Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum

Syphilis is a sexually transmitted disease caused by the spirochaete bacterium Treponema pallidum. This study has developed a multiplex High-Resolution Melt-curve Loop-mediated isothermal amplification (multiplex HRM-LAMP) assay targeting the marker genes polA and tprL to detect T. pallidum. The multiplex HRM-LAMP assay conditions were optimized at 65 degrees C for 45 min. Real-time melt-curve analysis of multiplex HRM-LAMP shows two melt-curve peaks corresponding to polA and tprL with a Tm value of 80 +/- 0.5 degrees C and 87 +/- 0.5 degrees C, respectively. The detection limit of multiplex HRM-LAMP was found to be 6.4 x 10(-4) ng/mu L (3.79 copies/mu L) of T. pallidum. The specificity was evaluated using seven different bacterial species, and the developed method was 100% specific in detecting T. pallidum. A total of 64 blood samples of T. pallidum suspected cases were used to validate the assay method. The clinical validation showed that the assay was 96.43% sensitive and 100% specific in detecting syphilis. Thus, the developed method was more rapid and sensitive than other available methods and provides a multigene-based diagnostic approach to detect T. pallidum.