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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPMENT OF DETECTION TECHNOLOGIES FOR TOXINS AND THEIR VALIDATION IN FOOD MATRICES Title: Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods

Authors
item Clotilde, Laurie
item Hartman, Gary -
item Lau, David -
item Carter, John

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 12, 2010
Publication Date: March 1, 2011
Citation: Clotilde, L.M., Hartman, G.L., Lau, D.K., Carter, J.M. 2011. Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods. Journal of Food Protection. 74(3):373-379.

Interpretive Summary: Shiga toxin-producing Exchericia coli (STEC) are disease-causing bacteria with high economic impact. In the US, the annual cost of health care related to STEC infections reaches up to $2 billion with 500 deaths. These bacteria cause disease by producing toxins. The well-known STEC 0157 bacteria can be detected by identification of these toxins as well as the 0157 "signature" molecule on the surface of the bacteria. Currently, there are no commercially available kits capable of simultaneously detecting STEC 0157 and its toxins in food such as ground beef, lettuce, and milk. Thus we developed such a test that provides results in less than 24 hours. Our test works with magnetic fluorescent microbeads capable of detecting both STEC 0157 and its toxins, while simplifying recovery of the bacteria from food. This is of special interest to regulatory agencies as they need to recover the bacteria before they can recall contaminated food. Moreover, because of its improved speed, our test is ideal for use in food processing plants to allow in-house testing of products prior to sale.

Technical Abstract: Shiga toxin–producing Escherichia coli (STEC) is a significant foodborne pathogen with great economic consequences. There has been an increased food safety concern with this organism since outbreaks of human illnesses caused by this pathogen were first reported in 1982. Therefore, developing a reliable, sensitive, and rapid assay capable of detecting E. coli O157 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 50-ml sample. Using commercially available monoclonal antibodies coupled to carboxylated magnetic microbeads, we developed an immunoassay capable of simultaneously serotyping E. coli O157 and detecting Stx1 and/or Stx2. The specificity and sensitivity of this immunoassay was tested against a collection of 34 E. coli isolates belonging to various O serogroups phenotypically different for Stx. The results were compared with microplate sandwich enzyme-linked immunosorbent assay (ELISA), and no cross-reactivity was observed for any of the monoclonal antibodies used. An increased sensitivity up to 1,000 times was observed in the microbead-based immunoassay when compared with the microplate sandwich ELISA. The results indicate that Luminex technology has the potential to simultaneously detect multiple targets without loss of specificity and/or sensitivity. A blind experiment was conducted with 48 samples of ground beef, lettuce, and milk spiked with less than or equal to 2 CFU/g E. coli. All the samples were correctly identified, with no false positives or false negatives. This microbead-based immunoassay could be extended to imultaneously detect additional foodborne pathogens and their toxic markers.

Last Modified: 10/31/2014
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