A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application

• DOI: 10.3389/fcimb.2023.1192134
• Publication Date: 2023-05-23T04:45:31Z
• Authors: Nan Jia; Chaohong Wang; Xiaming Liu; Xiaolan Huang; Fei Xiao; Jin Fu; Chunrong Sun; Zheng Xu; Guirong Wang; Juan Zhou; Yi Wang
• Keywords: 0301 basic medicine; multiple cross displacement amplification; Cas12a; COVID-19; Mycobacterium tuberculosis; Microbiology; Sensitivity and Specificity; QR1-502; 03 medical and health sciences; Cellular and Infection Microbiology; tuberculosis; CRISPR; Humans; Tuberculosis; CRISPR-Cas Systems; Pandemics

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified specific sdaA gene MTB MCDA, and MCDA results were then decoded detection, resulting in simple visual fluorescent signal readouts. A set standard primers, an engineered CP1 primer, quenched ssDNA reporter, gRNA designed targeting MTB. The optimal temperature pre-amplification 67°C. whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), reaction (40 CRISPR-Cas12a-gRNA (5 minutes). limit (LoD) assay 40 fg per reaction. does not non-tuberculosis mycobacterium (NTM) strains other species, validating its specificity. clinical performance was higher than that smear microscopy test comparable Xpert method. In summary, promising effective tool infection surveillance prevention, especially point-of-care (POC) field deployment source-limited regions.