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

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.

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.

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.

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.