Novel detection strategies of Escherichia. coli O157:H7 based on CRISPR/Cas12b- and LAMP- platform

Foodborne pathogens represent a major threat to food safety and public health. Escherichia coli O157:H7, widely present in sources such as bovine feces, undercooked ground beef, fresh produce, and drinking water, can cause diarrhea, hemorrhagic colitis, and even death upon infection. Therefore, developing sensitive and portable detection methods is crucial for food safety monitoring and outbreak response. This study established a detection method combining loop-mediated isothermal amplification (LAMP) with a CRISPR/Cas12b system for the highly sensitive and rapid detection of E. coli O157:H7. Using elaborately designed LAMP primers and Cas12b sgRNA targeting the FliC gene, we developed and optimized two assay formats: a two-tube method and a one-tube method. The two-tube method, involving separate LAMP amplification followed by CRISPR/Cas12b detection, demonstrated high sensitivity with a limit of detection (LOD) of 10 copies/μL for plasmid DNA. To prevent cross-contamination from tube opening, we further developed a one-tube system that integrates LAMP and CRISPR detection into a single closed-tube reaction, achieving an LOD of 10 copies/μL. At the same concentration, the two-tube method demonstrates a higher detection rate for plasmids, with both methods having the same limit of detection (LOD). Both methods exhibited excellent specificity, showing no cross-reactivity with other tested common foodborne bacteria. The established method is characterized by simple operation, low cost, rapid turnaround, and high accuracy, offering a reliable tool for on-site food safety monitoring and emergency detection. • Novel detection integrates LAMP amplification system and CRISPR/Cas12b system. • Due to sgRNA, the whole process overcome the defect of Cas9 in high miss rate. • Cas12b has lower tolerance, which improves the sensitivity and specificity. • The one-tube assay system can achieve accurate results without opening the cap.