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

Abstract

<i>Shigella flexneri</i> 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 <i>in vitro</i> 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 <i>S. flexneri</i>. The CRISPR/Cas12a-E-LAMP protocol conducts LAMP reaction for <i>S. flexneri</i> templates followed by CRISPR/Cas12a detection of predefined target sequences. LAMP primers and sgRNAs were designed to the highly conserved gene <i>hypothetical protein</i> (accession: AE014073, region: 4170556-4171,068) of <i>S. flexneri</i>. 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 <i>S. flexneri</i> was 4 × 10⁰ copies/μl plasmids and without cross-reaction with other six closely related non-<i>S. flexneri.</i> Therefore, the CRISPR/Cas12a-E-LAMP assay is a useful method for the reliable and quick diagnosis of <i>S. flexneri</i> and may be applied in other pathogen infection detection.