Development a rapid and accurate multiplex real time PCR method for the detection Chlamydia trachomatis and Mycoplasma hominis

Abstract

Background: Sexually transmitted diseases easily spread among sexually active people and often have no symptoms. Rapid and accurate method for detecting these infections are necessary in early stages. The traditional detection methods of them are difficult and time-consuming.

Methods: In this study, multiplex real time PCR was optimized for rapid identification of Chlamydia trachomatis and Mycoplasma hominis in a single tube and was performed with our designed primers. The sensitivity test was carried out to designed primers with diluted genomic DNA. To defined the specificity, non STD bacteria were used as DNA template.

Results: This study indicated that the developed multiplex real time PCR can be an effective alternative procedure to the conventional methods for rapid and accurate identification of C Chlamydia trachomatis and Mycoplasma hominis. Multiplex real-time PCR Results of them were checked with melting curves. The sensitivity of our designed primer by multiplex real time PCR for Chlamydia trachomatis and Mycoplasma hominis were 4.78×10¹⁰ and 8.35×10¹⁰ , respectively, Which the primers did not amplify any product from a non-STD species.

Conclusions: Multiplex real time PCR by our new primers and analysis of melting curves were successfully usable for rapid and accurate detection of Chlamydia trachomatis and Mycoplasma hominis. This assay instead of traditional culture method, has considerable potential to be rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous and direct detection.

Keywords: Chlamydia trachomatis; Mycoplasma hominis; multiplex real time PCR.

Figure 1

Melting curve analysis of multiplex real‐time PCR. Melting curves for the selected genes of the 2 STDs in a singleplex PCR reaction for Chlamydia trachomatis (A), Mycoplasma hominis (B) and Duplex of C. trachomatis and M. hominis (C)