|Silva, Christopher - Chris|
Submitted to: ARS Food Safety and Inspection Service Research Workshop
Publication Type: Abstract Only
Publication Acceptance Date: 3/4/2016
Publication Date: 3/7/2016
Citation: Wu, V.C., Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Patfield, S.A., He, X. 2016. Safe, rapid, and sensitive method of quantitating and distinguishing among Shiga toxins in complex media and human serum. ARS Food Safety and Inspection Service Research Workshop. ARS-FSIS.
Technical Abstract: Shiga toxins are primarily responsible for the virulence associated with Shiga toxin producing Escherichia coli (STEC) infections. The expression of the Shiga toxins is controlled by a phage that infects the host. More than one phage can infect a host and the host can inactivate infecting phages. This means that having an intact stx gene does not mean it can be expressed. It also means that a given host can produce more than one toxin. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the trypsin digestion of the B subunits. We produced a gene encoding a single protein that yields a set of relevant tryptic peptides. This gene was used to prepare 15N-labeled protein by growing the expressing bacteria in medium supplemented with 15NH4Cl. These 15N-labeled internal standards were used to detect, quantify and distinguish among the known Shiga toxins in the low attomole (10-18) range in complex media, including human serum. As new Shiga toxins are identified, this approach can be adapted to detect them. The reduction/alkylation/trypsin digestion necessary for this mass spectrometry-based analysis completely inactivates the toxins present in a 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. The analysis can be accomplished within 5 h.