Abstract
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.
Highlights
Shigella is a kind of Gram-negative bacilli, and it is the most common pathogen of human bacillary dysentery (Jehl et al, 2012), which is a frequently occurring disease in developing countries, and seriously endangers human health, especially the growth and development of children (Wanwu Li et al, 2021)
Once the loop-mediated isothermal amplification (LAMP) amplification was finished, CRISPR/Cas12a mixture was mixed with LAMP products for cleavage
To obtain a better cleavage activity of CRISPR/Cas12a, it was optimized from single-guide RNA (sgRNA), Cas12a, and singlestranded DNA (ssDNA) probe. sgRNA plays a critical role of guidance in CRISPR/Cas12a cleavage system. sgRNA was composed of a sequence for the recognition of the target sequence and a sequence that forms a complex with Cas12a by stem ring structure (Supplementary Figure S1)
Summary
Shigella is a kind of Gram-negative bacilli, and it is the most common pathogen of human bacillary dysentery (Jehl et al, 2012), which is a frequently occurring disease in developing countries, and seriously endangers human health, especially the growth and development of children (Wanwu Li et al, 2021). According to the survey of the bacterial distribution of Shigella, Shigella flexneri is the main pathogen of bacterial diarrhea in developing countries worldwide (Wang et al, 2014). Traditional bacterial culture method combined with biochemical experiments for the identification of S. flexneri is time-consuming (Weiss et al, 2019). With the development of molecular biology techniques, the pathogenic mechanism and detection methods of S. flexneri have been further studied and applied (Gentle et al, 2016). The development of sensitive, specific, convenient, and rapid detection kits for S. CRISPR/Cas12a-E-LAMP Visual Detection Method flexneri can provide technical guarantees for livestock and poultry breeding and provide technical support for food safety and human health. The rapid and accurate detection technology of S. flexneri is very important
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
