Simultaneous Dual-Gene Detection of Escherichia coli O157:H7 Based on a CRISPR/Cas13-Mediated Biosensor

Escherichia coli (E. coli) O157:H7 is a major foodborne pathogen that poses significant public health risks, requiring rapid and accurate detection methods. In this study, we developed a one-pot CRISPR/Cas13-based biosensor for the simultaneous detection of two key genetic markers: rfbE_O157 and fliC_H7. By leveraging the distinct collateral cleavage preferences of LwaCas13a and PsmCas13b, the biosensor establishes dual fluorescence channels (TEX and FAM), enabling serotype-specific detection while minimizing false positives. The entire detection process (target gene amplification, transcription, and CRISPR-mediated biosensing) was integrated into a single reaction tube, eliminating the need for complex equipment and reducing the risk of contamination. The biosensor demonstrated high sensitivity, achieving a detection limit of 54 CFU/mL by using recombinase polymerase amplification (RPA). To validate its real-world applicability, an E. coli O157:H7 infection model was established in mice, and fecal samples were analyzed, showing results consistent with the standard PCR method. Compared with conventional CRISPR-based diagnostics, this approach enhances serotype discrimination, improves specificity, and offers a simple, rapid, and field-deployable solution for pathogen detection. Overall, this dual-channel biosensor provides a promising tool for clinical diagnostics, food safety monitoring, and infectious-disease surveillance.