PAM-less Exonuclease-assisted Cas12a for visual detection of Vibrio Species

Foodborne pathogens, including Vibrio spp. and norovirus, cause substantial economic and healthcare burdens worldwide. Rapid and sensitive point-of-care testing on-farm or restaurants for batch inspection of pathogenic contamination in raw food products is essential. Here, we present an easy-to-design, cost-effective PAM-less Exonuclease-assisted Cas12A Nucleic-acid Detection (PECAN) assay paired with nucleic acid amplification systems for rapid and sensitive visual detection of 2 pathogenic Vibrio species: Vibrio parahaemolyticus (TDH) and Vibrio Cholerae (ctxA) without protospacer adjacent motif (PAM) site limitation. With T7 exonuclease, PAM-less detection could be achieved with a low concentration of cas12a, costing $0.8 USD per reaction. The system could also be adapted for PAM-less cas12a nucleic acid detection in-field or in-lab for sensitive DNA or RNA detection. We also constructed a low-cost reusable 3D printed heater chassis and reusable sodium acetate heat packs for field use without generating solid waste.