Cas12b-assisted one-step dual-target CRISPR system for Mycobacterium tuberculosis detection

Tuberculosis (TB) remains a persistent public health threat, while the efficiency of traditional diagnostic methods is restricted. To address this, we developed a CRISPR-Cas12b-based one-step dual-target detection system (TB-QUICKv2) for sensitive and specific detection of the Mycobacterium tuberculosis complex (MTBC). Specifically, Cas12b and a molecular beacon (MB) probe were used to detect the target MTBC IS6110 sequence and an internal control (IC), the Bacillus spizizenii 16S sequence, in one reaction system, respectively. The optimal reaction temperature for TB-QUICKv2 was 57 °C and the detection procedure can be completed within 25 min. TB-QUICKv2 is highly specific (100%) for MTBC with a limit of detection of 10.4 CFU per milliliter at a 95% confidence interval. TB-QUICKv2 was further validated with clinical sputum samples and the results showed remarkably higher sensitivity (41/74) than the traditional culture method (37/74) and acid-fast bacillus (AFB) testing (33/74). Therefore, this study demonstrates that TB-QUICKv2 is a promising method for the rapid and accurate detection of MTBC and other infectious pathogens in the future. • TB-QUICKv2 is a one-step dual-target CRISPR system for TB diagnosis. • The turnaround time of TB-QUICKv2 from DNA to results is within 25 min. • We propose a feasible approach for creating one-step dual-target CRISPR-Dx systems.