A Rapid Enzyme-Linked Immunomagnetic Electrochemical (ELIME) Assay for the Detection of Escherichia coli O26 in Raw Milk

In this work, we present a magnetic bead-based immunosensorss for the determination of Escherichia coli O26 in milk. The assay uses commercial Dynabeads (R) for the capture of the target bacteria from 6-h enrichment broth and horseradish peroxidase-labelled polyclonal antibodies and detection of the peroxidase activity by chronoamperometry. Bovine seroalbumin was used as blocking reagent for reducing the binding of non-specific antibodies to the beads and/or other bacteria. The effectiveness of the enzyme-linked immunomagnetic electrochemical (ELIME) assay to detect low number of E. coli O26 in milk was demonstrated using spiked milk samples from five different lots with concentrations of 9.00-18.80 or 0.90-1.88 CFU in 25 mL. Three repetitions for each concentration were analysed in parallel also by real-time PCR following the standard method ISO/TS 13136:2012, using 24-h enriched cultures. Results demonstrated that the ELIME assay can reduce the time for analyses to one working day and produce reliable results that can be confirmed by overnight cultures of the beads-bacteria complexes allowing further characterization of the virulence, including the presence of Shiga toxin-encoding genes, which is important to discriminate the enterohaemorrhagic strains.