Rapid and sensitive diagnosis of live Mycobacterium tuberculosis 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 Wang ¹
, Huihuang Lin ²^,³^,⁴^,⁵
, Anqi Yang ⁶
, Jiaming Huang ⁷
, Weicong Ren ⁸
, Jiajun Dong ¹^,⁹^,¹⁰
, Shaojie Wang ¹
, Wenxue Xu ¹
, Yu Pang ⁸
, Jieming Qu ²^,³^,⁴^,^†
, Jia Liu ¹^,¹⁰^,¹¹^,¹²^,^†

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¹. Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China

². Department of Pulmonary and Critical Care Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

³. Institute of Respiratory Diseases, Shanghai Jiao Tong University School of Medicine, Shanghai, China

⁴. Shanghai Key Laboratory of Emergency Prevention, Diagnosis and Treatment of Respiratory Infectious Diseases, Shanghai, China

⁵. Department of Pulmonary and Critical Care Medicine, the Second Affiliated Hospital of Fujian Medical University, Quanzhou, China

⁶. Department of Central Laboratory, the Second Affiliated Hospital of Fujian Medical University, Quanzhou, China

⁷. Department of Microbiology, the Second Affiliated Hospital of Fujian Medical University, Quanzhou, China

⁸. Department of Bacteriology and Immunology, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, China

⁹. Research Ridge (Shanghai) Biotechnology Ltd., Shanghai, China

¹⁰. Guangzhou Laboratory, Guangzhou International Bio Island, Guangzhou, China

¹¹. Shanghai Clinical Research and Trial Center, Shanghai, China

¹². Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai, China

jmqu0906@163.com

liujia@shanghaitech.edu.cn

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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 DOI:10.1002/VIW.20230109

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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.