Multiplex crRNAs powered CRISPRCas13a detection for rifampicin-resistant mycobacterium tuberculosis.

BACKGROUND: Rapid and accessible detection of Mycobacterium tuberculosis (MTB) and rifampicin resistance is essential for timely treatment and transmission control. However, widely used drug-resistance testing methods, including qPCR and gene sequencing, are time-consuming and require specialized instrumentation, limiting their utility for rapid testing in primary-care and resource-limited settings. Therefore, rapid, sensitive and readily deployable assays are urgently needed for the simultaneous diagnosis of tuberculosis (TB) and screening for drug resistance. METHODS: A multiplex crRNA system was integrated with isothermal amplification, lyophilized reagents and a lateral flow readout to enable detection of 4 common rpoB mutation sites in a single reaction. Analytical sensitivity and specificity were evaluated using mutant plasmids, rifampicin-resistant clinical isolates, clinically relevant bacterial pathogens and non-tuberculous mycobacteria (NTM). Clinical performance was assessed using sputum-derived nucleic acids, with qPCR and Sanger sequencing as reference methods. RESULTS: This detection assay can accurately identify nucleic acids from 10 clinical rifampicin-resistant isolates carrying four types of target mutations. For nucleic acids extracted from 21 clinical sputum samples, the assay achieved a sensitivity and specificity of 100%, with detection results fully consistent with those of qPCR and gene sequencing. The limit of detection (LOD) of the assay for all mutant plasmids was 10 copies/μL. In addition, this study realized highly sensitive and specific detection of MTB, with a LOD as low as 1 copy/μL, and successfully identified nucleic acids from 42 clinical TB samples. No cross-reactivity was observed between this assay and 5 pathogenic bacteria with clinical symptoms overlapping with MTB, as well as 8 NTM strains. Amplification and visual lateral flow readout were completed within 1 h. CONCLUSIONS: This multiplex CRISPR-Cas13a assay enables rapid, sensitive and visually interpretable detection of MTB and common rifampicin resistance-associated mutations with limited instrumentation, offers a novel detection method for rifampicin-resistant tuberculosis (RR-TB) and exhibits potential in improving TB diagnosis.