Multiplexed Detection of Foodborne Pathogens and the Antibiotic Resistance Genes Based on the CRISPR/Cas System with Engineered LwaCas13a

Abstract Foodborne pathogens represent a persistent threat to global public health, necessitating the development of point-of-care testing (POCT) platforms that integrate rapidity, ultrahigh sensitivity, cost effectiveness, and operational simplicity. Herein, we developed a system for high-specificity identification of foodborne pathogens and associated antibiotic resistance genes, which is a dual-modal diagnostic platform integrating recombinase polymerase amplification (RPA) with an engineered LwaCas13a variant. By leveraging the CRISPR/Cas13a system, the platform establishes a multiplex fluorescence array that simultaneously detects four major foodborne pathogens (Salmonella enterica serovar typhimurium, Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus) and their corresponding critical antibiotic resistance genes (ARGs), including mecA, blaCTX-M, mcr-1, ermB, and tet(M). Notably, the system retained robust trans-cleavage activity in complex biological matrices, achieving a remarkable limit of detection (LOD) at 100 copies/mL. Furthermore, the CRISPR/Cas13a system is coupled to an instrument-free lateral flow assay (LFA) for the detection of L. monocytogenes and tet(M), which effectively circumvents matrix-induced viscosity and ionic inhibition in egg and milk samples. Moreover, our developed platform was validated using samples from 40 household refrigerators, which drastically reduces assay time and thereby circumvents the inhibitory effects that constrain qPCR in food matrices and achieves 100% diagnostic agreement compared with the gold-standard methods. Collectively, this dual-modal strategy provides a promising paradigm with point-of-care capability for food safety surveillance and antimicrobial resistance monitoring.