Saikaew S.Sangboonruang S.Pongsararuk R.Srilohasin P.Butr-Indr B.Intorasoot S.Phunpae P.Tharinjaroen C.S.Arunothong S.Panyasit W.Chaiprasert A.Tragoolpua K.Wattananandkul U.Mahidol University2025-10-042025-10-042025-09-01Biosensors Vol.15 No.9 (2025)https://repository.li.mahidol.ac.th/handle/123456789/112438Tuberculosis (TB) remains a major global health challenge, particularly in resource-limited settings where access to rapid and reliable diagnostics is limited. Conventional diagnostic methods, such as smear microscopy and culture, are either time-consuming or lack adequate sensitivity. This study optimized recombinase polymerase amplification (RPA) using 16 primer combinations targeting IS6110 highly specific to the Mycobacterium tuberculosis complex (MTC). A novel naked-eye assay, TB-GoldDx, was developed by integrating RPA combined with gold nanoparticles (AuNPs), enabling equipment-free diagnostics. TB-GoldDx demonstrated a detection limit of 0.001 ng of MTB H37Rv DNA (~210 bacilli) per 25 µL reaction. Among 100 bacterial strains, it achieved 95.83% sensitivity and 100% specificity among 100 bacterial strains, comprising 72 MTB isolates and 28 nontuberculous bacterial species. In 140 sputum samples, the assay showed 81.43% sensitivity and 58.57% specificity versus acid-fast bacilli (AFB) smear microscopy, with sensitivity improving to 95.45% in high-load AFB 3+ specimens. Compared to a commercial line probe assay (LPA), TB-GoldDx exhibited slightly higher sensitivity (84.78% vs. 82.61%) but lower specificity (54.05% vs. 78.38%). Delivering rapid, visual results in under an hour, TB-GoldDx offers a low-cost, easily deployable solution for point-of-care tuberculosis detection, especially in underserved regions, reinforcing global End TB efforts.ChemistryBiochemistry, Genetics and Molecular BiologyPhysics and AstronomyEngineeringArticleSCOPUS10.3390/bios150906072-s2.0-1050171860282079637441002347