Development of a 3-plex droplet digital PCR for identification and absolute quantification of Salmonella and its two important serovars in various food samples

Abstract

Salmonella is one of the most common foodborne pathogens that cause diarrhea in human, in which Enteritidis and Typhimurium are the top serovars frequently isolated from foodstuffs. A novel 3-plex droplet digital PCR (ddPCR) was successfully developed in this study for the simultaneous identification and absolute quantification of Salmonella and its two important serovars (Enteritidis and Typhimurium). The specificity of all the primers and probes was validated by testing 24 strains of Salmonella and 10 strains of other bacteria. Meanwhile, the anti-interference ability of this method was also evaluated using high proportion of non-target strains or various bacterial combinations. The detection limit of this 3-plex ddPCR was as low as 5 fg/μL for gDNA and 101 CFU/mL for pure cultures, respectively. Moreover, compared with quantitative PCR (qPCR), the detection limit of this ddPCR was much lower when it was applied to the detection of artificially contaminated food samples, which was 101 CFU/mL for lettuce, and 102 CFU/mL for milk and chicken juice samples, respectively. Additionally, a total of 100 retailed food samples were tested by this validated 3-plex ddPCR, which was also compared with traditional cultivation method. Notably, 19 of 20 samples showed consistent positive results by both methods, while the rest with the lowest concentration of Salmonella cells was detectable only by ddPCR. These results demonstrated that the 3-plex ddPCR established in this study presented highly specific, sensitive, and absolute quantitative capability, which had the potential to identify Salmonella, and to differentiate the two important serovars Enteritidis and Typhimurium in a wide variety of food samples.