A one-pot biplex RPA-Cas assay for sensitive detection of Mycobacterium tuberculosis from tongue swabs

The global management of tuberculosis (TB) is limited by the infrastructure requirements of current molecular diagnostics and the logistical burdens of sputum-based testing. While isothermal nucleic acid amplification provides a decentralized alternative, its clinical utility is often limited by amplicon carryover contamination and complex multi-step protocols. In this study we developed a fully integrated, “one-pot” biplex assay that couples recombinase polymerase amplification (RPA) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‑associated protein Cas12a-mediated trans -cleavage within a single, homogeneous reaction. This single-pot chemistry that targets the IS 1081 and IS 6110 genes of Mycobacterium tuberculosis eliminates the need for post-amplification handling, addressing a primary failure mode of decentralized testing, while maintaining an analytical sensitivity of 1.1 genomes per 15-µL reaction. The assay delivers sample-to-result within 30 min, with no observed cross-reactivity with common nontuberculous mycobacteria and respiratory pathogens. We further validated the system using clinical tongue swab samples, a non-invasive sampling method recently recognized by the World Health Organization to improve diagnostic yield in sputum-scarce populations. Our results demonstrate that this one-pot RPA-Cas12a system provides a sensitive, specific, and practical solution for point-of-care TB diagnosis, offering a viable alternative to traditional laboratory-based methods.