Department of Microbiology, ESIC MC PGIMSR & MH, Rajajinagar, Bengaluru, 560010, Karnataka, India.
Department of Obstetrics and Gynaecology, ESIC MC PGIMSR & MH, Rajajinagar, Bengaluru, 10, India.
Indian J Med Microbiol. 2024 May-Jun;49:100610. doi: 10.1016/j.ijmmb.2024.100610. Epub 2024 May 14.
Trichomoniasis is a curable, non-viral, sexually transmitted infection. Early diagnosis and treatment of cases can prevent complications and further spread of infection. Rapid diagnostics tests, which can be performed on-site, will help in early diagnosis. The study aims to develop a rapid diagnostic test based on the principle of fluoro-colorimetric LAMP for detecting Trichomonas vaginalis (TV).
T. vaginalis was grown in a modified CPLM medium, and DNA was extracted. Three pairs of LAMP primers targeting the actin gene were designed using the primerexplorer V.5 online tool. The LAMP assay was standardized for temperature and time. To determine the LAMP assay's detection limit, diluted TV DNA and spiked urine samples were used. Conventional PCR was done using previously published primers and compared with LAMP results. The sensitivity and specificity to detect TV from clinical specimens were assessed.
The optimum performance of the LAMP assay was determined to be 63 °C for 60 min and terminated at 80 °C for 5 min. The LAMP assay could detect 60 fg/μl of diluted TV DNA and up to 1 parasite/ml of spiked samples. The assay was 1000 times more sensitive than PCR. The LAMP assay was 100% sensitive and specific with crude extract, and reactions were visually discernible.
INTERPRETATION & CONCLUSIONS: The LAMP assay developed in the study is easy to perform and interpret, affordable, rapid, and highly sensitive to detect T. vaginalis. It is ideally suited for the point-of-care test, as it fulfills WHO's recommended ASSURED characteristics.
滴虫病是一种可治愈的、非病毒性的性传播感染。早期诊断和治疗病例可以预防并发症和进一步传播感染。现场进行的快速诊断测试将有助于早期诊断。本研究旨在开发一种基于荧光比色环介导等温扩增（LAMP）原理的快速诊断测试，用于检测阴道毛滴虫（TV）。
在改良的 CPLM 培养基中培养 TV，并提取 DNA。使用在线工具 primerexplorer V.5 设计了针对肌动蛋白基因的三对 LAMP 引物。对 LAMP 检测进行了温度和时间的标准化。为了确定 LAMP 检测的检测限，使用稀释的 TV DNA 和掺入的尿液样本进行了检测。使用先前发表的引物进行了常规 PCR，并与 LAMP 结果进行了比较。评估了从临床标本中检测 TV 的灵敏度和特异性。
确定 LAMP 检测的最佳性能为 63°C 60 分钟，80°C 终止 5 分钟。LAMP 检测可以检测到 60fg/μl 的稀释 TV DNA 和高达 1 个寄生虫/ml 的掺入样本。该检测比 PCR 灵敏 1000 倍。LAMP 检测对粗提物具有 100%的灵敏度和特异性，且反应肉眼可辨。
本研究中开发的 LAMP 检测易于操作和解释，价格实惠，快速，对检测 TV 高度敏感。它非常适合即时检测，符合世卫组织推荐的 ASSURED 特性。
