Simultaneous molecular detection of Mycobacterium tuberculosis and multidrug resistance using CRISPR-AaCas12b-based nucleic acid assay

Objectives To address the unmet need for rapid, accurate diagnosis of Mycobacterium tuberculosis (MTB) and multidrug-resistant tuberculosis (MDR-TB), we developed and validated a clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas)-based diagnostic assay. Methods A multiplex-recombinase polymerase amplification (RPA) coupled CRISPR- Alicyclobacillus acidiphilus Cas12b (AaCas12b) assay was established for simultaneous detection of MTB by targeting the specific insertion sequence IS6110 and the two most common drug resistance mutations, rpoB 1349C>T for rifampicin resistance and katG 944G>C for isoniazid resistance. The assay supported dual-readout signal detection using both a fluorescent platform and lateral flow chromatography (LFC). Its diagnostic performance was evaluated in 48 clinical samples using WHO-recommended GeneXpert MTB/RIF, phenotypic drug susceptibility testing (pDST), and sequencing as reference standards. Results The multiplex-RPA CRISPR-AaCas12b assay showed a limit of detection (LoD) of 1.5 CFU/mL for MTB detection, with a sensitivity of 97.1% and a specificity of 100% using culture as the reference standard, and a total turnaround time of 30 min (20 min for RPA and 10 min for CRISPR cleavage). For MDR-TB-related mutations, the assay achieved a sensitivity of 94.1% and a specificity of 100% for rpoB 1349C>T, and 94.7% sensitivity and 93.1% specificity for katG 944G>C, using sequencing as the reference standard. Notably, the LFC-integrated assay maintained comparable diagnostic accuracy with a total turnaround time of 35 min (20 min for RPA, 5 min for CRISPR cleavage, and 10 min for lateral flow strip reading). Conclusion The established multiplex-RPA CRISPR-AaCas12b assay enables simple, accurate, and sensitive detection of MTB and common mutations associated with MDR-TB. With a rapid, simplified workflow and low resource requirements, this approach holds considerable potential for point-of-care testing in resource-limited settings, thus facilitating improved surveillance and control of TB and drug-resistant TB.