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United States Department of Agriculture

Agricultural Research Service

Research Project: Immunodiagnostics to Detect Prions and Other Important Animal Pathogens

Location: Produce Safety and Microbiology Research

Title: Safe and effective means of detecting and quantitating Shiga-like toxins in attomole amounts

Authors
item Silva, Christopher
item Erickson-Beltran, Melissa
item Skinner, Craig
item Dynin, Irina
item Hui, Colleen
item Patfield, Stephanie
item Carter, John
item He, Xiaohua

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 24, 2014
Publication Date: April 24, 2014
Citation: Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Dynin, I., Hui, C., Patfield, S.A., Carter, J.M., He, X. 2014. Safe and effective means of detecting and quantitating Shiga-like toxins in attomole amounts. Analytical Chemistry. DOI:10.1021/ac402930r.

Interpretive Summary: There are a variety of Shiga-like toxins (Stx) and they are the cause of the symptoms associated with some of the more serious food-borne illness. It is important to be able to detect these toxins at low levels and to discriminate among the Stx subtypes in order to identify the source of the outbreak and treat the patients. We have developed a sensitive test to detect the presence of these toxins and to distinguish among some of the subtypes. We are able to detect toxins at a level well below that necessary to cause disease. Furthermore, the sample preparation procedures necessary for this analysis completely inactivate the toxins. Thus, this approach is both a safe and effective method of detecting Shiga-like toxins, since it does not require the use of intact and active toxins.

Technical Abstract: Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with STEC infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B 'subunits. Stable-isotope-labeled analogs were prepared and used as internal standards to identify and quantitate these characteristic peptides. The calibration curves had excellent correlation coefficients (>0.99) and were linear over a > 100-fold range. We determined that this approach can be used to detect and quantitate Shiga toxins (Stx), Shiga-like toxin type 1 (Stx1), and type 2 (Stx2) subtypes. The limit of detection is in the low attomole range. Furthermore this approach can be used to distinguish among many of the known Stx1 and Stx2 subtypes. In addition we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins.

Last Modified: 12/20/2014
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