A ddPCR method for simultaneous detection and quantification of Salmonella enterica, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus in foods

The traditional methods for the detection and quantification of foodborne bacteria are time-consuming with the potential for false negative pathogen-free results. Rapid and effective detection and quantification of foodborne pathogenic microorganisms are crucial in prevention and control of foodborne diseases. In the last decade, the technique of droplet digital polymerase chain reaction (ddPCR) has been widely applied for the precise quantification of gene expression. The aim of this study is to establish a quadruplex ddPCR method to detect and quantify simultaneously four major bacterial pathogens, Salmonella enterica serotype Typhi, Staphylococcus aureus, Listeria monocytogenesand Bacillus cereus, potentially co-existing in instant food. A strong linear correlation (r²>0.999) was observed in the ranges of 33-21500 copies/20µL for S. Typhi, 28-18400 copies/20µL for S. aureus, 25-27000 copies/20µL for L. monocytogenes and 15-15600 copies /20µL for B. cereus respectively. For this established ddPCR method, the lower detection limits were 8 copies/20µL (S. Typhi), 7 copies/20µL (S. aureus), 9 copies/20µL (L. monocytogenes) and 7 copies/20µL (B. cereus) respectively. For its evaluation this method was also compared in parallel with the plate counting method using the artificially contaminated food samples with the added pathogens mentioned above. Unpaired t-test showed the results from the two methods were not statistically different but the ddPCR method is superior to the plate counting method with shorter turnaround time, lower detection limit, and more robust reproducibility.