Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA.

Cheryl Armstrong,Fernando Rubio,Andrew Gehring,Terence Strobaugh,Leah Ruth,Joseph Capobianco

doi:10.3390/toxins10110422

Abstract

Amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) is comprised of a bead-based immunoassay that is used for small molecule detection. In this study, a novel AlphaLISA was developed and optimized for the detection of Shiga-toxin 2 (Stx2). Efficacy and sensitivity trials showed the AlphaLISA could detect ≥0.5 ng/mL of purified Stx2, which was comparable to the industry-standard enzyme-linked immunosorbent assay (ELISA) tests for Stx2 detection. In addition, evaluation of Shiga toxin-producing Escherichia coli (STEC)-inoculated Romaine lettuce and ground beef samples demonstrated that both the AlphaLISA and the ELISA were able to discern uninoculated samples from 1× and 10× diluted samples containing ~10 CFU/mL of STEC enriched in modified tryptic soy broth with mitomycin C for 16 h. Overall, the increased signal-to-noise ratios indicated a more robust signal was produced by the AlphaLISA compared to the ELISA and the delineation of higher toxin concentrations without the need for sample dilution implied a greater dynamic range for the AlphaLISA. Implementation of the newly developed AlphaLISA will allow for more rapid analysis for Stx2 with less manual manipulation, thus improving assay throughput and the ability to automate sample screening while maintaining detection limits of 0.5 ng/mL.

Highlights

Food poisoning due to Shiga toxin-producing Escherichia coli (STEC) is a consistent cause for concern in the United States because of its association with hemorrhagic colitis
Hemorrhagic colitis is characterized by the onset of a variety of symptoms including nausea, vomiting, abdominal pain, diarrhea, and bloody stools with approximately 5% of cases progressing to a more severe form of clinical disease known as hemolytic uremic syndrome (HUS) [1]
Because treatment of STEC infections with antibiotics greatly increases the risk of serious complications resulting from the development of the condition known as hemolytic uremic syndrome (HUS) [4,5] and Shiga toxin is known to be essential for the development of HUS from STEC infections [6]; the production and subsequent release of Shiga toxin has been a topic of intense investigation

Summary

Introduction

Food poisoning due to Shiga toxin-producing Escherichia coli (STEC) is a consistent cause for concern in the United States because of its association with hemorrhagic colitis. Stopping STEC contamination in food production is critical for curtailing the number of infections and preventing full-scale outbreaks from occurring, while obtaining an accurate diagnosis upon infection is crucial for ensuring proper care for patients. Production of Shiga toxin is both a distinguishing feature and an important virulence factor for STEC strains. It can be expressed by strains carrying the stx gene, which is encoded for by a lambdoid bacteriophage [7,8]. Current research separates Shiga toxin into two main antigenically distinct groups, Shiga toxin 1 (Stx1) and

Methods

Results

Conclusion