Development of TaqMan Duplex Real-time PCR for Simultaneous Detection of Chlamydia Trachomatis and Mycoplasma Genitalium

Background and Objective: Sexually infections transmitted by bacteria are one of thetherapeutic and social problemsworldwide. The Real-time PCR assay is one of the most sensitive diagnostic and screening methods for these infections. The purpose of this study wassimultaneous detection of Chlamydia trachomatis and Mycoplasma genitaliumusing the TaqMan duplex real-time polymerase chain reaction. Materials and Methods: In the present study, we used extracted DNA from women with vaginal infections available in the bank ofthe molecular and medicalresearch center, Arak University of Medical Sciences, Arak, Iran. The duplex real-time PCR was evaluated to detectChlamydia trachomatis and Mycoplasmagenitalium in 110 vaginal andendocervical samples. To validate the diagnostic performance of the designedmethod, the tests were compared with a commercial diagnostic kit. Results: The size of the ampliconsOmpA, 135 bp and MgPa, 145bp,indicated the accuracy of the primers designing procedures. The analysis of a panel of human STI revealed no cross-reactivity in the method. The diagnostic sensitivity and specificity of developedTaqMan duplex real-time PCR were 97% and 75%, respectively,compared to a commercial diagnostic kit. Conclusion: The results indicated that the used TaqMan duplex real-time PCR technique is specific and cost-effective and can be an excellent alternative to commercial kits for simultaneously detecting C. trachomatis and M. genitalium. Although the disadvantage of this method is its high cost, this disadvantage has significantly been resolved by the multiplex method.