新型二聚体突变荧光引物定量检测沙眼衣原体

A novel quantitative real-time PCR assay using self-reporting duplex mutation primers for detection of Chlamydia trachomatis

目的以二聚体突变荧光引物技术为基础建立对沙眼衣原体进行定量检测的新方法。方法以沙眼衣原体主要外膜蛋白(MOMP)基因构建重组质粒作为DNA标准品,设计二聚体突变荧光引物,优化定量PCR体系并进行性能评价;同时对148例临床生殖道标本进行检测。结果建立的二聚体突变荧光引物的定量PCR方法,其线性范围为101～109 copies/μL,灵敏度为10 copies/μL;低浓度样品的批内变异系数(CV)为4.71%,批间CV为5.57%;高浓度样品的批内CV为3.20%,批间CV为3.66%;常见生殖道病原菌检测结果均为阴性;对确诊的148例患者标本检测准确率为99.32%。结论建立的沙眼衣原体定量PCR检测方法具有快速、准确、结果可靠等特点,可为沙眼衣原体感染的诊断、治疗监测和流行病学调查提供较好的技术支持。

Objective To establish a novel real-time fluorescence PCR method to detect Chlamydia trachomatis（CT） using self-reporting duplex mutation primers.Methods The recombinant vector was constructed with major outer membrane protein（MOMP） gene of CT and was used as the standard template.The self-reporting duplex mutation primers were designed according to the cloned gene sequence.The quantitative PCR reaction system was optimized and the experimental performance of real-time PCR was evaluated.A total of 148 clinical samples from tractus genitalis were detected.Results The developed method showed a wide range of linearity from 101 to 109 copies/μL and high sensitivity（10 copies/μL）.The intra-assay and inter-assay coefficient of variation（CV） in the low concentration of samples were 4.71% and 5.57% respectively,while they were 3.20% and 3.66% in the high concentration of samples.The results of common pathogenic bacteria in tractus genitalis detected by the established method were all negative,while 99.32% were positive in 148 clinical samples which were identified as Chlamydia trachomatis infection.Conclusions The established real-time quantitative PCR was reliable,accurate and feasible for detection of Chlamydia trachomatis.The assay provided complete data for diagnosis,therapeutic monitoring and epidemiologic survey of Chlamydia trachomatis infection.