Abstract
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 °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 °C and 87 ± 0.5 °C, respectively. The detection limit of multiplex HRM-LAMP was found to be 6.4 × 10–4 ng/μL (3.79 copies/μL) 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.
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Acknowledgements
The authors acknowledge the SRM Institute of Science and Technology, Kanchipuram, Tamil Nadu, for providing the research facilities.
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SRM Institute of Science and Technology, Kanchipuram, provided financial support for this project.
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Communicated by Erko Stackebrandt.
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Priya, K., Rathinasabapathi, P., Arunraj, R. et al. Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum. Arch Microbiol 204, 355 (2022). https://doi.org/10.1007/s00203-022-02973-z
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DOI: https://doi.org/10.1007/s00203-022-02973-z