Fast and sensitive detection of viable Escherichia coli O157:H7 using a microwell-confined and propidium monoazide-assisted digital CRISPR microfluidic platform

Escherichia coli O157:H7 is a major foodborne pathogen that poses a significant threat to food safety and human health. Rapid and sensitive detection of viable Escherichia coli O157:H7 can effectively prevent food poisoning. Here, we developed a microwell-confined and propidium monoazide-assisted digital CRISPR microfluidic platform for rapid and sensitive detection of viable Escherichia coli O157:H7 in food samples. The reaction time is significantly reduced by minimizing the microwell volume, yielding qualitative results in 5 min and absolute quantitative results in 15 min. With the assistance of propidium monoazide, this platform can eliminate the interference from 99% of dead Escherichia coli O157:H7. The direct lysis method obviates the need for a complex nucleic acid extraction process, offering a limit of detection of 3.6 × 10¹ CFU mL^-1 within 30 min. Our results demonstrated that the platform provides a powerful tool for rapid detection of Escherichia coli O157:H7 and provides reliable guidance for food safety testing.