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Updated: Aug 31, 2025
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Published on: April 25, 2025
Sensitive and Rapid Detection of Escherichia coli O157:H7 From Beef Samples Based on Recombinase Aided Amplification
Taisong Fang1, Jinling Shen1, Junxin Xue1
1Technology Center for Animal Plant and Food Inspection and Quarantine of Shanghai Customs, Shanghai 200135, China.
Background:
Escherichia coli O157:H7, being the cause of hemorrhagic colitis in humans, is recognized as one of the most dangerous and widespread foodborne pathogens. A highly specific, sensitive, and rapid E. coli O157:H7 detection method needs 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:
First, the feasibility (components of CRISPR/Cas12a system) of the developed method was evaluated. Then a total of 34 bacterial strains were used for the specificity test, and gradient dilutions of extracted DNA and bacterial solutions of E. coli O157:H7 were prepared for the sensitivity test. Third, 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 the 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 CFU/25 g. The detection results of the 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.
