Simultaneous quantitative detection of viable Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. using sodium deoxycholate-propidium monoazide with multiplex real-time PCR.

Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. are common food-borne pathogens in milk that may cause serious diseases. In the present study, we established a simple, rapid, and specific method to simultaneously detect viable E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk. Three specific genes, fliC from E. coli O157:H7, cgcA from Cronobacter spp., and invA from Salmonella spp., were selected and used to design primers and probes. False-positive results were eliminated with the use of a combined sodium deoxycholate (SD) and propidium monoazide (PMA) treatment. Using the optimized parameters, this SD-PMA treatment combined with multiplex real-time PCR (SD-PMA-mRT-PCR) detected E. coli O157:H7, Cronobacter spp. and Salmonella spp. respectively, at 10 cfu/mL in pure culture or artificially spiked skim milk samples. A reasonable recovery rate (from 100 to 107%) for detection of viable bacteria using the SD-PMA-mRT-PCR assay was obtained in the presence of dead bacteria at 10 cfu/mL. The SD-PMA-mRT-PCR method developed in this study can accurately detect and monitor combined contamination with E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk and milk products.