Detection of enterohaemorrhagic Escherichia coli in food by droplet digital PCR to detect simultaneous virulence factors in a single genome

Abstract

Shiga toxin producing E. coli are a problem for food producers. STEC's require a combination of virulence factors to cause disease, so ideally detection techniques should detect the presence of multiple virulence factors in a single cell directly from food. Droplet Digital PCR (ddPCR) is commonly used to quantify the number of copies of a gene in a sample, moreover it is able to link two genes to the same piece of DNA. Here stx and an O-antigen specific gene are detected simultaneously with taqman probes confirming that the cells are intact as well as distinguishing between strains based on their genotype. Using ddPCR E. coli O157:H7 and O104:H4 are quantified from apple juice, milk and spinach washings without an enrichment step, the detection limit of ddPCR in apple juice was 2 cfu/mL. Also, ddPCR was used to detect pathogenic bacterial cells in the presence of background strains which shared one or none of the target genes, including avirulent strains. Whole cell ddPCR is compared to several DNA extraction techniques demonstrating that whole cell ddPCR is more reliable for linking genes within an organism. Whole cell ddPCR is a promising technique for the rapid and specific detection of foodborne pathogens.