Sensitive and Rapid Detection of Escherichia coli O157:H7 From Beef Samples Based on Recombinase Aided Amplification Assisted CRISPR/Cas12a System

Abstract

Background Escherichia coli O157: H7, being the cause of hemorrhagic colitis in human, is recognized as one of the most dangerous and widespread foodborne pathogens. A highly specific, sensitive and rapid E. coli O157: H7 detection method need to be developed since the traditional detection methods are complex, costly and time-consuming. Objective In this study, a recombinase aided amplification (RAA) assisted CRISPR/Cas12a (RAA-CRISPR/Cas12a) fluorescence platform for specific, sensitive and rapid nucleic acid detection of E. coli O157: H7 was introduced. Methods Firstly, the feasibility (components of CRISPR/Cas12a system) of the developed method was evaluated. Then, a total of 34 bacterial strains were used for specificity test, and gradient dilutions of extracted DNA and bacterial solutions of E. coli O157: H7 were prepared for sensitivity test. Thirdly, a real-time PCR assay for detection of the specific wzy gene of E. coli O157: H7 (FDA’s Bacteriological Analytical Manual) was used for sensitivity comparison. Finally, analysis of RAA-CRISPR/Cas12a detection in spiked and 93 real ground beef samples was carried out. Results The developed RAA-CRISPR/Cas12a method showed high specificity and the detection could be completed within 30 min (after 4 h enrichment in spiked ground beef samples). The limit of detection (LOD) of bacterial concentrations and genomic DNA was 5.4 × 102 CFU/mL and 7.5 × 10−4 ng/μL, respectively, which exhibited higher sensitivity than RAA-gel electrophoresis and RT-PCR methods. Furthermore, it was shown that E. coli O157: H7 in ground beef samples could be positively detected after 4 h enrichment when the initial bacterial inoculum was 9.0 × 10° CFU/25 g. The detection results of RAA-CRISPR/Cas12a method were 100% consistent with those of the RT-PCR and traditional culture-based methods while screening the E. coli O157: H7 from 93 local collected ground beef samples. Conclusions The developed RAA-CRISPR/Cas12a method showed high specificity, high sensitivity, and rapid positive detection of E. coli O157: H7 from ground beef samples. Highlights The RAA-CRISPR/Cas12a system proposed in this study provided an alternative molecular tool for quick, specific, sensitive and accurate detection of E. coli O157: H7 in foods.

Advances in the application of recombinase-aided amplification combined with CRISPR-Cas technology in quick detection of pathogenic microbes

“…The sensitivity of the RAA-Cas13a method can reach 10 copies of RNA molecules/reaction (Li et al, 2022). It summarizes all important reports about the application of RAA combined with CRISPR-Cas12a/Cas13a in the quick detection of pathogenic microbes (Table 1) (Li et al, 2021;Wang et al, 2022c;Zhang et al, 2022b;Chen et al, 2022;Fang et al, 2022;Qian et al, 2022;Wei et al, 2022;Xing et al, 2023;Zhao et al, 2023).…”

Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry

“…Additionally, the RPA-CRISPR/Cas12a strategy has been used for the detection of other foodborne pathogenic bacteria ( Listeria monocytogenes , Yersinia enterocolitica , Staphylococcus aureus , Campylobacter jejuni , Escherichia coli , etc.) in food production animals at attomolar level [ 96 , 97 , 98 , 99 , 100 ].…”