Rapid accurate platform detection of Mycobacterium tuberculosis using Cas12a and Cas13a

Tuberculosis remains a major global health challenge, underscoring the need for rapid and accessible diagnostic tools suitable for resource-limited settings. In this study, we developed and clinically evaluated two LAMP-based CRISPR diagnostic pipelines targeting the IS6110 sequence of Mycobacterium tuberculosis . Two two-pot LAMP-CRISPR diagnostic pipelines were developed and evaluated: one based on Cas12a-mediated DNA detection and one based on Cas13a-medaited RNA detection following in vitro transcription (IVT). Analytical sensitivity was determined using serial dilutions of commercial M. tuberculosis DNA controls. Clinical validation was performed using 76 culture-characterized clinical spiecimens, including respiratory and extra-pulmonary samples, and assay performance was assessed using fluorescence and lateral flow readouts. Both pipelines achieved a limit of detection of 10 copies/µL and showed high specificity (100%) against non-tuberculous mycobacteria. When tested on 76 clinical samples, the LAMP–Cas12a assay demonstrated higher global diagnostic sensitivity than the LAMP–IVT–Cas13a pipeline (89% vs. 76%), likely due to the additional IVT step required for Cas13a. Both assays were compatible with lateral flow readouts, supporting point-of-care use. Overall, LAMP/CRISPR diagnostics represent a promising alternative to conventional TB tests, with Cas12a currently offering superior performance in two-pot formats, while RNA-targeting Cas13a remains of interest for future applications aimed at identifying active disease.