Campylobacter jejuni is the major cause of campylobacteriosis, one of the most common foodborne illnesses worldwide. Here, we report the development of RAA-exo-probe and RAA-CRIPSR/Cas12a assays for the detection of C. jejuni in food samples. The two assays were found to be highly specific to C. jejuni and highly sensitive, as they were one log more sensitive compared to the traditional culture method, with detection thresholds of 9 and 5 copies per reaction, respectively. These assays successfully detected C. jejuni in spiked chicken samples and natural meat samples (chicken, beef, mutton, etc.) and were overall less dependent on expensive equipment, only requiring a fluorescent reader. Their ease of use compared to other nucleic acid amplification-based methods indicates that these assays could be adapted for the rapid, routine surveillance of C. jejuni contamination in food samples, particularly for work done in the field or poorly equipped labs.

Background Shiga toxin-producing Escherichia coli (STEC) is a significant cause of foodborne illness causing various gastrointestinal diseases including hemolytic uremic syndrome (HUS), the most severe form, which can lead to kidney failure or even death. Objective Here, we report the development of RAA (Recombinase Aided Amplification)-exo-probe assays targeting the stx1 and stx2 genes for the rapid detection of STEC in food samples. Results These assays were found to be 100% specific to STEC strains and were also highly sensitive with a detection limit of 1.6 × 103 CFU/mL or 32 copies/reaction. Importantly, the assays were able to successfully detect STEC in spiked and real food samples (beef, mutton, and pork), with a detection limit as low as 0.35 CFU/25g in beef samples after an overnight enrichment step. Conclusion Overall, the RAA assay reactions completed within ∼20 minutes and were less dependent on expensive equipment, suggesting they can be easily adopted for in field testing requiring only a fluorescent reader. Highlights As such, we have developed two rapid, sensitive, and specific assays that can be used for the routine monitoring of STEC contamination in food samples, particularly in the field or in poorly equipped labs.

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.