AUTHOR=Shi Yaoqiang , Kang Lan , Mu Rongrong , Xu Min , Duan Xiaoqiong , Li Yujia , Yang Chunhui , Ding Jia-Wei , Wang Qinghua , Li Shilin 

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

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=Volume 10 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.845688

DOI=10.3389/fbioe.2022.845688

ISSN=2296-4185

ABSTRACT=Shigella flexneri is a serious threat to global public health and a rapid detection method is urgently needed. CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system is widely used in gene editing, gene therapy, and in vitro 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 Shigella flexneri. The CRISPR/Cas12a-E-LAMP protocol conducts LAMP reaction for S. flexneri templates followed by CRISPR/Cas12a detection of predefined target sequences. LAMP primers and sgRNAs were designed to the highly conserved gene hypothetical protein (Accession: AE014073, Region: 4170556-4171068) of S. flexneri. 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 S. flexneri was 4*100 copies/μL plasmids, and without cross-reaction with other 6 closely related non-S. flexneri. Therefore, the CRISPR/Cas12a-E-LAMP assay is a useful method for the reliable and quick diagnosis of S. flexneri and may be applied in other pathogens infection detection.