Detection of rifampin-resistant Mycobacterium tuberculosis using CRISPR/Cas14a-enabled molecular techniques

The emergence of drug-resistant Mycobacterium tuberculosis (MTB) strains highlights the urgent need for precise and timely diagnostic methods to prevent prolonged and complex treatment regimens. This study aims to develops a CRISPR/Cas14a-based assay for accurate identification of MTB and rifampin-resistant MTB (RR-MTB) strains. The 16 S rDNA sequence and rifampin resistance-determining region (RRDR) of rpoB gene were chosen for the detection of MTB and RR-MTB, respectively. Several sgRNAs were designed for each target and evaluated for their performance. The platform was then systematic optimized by adjusting the concentrations of different components, followed by the evaluation of its sensitivity for the detection of MTB and RR-MTB. The system’s efficacy was further validated through a double-blind test on 16 clinical MTB isolates, and the results were compared with genomic sequencing. Through a meticulous screening process, we identified optimal single-guide RNAs (sgRNAs) capable of distinguishing MTB from nontuberculous mycobacteria (NTM) and the eight predominant mutation types associated with rifampin resistance. Our refined CRISPR/Cas14a platform demonstrated a remarkable sensitivity, with a limit of detection (LOD) of 200 copies/µL for MTB and 2 copies/µL for RR-MTB, respectively. This platform demonstrated a 100% accuracy rate in identification of RR-MTB using clinical MTB isolates. The CRISPR/Cas14a-based platform we developed exhibited superior performance for the detection of MTB and RR-MTB, with significant implications for the diagnosis and management of tuberculosis, particularly in regions with high prevalence of drug-resistant strains.