密螺旋体
多路复用
环介导等温扩增
梅毒
熔化曲线分析
分子生物学
高分辨率熔体
生物
微生物学
基因
病毒学
实时聚合酶链反应
遗传学
聚合酶链反应
DNA
人类免疫缺陷病毒(HIV)
作者
Krishnamoorthy Priya,Pasupathi Rathinasabapathi,Rex Arunraj,Sugapriya Dhanasekaran,Mohandass Ramya
标识
DOI:10.1007/s00203-022-02973-z
摘要
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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