A Simplified Amplification‐Free Strategy with Lyophilized CRISPR‐CcrRNA System for Drug‐Resistant Salmonella Detection

Abstract Drug resistance in pathogenic bacteria has become a major threat to global health. The misuse of antibiotics has increased the number of resistant bacteria in the absence of rapid, accurate, and cost‐effective diagnostic tools. Here, an amplification‐free CRISPR‐Cas12a time‐resolved fluorescence immunochromatographic assay (AFC‐TRFIA) is used to detect drug‐resistant Salmonella . Multi‐locus targeting in combination crRNA (CcrRNA) is 27‐fold more sensitive than a standalone crRNA system. The lyophilized CRISPR system further simplifies the operation and enables one‐pot detection. Induction of nucleic acid fixation via differentially charged interactions reduced the time and cost required for flowmetric chromatography with enhanced stability. The induction of nucleic acid fixation via differentially charged interactions reduces the time and cost required for flowmetric chromatography with enhanced stability. The platform developed for the detection of drug‐resistant Salmonella has an ultra‐sensitive detection limit of 84 CFU mL −1 within 30 min, with good linearity in the range of 10 2 –10 6 CFU mL −1 . In real‐world applications, spiked recoveries range from 76.22% to 145.91%, with a coefficient of variation less than 10.59%. AFC‐TRFIA offers a cost‐effective, sensitive, and virtually equipment‐independent platform for preventing foodborne illnesses, screening for drug‐resistant Salmonella , and guiding clinical use.