Phage amplification-coupled CRISPR/Cas12a system for selective detection of viable E. coli in fresh produce

Rapid and specific detection of viable foodborne pathogens is critical for ensuring the safety of the food supply chain and preventing the risk of foodborne illness outbreaks. In this study, bacteriophage T7 amplification was integrated with a CRISPR/Cas12a-based assay for detecting viable E. coli in model systems, including fresh produce homogenate, using minimal instrumentation and without requiring conventional nucleic acid amplification steps. The CRISPR/Cas12a reaction condition was optimized for the detection of bacteriophage T7 DNA amplification upon infection of viable bacterial cells. The optimal concentrations of Cas12a and crRNA were 250 and 500 nM, respectively, with an optimal isothermal temperature of 50°C. The developed detection approach enabled the detection of viable E. coli at concentrations as low as 1 CFU/mL in culture broth and 10 2 CFU/mL in spinach homogenate within an 8-h-15-min timeframe, without the need for additional DNA amplification. The developed approach exhibited high specificity for E. coli detection in the presence of a mixture of non-targeted bacteria. This work highlights the potential of phage-assisted CRISPR/Cas12a systems for rapid, low-cost, and reliable detection of viable foodborne pathogens in complex food matrices.