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The bacterium Escherichia coli of serotype 0157:H7 is a major human intestinal pathogen being responsible for the Jack in The Box hamburger outbreak in 1993 in the State of Washington and recently was found to be the cause of over 9,000 cases and 11 deaths in Japan, with hamburger again implicated. In order to identify contaminated meat before distribution from slaughter houses the development of methods for the rapid detection of specific numbers of this bacterium is essential.
Methodology will be developed for the direct detection of low numbers of E. coli 0157:H7 in ground beef within an 8 hour period. Ground beef (20 g) will be seeded with a varying number of E. coli 0157:H7 and the beef then added to 180 ml of 0.85% sodium chloride with the addition of 50 ml of corn oil. A motorized tissue disintegrator will then be used to disrupt and homogenize the tissue which we have shown does no damage to the bacterial cells. The corn oil serves to dissolve the fat content so as not to form a protein-fat-water emulsion. We have found that low speed centrifugation results in an optically clear middle water phase containing nearly all of the bacterial cells, with fat and oil at the top and tissue particles at the bottom. If the middle water phase is then subjected to high speed centrifugation, nearly all bacterial cells form a pellet at the bottom of the tube. The bacterial pellet will then be suspended in 2 ml of 0.01M phosphate buffer and glass immuno-magnetic beads coated with antibody to E. coli 0157 will be added for selective capture of the targeted bacterial cells. The magnetic immuno beads will then be used directly to amplify and detect the sequence of DNA coding for VT1 and VT2 toxins by applying the polymerase chain reaction to the captured bacterial cells on the surface of the beads. The quantity of amplified DNA will then be used to determine the original number of E.coli 0157:H7 cells.