Development of a Rapid Method to Quantify Salmonella Typhimurium Using a Combination of MPN and qPCR with a Shortened Enrichment Time

Introduction: Since Salmonella spp. are one of the more prevalent pathogens causing foodborne disease in humans, thus it is of public health considerable concern to the food industry. Conventional plating methods based on selective and differential media or most-probable-number (MPN) methods to quantify Salmonella are labor and time intensive, therefore, it is critical to develop more rapid and accurate detection methods that allow for quantification.

Purpose: In this study, a detection method was developed to quantify Salmonella Typhimurium using MPN combined with qPCR and a shortened time enrichment (MPN-qPCR-STE).

Methods: For Salmonella Typhimurium enumeration, 10-fold dilutions of cell suspension was transferred into three wells on a microtiter plate (same as three-tube MPN assay) and the plate was subsequently incubated at 37º for 4 h. After nonselective enrichment with nutrient broth (tryptic soy broth), the presence of Salmonella Typhimurium in each well was identified using a qPCR and bacterial populations were determined based on calculating MPN. Bacterial cell populations were also determined with various quantification methods including conventional MPN, plating methods, and qPCR alone to compare across independent methods.

Results: The coefficient of determination (r²) between MPN-qPCR-STE and the conventional MPN exhibited a high level of correlation (0.9752), suggesting that the MPN-qPCR-STE offers a reliable alternative method for Salmonella Typhimurium quantification. Although conventional plating and qPCR were limited in their ability to detect low population levels of Salmonella Typhimurium (e.g., 0.18 log MPN/ml), these levels could be successfully detected with MPN-qPCR-STE.

Significance: The obvious strengths of  the MPN-qPCR-STE are that 1) further confirmation step is not required, 2) the detection limit is as low as conventional MPN, but 3) is more rapid, requiring approximately 8 h to simultaneously complete identification and quantification. This method can be utilized to rapidly quantify Salmonella Typhimurium saving both time and labor and may be particularly useful for the food industry and related applications where quantification is important.