Abstract：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.