|CLARK, MIKE - Bio-Rad Laboratories|
|CARIOU, ASTRID - Bio-Rad Laboratories|
|LEVEAU, ADELAIDE - Bio-Rad Laboratories|
|PIERRE, SOPHIE - Bio-Rad Laboratories|
|FRATAMICO, PINA - Collaborator|
Submitted to: International Journal of Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/24/2019
Publication Date: 12/28/2019
Citation: Capobianco Jr, J.A., Armstrong, C.M., Clark, M., Cariou, A., Leveau, A., Pierre, S., Fratamico, P., Strobaugh Jr, T.P. 2019. Detection of Shiga toxin producing Escherichia coli (STEC) in beef products using droplet digital PCR. International Journal of Food Microbiology. 319(2020):108499.
Interpretive Summary: Consumption of most Escherichia coli will not lead to illness. However, human consumption of Shiga toxin producing Escherichia coli (STEC) may induce serious illness, or even death. Routine inspection of food and food production facilities helps reduce the probability of STEC contaminated foods from reaching consumers. The assessment of foods relies heavily on testing methods. The currently validated microbiological methods used in STEC detection, employs a screening step to differentiate between pathogenic and benign Escherichia coli. The purpose of the screening step is to help maximize the opportunity cost of regulators and producers alike by identifying only samples which merit further testing. A screening assay based upon droplet digital polymerase chain reaction (ddPCR) was developed and validated which demonstrates a greater degree of efficiency compared to currently used systems. This detection platform will offer significant cost-savings through the reduction of the costs associated with assessing samples that do not contain the foodborne pathogen, and enable more samples to be analyzed.
Technical Abstract: Many of the current accredited methods for the molecular detection of Shiga toxin producing Escherichia coli (STEC) in foods rely on a PCR-based screen for the pathotype-specific genetic markers stx and eae. Unfortunately, these methods often suffer from a high rate of false-positive results, partly due to the inability of the methods to determine if both stx and eae originated from a single organism as opposed to a mixture of organisms. This study was undertaken to evaluate a droplet digital PCR (ddPCR)-based method that could reduce the false positive rate associated with current STEC screening methods by confirming that both stx and eae reside within the same cell. The ddPCR system used in this study, dd-Check STEC Solution (Bio-Rad), works by partitioning intact cells into emulsion droplets, which subsequently undergo multiplexed endpoint PCR. This enables the assay to differentiate between samples where a single organism contains both stx and eae from samples in which stx and eae reside in different organisms. Comparison were made between the dd-Check STEC Solution and two commercially available real-time PCR assays using over 35 unique simulations of STEC contamination in ground beef with the dd-Check STEC Solution demonstrating an equivalent sensitivity to the established PCR screening techniques. Moreover, the results indicated that the dd-Check STEC Solution has the potential to reduce the number of false-positive results when screening for STEC because the assay could distinguish between the co-existence of multiple genes within the same cell from a mixed microbial population; specifically, the stx and eae genes. Ultimately, this system will result in cost-savings by reducing the man-hours and testing expenses associated with the evaluation of false-positive samples.