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Updated: Sep 21, 2025
Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1
Published on: March 13, 2018
Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection
Krishnamoorthy Priya1, Pasupathi Rathinasabapathi1, Rex Arunraj1
1Molecular Genetics Laboratory, Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur, Kanchipuram District, Chennai, Tamil Nadu, 603203, India.
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.
