Novel Target for Pathogenic Leptospira Detection via Colorimetric Loop‐Mediated Isothermal Amplification ( LAMP ): Development, Validation, and Comparative Analysis

ABSTRACT Background Leptospirosis is a globally prevalent zoonotic disease that remains challenging to diagnose due to its nonspecific symptoms and the limitations of current diagnostic methods and targets. To address these challenges, we developed and validated a naked‐eye loop‐mediated isothermal amplification (LAMP) assay targeting the newly identified orpH region for the rapid detection of pathogenic Leptospira species. Methods Comparative genomic analysis identified orpH as a conserved sequence among pathogenic Leptospira. A colorimetric LAMP assay (LAMP‐orpH) was developed and evaluated using reference strains and serum samples obtained from patients with suspected leptospirosis. The diagnostic performance of the assay on these serum samples was evaluated in comparison with conventional PCR and the microscopic agglutination test (MAT), with the real‐time PCR assay targeting lipL32 designated as the reference standard. Results The LAMP‐ orpH assay demonstrated 87.2% sensitivity and 94.8% specificity among 76 suspected cases and 29 controls, showing strong agreement with real‐time PCR ( κ = 0.826). It outperformed MAT (63.8% sensitivity) and conventional PCR (23.4% sensitivity), achieving a detection limit of 10 2 cells/mL in spiked serum. Conclusion The LAMP‐ orpH assay offers a rapid, sensitive, and specific tool for early leptospirosis diagnosis. Its simple colorimetric readout and minimal equipment requirements make it ideal for use in rural and resource‐limited settings, providing a practical alternative to MAT, conventional PCR, and qPCR.