Multiplex Loop-Mediated Isothermal Amplification Coupled Lateral Flow Assay for Point-of-Care Detection of Syphilis

Syphilis is a re-emerging sexually transmitted disease caused by the pathogenic spirochete T. pallidum. Every year more than 5 million cases are reported globally. The current diagnostic methods are primarily based on serological assays, which are less sensitive at an early stage of infection. To improve the disease diagnosis, there is a need to develop a rapid, simple, sensitive, and cost-effective point-of-care application, which plays an effective role in the detection of syphilis infection. In this study, we developed a multiplex loop-mediated isothermal amplification coupled lateral flow assay (multiplex LAMP-LFA) for the detection of syphilis. Two different genes, the target amplicon (polA) and the internal control amplicon (human RNase P) were amplified using multiplex LAMP assay. The amplified products were detected using LFA strips coated with Anti-FITC and Anti-DIG antibodies within 5 minutes of flowthrough. Multiplex LAMP LFA detection limit was found to be 3.8 × 10³ copies/mL with high specificity. The developed strip was tested with 130 clinically suspected cases and 50 healthy individuals. With the clinical samples, the method shows a sensitivity of 93.84% and a specificity of 100%. The Multiplex LAMP LFA has the potential to overcome the limitations of both Non Treponemal tests and Treponemal tests which are prone to prozone effects and expensive reagents respectively. The proposed method holds promise for sensitive, rapid, and visual detection of T. pallidum, thereby offering a facile and affordable alternative to existing diagnostic methods. This approach is poised to advance the development of point-of-care diagnostics, addressing a critical need in public healthcare, particularly in resource-limited settings.

The online version contains supplementary material available at 10.1007/s12088-024-01308-4.