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.

Many food safety incidents are reported worldwide every year, and the diseases caused by these incidents have seriously threatened world public health. In 2020, the World Health Organization (WHO) reported that 550 million people worldwide had diarrhea caused by consuming contaminated food, among which about 230,000 people died (at a death rate of 0.04%). Foodborne diseases have become the most harmful disease to public health. There are dozens of known foodborne pathogens, among which the most prominent are Salmonella spp., S. aureus, L. monocytogenes, B. cereus, Escherichia coli O157:H7, Shiga toxin-producing E. coli, Vibrio spp. and etc. Among those foodborne pathogens, Salmonella spp. are significant cause agents of the global zoonosis. WHO reported that among 582 million cases of 22 foodborne diseases during 2010 to 2015, there were 351,000 deaths and 52,000 deaths were originated by Salmonella spp. making a death ratio of 14.8% and this type of death ranking in the first place. Foodborne Salmonella spp. infection is reported every year around the globe after consuming contaminated food such as contaminated milk powder, meat, crayfish, eggs and melon. Foodborne Salmonella spp. infection is so important but currently the food surveillance of this pathogen has not been sufficiently implemented yet. For example, the detection rate of Salmonella spp. within retail meat products in Shanghai was only 26.1% in 2016. S. aureus is ubiquitously found in nature and produces heat-resistant enterotoxins during its proliferation in food. When the enterotoxin content in 100 g of food reaches 18 µg, food poisoning could be caused. In recent years, the foodborne disease outbreaks caused by S. aureus and its enterotoxins have made this infection among the top three ranked in China. Food poisoning incidents caused by B. cereus have been reported worldwide. This bacterial contaminant is generally found in rice and noodle products but also detected in eggs, meat, dairy products, and aquatic products with a detection rate of 25.5%. According to the U.S. Center for Disease Control and Prevention, L. monocytogenes can survive and proliferate in refrigerated food for an extended period and is the third dominant cause of death from foodborne diseases in the United States, characterized by insufficient available medical treatments and high mortality. Wang, et al.. found that the contamination rate of L. monocytogenes in raw materials and intermediate product samples in instant food factories was 16.1%, which shows this pathogen is a significant safety hazard. The current detection of the above four foodborne pathogens mainly relies on the techniques such as selective cultivation and biochemical identification, which are complicated and time-consuming with low accuracy and a high risk of misdetection, especially for the detection with instant food. Therefore, a more rapid, accurate, and high throughput method is urgently needed to monitor the pathogenic risks to food safety.

ddPCR is considered a rapid, accurate, and absolute DNA quantitative PCR method developed in recent years as the third-generation nucleic acid amplification technology. In its reactions, the individual target DNA molecules are firstly distributed into a such large quantity of droplets as micro-reaction units, to maintain one or no copy of the target DNA molecule in each micro-reaction unit, then the DNA amplification and fluorescence signal detection are carried out, and finally the absolute quantitation of the target DNA molecule is achieved based on the Poisson distribution principle without the need for standard curves. At present, ddPCR is mainly used for medical gene mutation analysisfood-derived component analysistransgenic analysisand the quantitative detection of some foodborne pathogens. In the literature so far with the best of our understanding, ddPCR has been reported mostly as a singleplex or duplex method for the quantitative detection of pathogens and there are no reports on the simultaneous detection of four bacterial pathogenic bacteria using a quadruplex ddPCR technology for the detection of multiple target genes in the same PCR reactions with a variety of samples of food origins.

In this study, we developed a quadruplex ddPCR-based method for simultaneous and sensitive detection and quantitation of four health-related pathogens of generic interest: S. Typhi, S. aureus, L. monocytogenes, and B. cereus using a two-channel ddPCR instrument (QX200, Bio-Rad). We demonstrated that this quadruplex ddPCR method is equivalent to the conventional plate counting method in reliability but less time- and labor-consuming.