IDENTIFICATION OF MYCOBACTERIUM TUBERCULOSIS USING CRISPR-CAS12A

Abstract

Tuberculosis (TB) is a global health concern and a communicable disease. It was the leading cause of death from a single infectious agent before the COVID-19 pandemic, surpassing HIV/AIDS. Mycobacterium tuberculosis (MTB) is the causative agent of TB, which spreads through airborne transmission, primarily via coughing. However, the conventional method for identifying MTB is time-consuming and limited to large hospitals, resulting in delays in targeted treatment and challenges in reaching affected communities. Therefore, there is an urgent need for a rapid and easy-to-use approach to detect MTB, especially in resource-limited areas. In this study, we investigated the use of CRISPR-Cas12a-based detection as an alternative and promising method for TB screening. We developed a specific detection method for MTB using the CRISPR system combined with recombinase polymerase amplification (RPA) to enhance signal amplification. The results demonstrated successful amplification and detection of the rpoB target gene of MTB using the designed primers and crRNA. Importantly, these assays exhibited no cross-reactivity with other Mycobacterium species, and this technique had a limit of detection (LOD) as low as 103 copies/µL. In conclusion, the RPA combined with CRISPR-Cas12a assay proved to be an effective screening method for MTB, providing results in less than 1 hour from extracted DNA. This assay holds significant potential as a portable, rapid, and cost-effective MTB detection tool, especially in low-resource settings