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

<title>Abstract</title> To establish a 4-plex ddPCR method for quantitatively detecting the four major pathogenic bacteria: <italic>Salmonella</italic>, <italic>Staphylococcus aureus</italic>, <italic>Listeria monocytogenes</italic>, and <italic>Bacillus cereus</italic> 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&gt;0.999). The linear dynamic ranges were: 33-21504 copies/20µL for <italic>Salmonella</italic>, 28-18404 copies/20µL for <italic>Staphylococcus aureus</italic>, 25-27012 copies/20µL for <italic>Listeria monocytogenes</italic> and 15-15560 copies /20µL for <italic>Bacillus cereus</italic>. 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 (<italic>Salmonella</italic>), 7 copies/20µL (<italic>Staphylococcus aureus</italic>), 9 copies/20µL (<italic>Listeria monocytogenes</italic>) and 7 copies/20µL (<italic>Bacillus cereus</italic>). 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.