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

Frontiers in Bioengineering and Biotechnology

Jia-Wei Ding¹  Qinghua Wang²  Yaoqiang Shi³  Yujia Li³  Shilin Li³  Lan Kang³  Min Xu³  Xiaoqiong Duan³  Rongrong Mu³  Chunhui Yang³ 

[1] Clinical Laboratory Department, Yan’an Hospital Affiliated to Kunming Medical University, Kunming, China;Department of Emergency, The Traditional Chinese Medicine Hospital of Wenjiang District, Chengdu, China;Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China;

关键词: CRISPR/Cas12a;    loop-mediated isothermal amplification;    Shigella flexneri;    point of care test;    visual detection;   

DOI  :  10.3389/fbioe.2022.845688

来源: DOAJ

【 摘 要 】

Shigella flexneri 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 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 S. 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: 4170556–4171,068) 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 six 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 pathogen infection detection.

【 授权许可】

Unknown