title: Validation of a cell-free translation assay for detecting shiga toxin 2 in bacterial culture.
abstract: A cell-free translation (CFT) assay for detecting Shiga toxin (Stx) at different levels of purity has been validated. The limits of detection for pure Stx2 (PStx2) and partially pure Stx2 (PPStx2) in water reached 20 and 3.5 pg/microL, respectively, without the artificial process of proteolytic activation and reduction of the pro-toxin. The specific detection of Stx2 was confirmed by a neutralization test using Stx2-specific mouse monoclonal antibody. This assay can be used for differentiation of Stx-producing Escherichia coli from non-Stx-producing E. coli. Four E. coli O157:H7 strains genotypically different for Stx were tested. The translational inhibition of Stx-producing E. coli was significantly higher than that of non-Stx-producing E. coli when bacterial culture supernatants were used for the analysis. Inhibition occurred even with supernatants diluted 1000-fold. The thermal stability of Stx2 was studied using the CFT assay, and significant differences were observed among three Stx2 preparations heated at 70 degrees C for 60 min. It was concluded that the CFT assay is a rapid, specific, and sensitive method for detecting Stx2 activity.
authors.affiliation: US Department of Agriculture, Western Regional Research Center, Agricultural Research Service, Albany, California 94710, USA. xiaohua.he@ars.usda.gov
authors.forename: Xiaohua
authors.initials: X
authors.lastname: He
authors.forename: Beatriz
authors.initials: B
authors.lastname: Quiñones
authors.forename: John Mark
authors.initials: JM
authors.lastname: Carter
authors.forename: Robert E
authors.initials: RE
authors.lastname: Mandrell
mesh_terms: D001681:Biological Assay / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019453:Escherichia coli O157 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects*; D055550:Protein Stability; D022641:Shiga Toxin 2 / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D022641:Shiga Toxin 2