Cas12a/Guide RNA-Based Platform for Rapid and Accurate Identification of Major <i>Mycobacterium</i> Species

Abstract

<i>Mycobacterium tuberculosis</i> infection and nontuberculous mycobacteria (NTM) infections exhibit similar clinical symptoms; however, the therapies for these two types of infections are different. Therefore, the rapid and accurate identification of <i>M. tuberculosis</i> and NTM species is very important for the control of tuberculosis and NTM infections. In the present study, a Cas12a/guide RNA (gRNA)-based platform was developed to identify <i>M. tuberculosis</i> and most NTM species. By designing species-specific gRNA probes targeting the <i>rpoB</i> sequence, a Cas12a/gRNA-based platform successfully identified <i>M. tuberculosis</i> and six major NTM species (<i>Mycobacterium abscessus</i>, <i>Mycobacterium intracellulare</i>, <i>Mycobacterium avium</i>, <i>Mycobacterium kansasii</i>, <i>Mycobacterium gordonae</i>, and <i>Mycobacterium fortuitum</i>) without cross-reactivity. In a blind assessment, a total of 72 out of 73 clinical <i>Mycobacterium</i> isolates were correctly identified, which is consistent with previous <i>rpoB</i> sequencing results. These results suggest that the Cas12a/gRNA-based platform is a promising tool for the rapid, accurate, and cost-effective identification of both <i>M. tuberculosis</i> and NTM species.