According to the latest figures from the Centers for Disease Control (2011) foodborne illnesses caused by Shiga toxin-producing Escherichia coli (STEC) are responsible for 265,000 cases of food-borne illness, 3600 hospitalizations, and 30 deaths each year in the US. Of these, 96,000 infections are caused by E. coli O157:H7, while non-O157 STECs cause another 169,000 infections. Rapid detection of live non-O157 STEC is an important unmet requirement for food safety.Using a fluorescent magnetic microbead assay platform (Luminex Magpix) we formatted a multiplex sandwich immunoassay targeting 10 of the clinically most common STEC serogroups, including: O26, O45, O91, O103, O104, O111, O113, O121, O145, and O157. We validated the assay against three distinct isolates of each of these 10 STEC serotypes, spiked into milk and culture-amplified overnight.Positive samples gave signals ranging from 2000 to 55,000 (Mean Fluorescent Intensity units, MFI), while mismatched STEC controls gave signals of 100 to 12,000 MFI. Both absolute range and signal-to-noise ratio varied between the different assays and analytes, suggesting different positive threshold values for the different assays in the multiplex. In particular we noted that our assays for O91 and O104 gave relatively weaker signals for both positive and negative samples. We also noted that our three O104 strains produced high background cross reactions in the other assays. High non-specific binding may be a property inherent to O104, and we plan to look at more strains of this serovar. We also plan to validate the multiplex assay in spiked lettuce and ground beef.

Multiplex microbead immunoassay for detection in food of the 10 most common Shiga toxin producing E. coli pathogen serotypes

248th National Meeting (2014)

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