CRISPR-Cas12a-based detection and differentiation of Mycobacterium spp.

Abstract

Mycobacterium species cause several vital human diseases, including tuberculosis (TB) and non-tuberculous mycobacterial (NTM) infections. TB is a chronic infectious disease caused by the Mycobacterium tuberculosis complex (MTBC). Additionally, non-tuberculous mycobacteria infections are caused by major NTM species, including Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium kansasii, Mycobacterium gordonae, and Mycobacterium intracelulare, are medically necessary. Both groups of infections can lead to outbreaks in immunocompromised individuals and AIDS patients. It is crucial to differentiate between these two groups of mycobacterial infections, as they require distinct treatment approaches. However, initial diagnosis poses limitations in terms of time and diagnostic tools. Therefore, accurate and rapid diagnosis is essential for effective treatment and controlling the spread of diseases. This research, the CRISPR-Cas12a technique was applied to develop a detection and classification method for mycobacteria at the species level. The testing results showed that the combination of the RPA technique with CRISPR-Cas12a for mycobacterial detection at the species level achieved a Limit of Detection (LOD) of 1 and 10 copies/µl, providing results consistent with standard detection methods for cultured samples. In summary, the CRISPR-Cas12a technique for the detection Mycobacterium spp. proved to be an efficient screening method, utilizing simple tools and delivering rapid results within one hour.