Multiplex Microbead Immunoassay for Detection of the Ten Most Common Shiga Toxin-Producing Escherichia coli Pathogen Serogroups in Food

Introduction:  Non-O157 Shiga Toxin-producing Escherichia coli (STEC) are a growing concern, as they have been linked to many recent outbreaks. Although six STEC serogroups are now regulated as adulterants in the US, important emerging threats include highly virulent serogroups (e.g., O104) that are not included in the “Top Six.”

Purpose:  We have generated a 10-plex immunoassay for rapid detection and identification of the 10 serogroups most commonly involved in United States outbreaks of STEC: O26, O45, O91, O103, O104, O111, O113, O121, O145 and O157.

Methods:  We used antibodies (Ab) specific for the LPS O-antigens of STEC, in a sandwich immunoassay format, using the same polyclonal Ab for both capture and detection. We used the Luminex MAGPIX® fluorescent magnetic microsphere platform, which facilitates assay multiplexing. After developing the assay with pure cultures grown overnight in BHI, we validated it in milk, lettuce, and ground beef spiked with 3 strains of each serogroup.

Results:  Our assay yielded 100% sensitivity and specificity for the 30 strains tested, for all 3 foods with all 10 individual assays comprising the 10-plex. Background was low, and S/N ranged from 62 to 251. We observed limited cross-contamination of samples, which was verified via multiplex PCR STEC assay.

Significance:  This assay may be used for detection and identification of the 10 most common STEC serogroups in contaminated/adulterated milk, lettuce, and ground beef. It can be used to accelerate outbreak identification and trace-back. Importantly, the Luminex format provides immunomagnetic bead isolation of live bacteria, which may be used for subsequent confirmation and additional characterization.