A 4-plex droplet digital PCR (ddPCR) to quantity four food-borne pathogenic bacteria simultaneously

To establish a 4-plex ddPCR method for quantitatively detecting the four major pathogenic bacteria: Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus in instant food simultaneously, primers, probes, and PCR conditions were optimized. For the 4-plex ddPCR assay, four target pathogens demonstrated a good linear correlation to the readouts (r²>0.999). The linear dynamic ranges were: 33-21504 copies/20µL for Salmonella, 28-18404 copies/20µL for Staphylococcus aureus, 25-27012 copies/20µL for Listeria monocytogenes and 15-15560 copies /20µL for Bacillus cereus. The relative standard deviations (RSD) at different concentrations of the target pathogens were all less than 25% (n = 5) indicating the good repeatability of the assay. The detection limits were 8 copies/20µL (Salmonella), 7 copies/20µL (Staphylococcus aureus), 9 copies/20µL (Listeria monocytogenes) and 7 copies/20µL (Bacillus cereus). The established 4-plex ddPCR assay was evaluated in parallel with the classical plate colony-counting method to detect the target pathogens in the four types of simulated samples. The results from the two assays are similar with deviations less than 9%. Still, the PCR assay appears superior to the plate method, evidenced by the short turnaround time, lower detection limit, and robust reproducibility.