An esterase activity-based biosensor for rapid and sensitive detection of viable Escherichia coli O157:H7 in milk

Abstract

Escherichia coli O157:H7 is harmful to humans by producing toxins that cause thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome. Real-time recombinase-aided amplification (rRAA) can generate real-time fluorescence signals to achieve sensitive detection of pathogens within 20 min but cannot distinguish dead bacteria from viable bacteria, leading to false-positive results. Thiazole orange monoazide (TOMA) is an activity-labile nucleic acid-modifying compound, permeating into bacterial cells and, in dead cells, covalently linking to genomic DNA to suppress DNA amplification. In viable cells, however, TOMA is cleaved by active esterase and so inhibition of DNA amplification would not occur. A rRAA assay integrated with TOMA was successfully developed potentially enabling rapid and sensitive identification of viable E. coli O157:H7 in milk. The limit of detection was less than 10 cfu mL−1 in both pure culture and skimmed milk. Moreover, this assay could resist interference from non-target bacteria and high concentrations of dead bacteria.