Introduction: Foodborne pathogens could trigger foodborne diseases and have been a major threat to food safety. In the United States, FoodNet has reported more than 25,000 infections each year, resulting in 6000 hospitalizations, and 120 deaths annually from 2016 to 2020. The appearance of antibiotic-resistant bacteria can worsen the condition of infection. Phage-mediated technologies have evolved to be sensitive and rapid methods for the detection of a broad range of pathogens in recent years. Phages are uniquely suited for bacteria detection, which could not only act as probes to specifically recognize viable bacterial cells, but also be signal amplifiers through phage amplification (PAA)-based analysis.

Objective: Hence we proposed PAA-mediated strategies for the quantification of foodborne pathogens as well as the detection of viable antibiotic-resistant strains with high specificity, sensitivity, and accuracy.

Methodology: Our scheme was as follows: (1) Phage with a broad host range and high lysis ability was carefully selected; (2) Then PAA strategy was established and applied for the quantification of foodborne pathogens in foods, as well as assessment of potential antibiotic resistance in different food matrices; (3) Finally, the detection time of this assay was further decreased by combining with a multiplexed qPCR step for the quantification of progeny phage.

Results: Under the optimized conditions, this assay has been successfully applied to quantify viable Salmonella enterica and Staphylococcus aureus in food matrices such as milk and lettuce, with a detection limit of 10 CFU/mL. When combined with multiplex qPCR assay, PAA-qPCR further reduced the detection time from 6.5 h to 3.5 h, without complicating the DNA extraction or purification process.

Conclusions: The proposed PAA-based multiplex qPCR assay provides an effective and promising strategy for the simultaneous detection of viable bacteria, with rapidity, high sensitivity, and specificity with the ability to distinguish live bacteria.