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

Autor:	Jia N; Experimental Research Center, Capital Institute of Pediatrics, Beijing, China., Wang C; Department of Clinical Laboratory, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China., Liu X; The Second Department of Geriatrics, Handan Central Hospital, Handan, Hebei, China., Huang X; Experimental Research Center, Capital Institute of Pediatrics, Beijing, China., Xiao F; Experimental Research Center, Capital Institute of Pediatrics, Beijing, China., Fu J; Experimental Research Center, Capital Institute of Pediatrics, Beijing, China., Sun C; Experimental Research Center, Capital Institute of Pediatrics, Beijing, China., Xu Z; Experimental Research Center, Capital Institute of Pediatrics, Beijing, China., Wang G; Department of Clinical Laboratory, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China., Zhou J; Experimental Research Center, Capital Institute of Pediatrics, Beijing, China., Wang Y; Experimental Research Center, Capital Institute of Pediatrics, Beijing, China.
Jazyk:	angličtina
Zdroj:	Frontiers in cellular and infection microbiology [Front Cell Infect Microbiol] 2023 May 23; Vol. 13, pp. 1192134. Date of Electronic Publication: 2023 May 23 (Print Publication: 2023).
DOI:	10.3389/fcimb.2023.1192134
Abstrakt:	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 tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2023 Jia, Wang, Liu, Huang, Xiao, Fu, Sun, Xu, Wang, Zhou and Wang.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu