Work

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

Year: 2026

Type: article

Abstract: 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 IS1081 and IS6110 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.

Source: Scientific Reports

DOI: 10.1038/s41598-026-59825-9