CRISPR-Cas12a-Based Rapid and Sensitive Detection of rpoBL378R in Mycobacterium tuberculosis

Abstract

Rifampin is the most effective drug in the treatment of tuberculosis, whose major pathogen is Mycobacterium tuberculosis (MTB), whereas there are still certain MTB strains resistant to the therapy of rifampin. The rpoB mutations play a central role in MTB resistance to the rifampin therapy, so it is crucial to identify these mutations in order to discover novel therapeutic approaches to these drug-resistant MTB strains. Here we show that a CRISPR-Cas12a-based detection platform with recombinase polymerase amplification and fluorescence reporter can be utilized to detect and visualize an MTB drug-resistant point mutation (rpoBL378R) from its rpoB wild type. Notably, this detection system is highly specific because it did not cross-react with contrived reference samples containing the genomes of MTB H37Rv, Mycobacterium smegmatis (M. smegmatis), Mycobacterium aureus (M. aureus), and Escherichia coli (E. coli). Collectively, this strategy based on CRISPR-Cas12a that we show in this report is simple, sensitive as well as specific for detection of the rifampin-resistant MTB H37Rv with the rpoBL378R mutation, indicating that this CRISPR-Cas12a-based detection platform has high potential to be exploited for clinic application to identify MTB mutations.