CRISPR/Cas12a-Enhanced Loop-Mediated Isothermal Amplification for the Visual Detection of Shigella flexneri

• Published in: Frontiers in bioengineering and biotechnology 2022-02, Vol.10, p.845688-845688
• Main Authors: Shi, Yaoqiang, Kang, Lan, Mu, Rongrong, Xu, Min, Duan, Xiaoqiong, Li, Yujia, Yang, Chunhui, Ding, Jia-Wei, Wang, Qinghua, Li, Shilin
• Format: Article
• Language: English
• Subjects: Bioengineering and Biotechnology; CRISPR/Cas12a; loop-mediated isothermal amplification; point of care test; Shigella flexneri; visual detection
• ISSN: 2296-4185; 2296-4185
• DOI: 10.3389/fbioe.2022.845688

is a serious threat to global public health, and a rapid detection method is urgently needed. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system is widely used in gene editing, gene therapy, and diagnosis. Here, we combined loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a to develop a novel diagnostic test (CRISPR/Cas12a-E-LAMP) for the diagnosis of . The CRISPR/Cas12a-E-LAMP protocol conducts LAMP reaction for templates followed by CRISPR/Cas12a detection of predefined target sequences. LAMP primers and sgRNAs were designed to the highly conserved gene (accession: AE014073, region: 4170556-4171,068) of . After the LAMP reaction at 60°C for 20 min, the pre-loaded CRISPR/Cas12a regents were mixed with the LAMP products in one tube at 37°C for 20 min, and the final results can be viewed by naked eyes with a total time of 40 min. The sensitivity of CRISPR/Cas12a-E-LAMP to detect was 4 × 10  copies/μl plasmids and without cross-reaction with other six closely related non- Therefore, the CRISPR/Cas12a-E-LAMP assay is a useful method for the reliable and quick diagnosis of and may be applied in other pathogen infection detection.