Rapid and sensitive diagnosis of live Mycobacterium tuberculosis using clustered regularly interspaced short palindromic repeat‐Cas13a point‐of‐care RNA testing

• Published in: View (Beijing, China) Vol. 5; no. 3
• Main Authors: Wang, Yu, Lin, Huihuang, Yang, Anqi, Huang, Jiaming, Ren, Weicong, Dong, Jiajun, Wang, Shaojie, Xu, Wenxue, Pang, Yu, Qu, Jieming, Liu, Jia
• Format: Journal Article
• Language: English
• Published: Wiley 01.06.2024
• Subjects: CRISPR‐Cas13a; lateral flow; limit of detection (LOD); live mycobacterium tuberculosis; point‐of‐care testing; single‐stranded RNA (ssRNA)
• ISSN: 2688-3988; 2688-268X; 2688-268X
• DOI: 10.1002/VIW.20230109

Mycobacterium tuberculosis (MTB) is the causal pathogen of tuberculosis (TB). Rapid and accurate detection of live MTB is important for transmission control and patient treatment. Here, we described a clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas13a‐based molecular diagnosis approach for rapid and specific detection of live MTB. This detection method, which we termed CRISPR‐Live‐MTB, contained two consecutive reactions including nuclear acid sequence‐based amplification (NASBA) and CRISPR‐Cas13a collateral cleavage reaction. CRISPR‐Live‐MTB could efficiently detect MTB single‐stranded RNA (ssRNA) in 2 hours with high specificity over double‐stranded DNA (dsDNA). Importantly, CRISPR‐Live‐MTB exhibited a limit of detection of 2.4 copies for MTB ssRNA, which was 1000 times lower than that of the clinically used NASBA method. Moreover, lateral flow was integrated into the CRISPR‐Live‐MTB method to enable point‐of‐care testing application with a sensitivity of 95% and a specificity of 100%. Overall, our study demonstrated the feasibility of CRISPR‐Live‐MTB as a rapid, sensitive, and specific approach for live MTB detection. Clustered regularly interspaced short palindromic repeat (CRISPR)‐Live‐Mycobacterium tuberculosis (MTB) can complete live MTB diagnosis in 2 h as compared to 8 weeks with conventional culturing methods. CRISPR‐Live‐MTB has over 1000‐fold higher sensitivity than the clinically used molecular diagnosis method of nuclear acid sequence‐based amplification.