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United States Department of Agriculture

Agricultural Research Service

Research Project: Technologies for Detecting and Determining the Bioavailability of Bacterial Toxins

Location: Foodborne Toxin Detection and Prevention

Title: Microbead based assays for detection and characterization of E. coli in pure cultures

Authors
item Bernard Iv, Clay
item Clotilde, Laurie -
item Lin, Andrew -
item Hartman, Gary -
item Salvador, Alexandra -
item Lauzon, Carol -
item Muldoon, Mark -
item Lindpaintner, Klaus -
item Lau, David -
item Carter, John

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 17, 2012
Publication Date: N/A

Technical Abstract: Recently, the Centers for Disease Control and Prevention (CDC) has identified that the top 6 non-O157 Shiga toxin-producing E. coli (STEC) are emerging pathogens presenting significant risks of disease. These, as well as the infamous E. coli O157, cause an estimated 265,000 infections each year in the US, though most of these infections go unreported. Conventional enzyme linked immunosorbent assay (ELISA) is a gold standard for detecting antigen. However, a significant limitation is that only one analyte can be assessed per microplate well. Therefore, a reliable, sensitive, and rapid assay capable of simultaneously detecting E. coli and the main toxins produced by STEC (i.e., Shiga toxins 1 [Stx1] and 2 [Stx2]) will directly benefit regulatory agencies by minimizing analysis time. Here, we use Luminex technology to detect multiple analytes in a single 100 µL sample. Using fluorescent paramagnetic microbeads (Luminex), we developed two assays. One is a 9-plex immunoassay capable of simultaneously serotyping the top 7 E. coli: O26, O45, O111, O103, O121, O145 and O157, as well as detecting Stx1 and/or Stx2. The other is a 10-plex PCR assay capable of simultaneously serotyping the top 7 E. coli above, as well as serogroups O91, O113, and O128. When we tested the specificity of our assays against E. coli strains belonging to the 7 most virulent serogroups; >95% accuracy was observed. This microbead-based immunoassay clearly works in pure cultures, and we plan to validate them for detection of these pathogens and toxins in ground beef, lettuce, and milk.

Last Modified: 4/21/2014
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