Rapid and sensitive diagnosis of live <i>Mycobacterium tuberculosis</i> using clustered regularly interspaced short palindromic repeat-Cas13a point-of-care RNA testing

Yu Wang; Huihuang Lin; Anqi Yang; Jiaming Huang; Weicong Ren; Jiajun Dong; Shaojie Wang; Wenxue Xu; Yu Pang; Jieming Qu; Jia Liu

doi:10.1002/VIW.20230109

VIEW ›› 2024, Vol. 5 ›› Issue (3) : 20230109. DOI: 10.1002/VIW.20230109

RESEARCH ARTICLE

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

Yu Wang¹ ,
Huihuang Lin²^,³^,⁴^,⁵ ,
Anqi Yang⁶ ,
Jiaming Huang⁷ ,
Weicong Ren⁸ ,
Jiajun Dong¹^,⁹^,¹⁰ ,
Shaojie Wang¹ ,
Wenxue Xu¹ ,
Yu Pang⁸ ,
Jieming Qu²^,³^,⁴ ,
Jia Liu¹^,¹⁰^,¹¹^,¹²

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Abstract

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-MTBmethod 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.

Keywords

CRISPR-Cas13a / lateral flow / limit of detection (LOD) / livemycobacterium tuberculosis / point-of-care testing / single-stranded RNA (ssRNA)

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Yu Wang, Huihuang Lin, Anqi Yang, Jiaming Huang, Weicong Ren, Jiajun Dong, Shaojie Wang, Wenxue Xu, Yu Pang, Jieming Qu, Jia Liu. Rapid and sensitive diagnosis of live Mycobacterium tuberculosis using clustered regularly interspaced short palindromic repeat-Cas13a point-of-care RNA testing. VIEW, 2024, 5(3): 20230109 https://doi.org/10.1002/VIW.20230109

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2024 2024 The Authors. View published by Shanghai Fuji Technology Consulting Co., Ltd, authorized by Professional Community of Experimental Medicine, National Association of Health Industry and Enterprise Management (PCEM) and John Wiley & Sons Australia, Ltd.