Detection and colocalization of STEC genetic markers in wheat flour using whole cell digital droplet PCR.

INTRODUCTION: Shiga toxin-producing Escherichia coli (STEC) poses a significant food safety risk in wheat-based products. The detection of these pathogens in complex food matrices is challenging due to the labor-intensive nature of conventional methodologies and the variability in definitions across different countries. This study investigated whole cell digital droplet PCR (ddPCR) to detect the co-occurrence of critical STEC genetic markers in inoculated and naturally contaminated wheat flour. METHODS: We optimized the ddPCR protocol for the detection and colocalization of critical STEC genetic markers, namely stx1, stx2, eae, and the Top 7 serogroups. Furthermore, we developed a novel quantitative PCR (qPCR) assay to serve as a generic marker for E. coli, specifically targeting the yecE gene, for which we demonstrated inclusivity for E. coli and exclusivity against non-target species, both in silico and in vivo. This assay was used in the ddPCR colocalization tests performed throughout the study. RESULTS: Both qPCR and ddPCR analytical workflows were shown to have a similar LOD50 around 0.9 CFU/g in wheat flour under the tested conditions. Our colocalization analysis was successful in wheat flour inoculated with single strains or a pool of two strains with different genetic make-ups at different inoculation levels. Furthermore, we were able to show detection and genetic association patterns by ddPCR across naturally contaminated retail flour samples. DISCUSSION: This flexible whole cell ddPCR approach addresses significant limitations of existing STEC detection methods and provides a useful data set to support the development of a more sophisticated molecular analytical method which can be applied to wheat flour to detect and characterize STEC strains.