema with high sensitivity, specificity using the MGB Taqman probe. Method The recombinant vector pMD 18-T-HCV TP was used as the standard template. The MGB Taqman probe was designed according to the cloned gene sequence. And then the PCR reaction system was optimized and evaluated. Results 1） The recombinant vector was cloned successfully. 2） Methodological analysis showed the MGB Taqman probe detecting system was well established with wide detecting range, high sensitivity, specificity.3） With the results of clinical experiments, this novel fluorescent quantitative method had more advantages than serological methods. Conclusion This novel real time fluorescence quantitative PCR method using MGB Taqman probe for TP detection is more sensitive, specific and simple, which meets the need for clinical aonlieation.