Validation of a Cell-Free Translation Assay for Detecting Shiga Toxin 2 in Bacterial Culture

Authors: He, Xiaohua; Quinones, Beatriz; Carter, John; Mandrell, Robert

Submitted to: Journal of Agriculture and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/31/2009
Publication Date: 5/14/2009
Citation: He, X., Quinones, B., Carter, J.M., Mandrell, R.E. 2009. Validation of a Cell-Free Translation Assay for Detecting Shiga Toxin 2 in Bacterial Culture. Journal of Agriculture and Food Chemistry. 57(11):5084-5088

Interpretive Summary: This research provides validation of a rapid, sensitive, and specific assay to detect a foodborne toxin responsible for hundreds of illnesses and numbers of fatalities in the U.S. and elsewhere in recent years. Scientists from two food safety teams at the Agricultural Research Service's Western Regional Research Center in Albany, CA, have collaborated to develop a test-tube assay that detects the activity of the most important Shiga toxin (Stx) produced by the bacterium E. coli O157:H7. Known as Stx2, this toxin prevents cells from making protein and is important in determining the pathogenicity of this bacterium. E. coli O157:H7 has been found in about 1 in 600 samples of ground beef, the most common source of human infection in this country. Methods such as this "cell-free translation assay" are needed for effective monitoring of the nation's food supply, to prevent further outbreaks of life-threatening hemorrhagic colitis and hemolytic uremic syndrome associated with consumption of contaminated food. In addition to providing a tool for monitoring foods, the new assay also permits food scientists to determine the precise conditions for destroying toxin activity in processed foods.

Technical Abstract: We have validated a cell-free translation (CFT) assay for detecting Shiga toxin (Stx). The limit of detection (LOD) for pure Stx2 (PStx2) and partially pure Stx2 (PPStx2) in water reached 20 pg/µl and 3.5 pg/µL respectively without the artificial process of proteolytic activation and reduction of the pro-toxin. The LOD for PStx2 and PPStx2 in ground beef were 80 pg/µL and 5 pg/µL, respectively. The toxicity of Stx2 could be neutralized by a specific mouse monoclonal antibody. We demonstrated that this assay could be used for identification of Stx-producing Escherichia coli (E. coli) with the presence of beef extract. Four E. coli O157:H7 strains different genotypically for the Stx were tested. The translational inhibition of Stx-producing E. coli was significantly higher than that of non Stx-producing E. coli using bacterial culture supernatants for the analysis. Inhibition occurred even with supernatants diluted 1000-fold. Differences in the thermal stability of Stx2 depending upon the purity and food matrix of the preparation were indicated by no loss of activity with PStx2 and absence of activity with PPStx2 heated 71ºC for 60 min; and 50% decrease in activity in the bacterial culture supernatant after 20 min at 71ºC, with minimal decrease with further heating. However, in the beef extract the toxicity of bacterial culture supernatant was reduced by 90% after 20 min of heating. We conclude that the CFT assay is a rapid and sensitive method for measuring activity of Stx2 in ground beef and that PStx2 is more stable than PPStx2 to heat.