CRISPR-Cas12a-Based Rapid and Sensitive Detection ofrpoBL378R inMycobacterium tuberculosis

Abstract

AbstractRifampin is the most effective drug in the treatment of tuberculosis, whose major pathogen isMycobacterium tuberculosis(MTB), whereas there are still certain MTB strains resistant to the therapy of rifampin. TherpoBmutations 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 itsrpoBwild type. Notably, this detection system is highly specific because it did not cross-react with contrived reference samples containing the genomes of MTBH37Rv,Mycobacterium smegmatis(M. smegmatis),Mycobacterium aureus(M. aureus), andEscherichia 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 MTBH37Rvwith therpoBL378R mutation, indicating that this CRISPR-Cas12a-based detection platform has high potential to be exploited for clinic application to identify MTB mutations.