Real-time recombinase-aided amplification with improved propidium monoazide for the rapid detection of viable Escherichia coli O157:H7 in milk.

Escherichia coli O157:H7, the causative agent of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in humans, generates a effective harm to community health because of its high pathogenicity. A real-time recombinase-aided amplification (rRAA) is an emerging method for nucleic acid detection. However, genomic DNA of bacteria could exist in food and the environment for a long time after death and could be amplified by rRAA assay, resulting in false-positive signal; thus, developing a fast and sensitive method is necessary to detect viable foodborne pathogens in food products. In our research, rRAA assay coupled with an enhanced nucleic acid binding dye named improved propidium monoazide (PMAxx) was established and applied in viable E. coli O157:H7 identification in skim milk. The PMAxx could eliminate interference from dead bacteria by permeating impaired membranes and covalently linking to DNA to prevent DNA amplification. The PMAxx-rRAA assay was performed with high sensitivity and good specificity. The PMAxx-rRAA assay could detect as low as 5.4 × 100 cfu/mL of viable E. coli O157:H7 in pure culture, and 7.9 × 100 cfu/mL of viable E. coli O157:H7 in skim milk. In addition, the PMAxx-rRAA assay was performed in the presence of a high concentration of dead bacteria or nontarget bacteria in skim milk to verify the capacity to resist interference from dead bacteria and nontarget bacteria. Therefore, the established PMAxx-rRAA assay is a valuable tool for the identification of viable E. coli O157:H7 in complex food matrix.