Precision detection of rifampicin-resistant <i>rpoB</i>_L378R mutation in <i>Mycobacterium tuberculosis</i> with CRISPR-Cas12a

Rifampicin is one of the most effective anti-tuberculosis drugs. However, certain strains of Mycobacterium tuberculosis (MTB) have developed resistance to rifampicin, making it crucial to identify alternative drugs for treating rifampicin-resistant MTB infections. Mutations in the rpoB gene play a pivotal role in MTB's resistance to rifampicin. Therefore, identifying these mutations is essential for effectively managing rifampicin-resistant MTB strains. Here, we developed a CRISPR-Cas12a platform integrated with recombinase polymerase amplification (RPA) and fluorescence detection, which was specifically designed to identify the rpoB _L378R mutation associated with rifampicin resistance in MTB. Our findings indicated that this detection technique exhibited high specificity and did not cross-react with reference samples constructed from the genomes of MTB H37Rv, Mycobacterium smegmatis , Mycobacterium aurum , and Escherichia coli . The RPA-CRISPR-Cas12a-based platform established in this research was simple, sensitive, and specific for detecting the rifampicin-resistant MTB strain with the rpoB _L378R mutation. These results suggest its potential applicability in clinical diagnosis for identifying the MTB rpoB _L378R mutation.