| Enzyme assays were developed for the measurement of β-D-glucuronide (GUS) as a rapid detection method for E. coli in food. A fluorescent assay utilizing 4-methylumbelliferyl-β-D-glucuronide as substrate was optimized and tested with growth cultures of E. coli. A relationship between E. coli and the level of GUS activity was established. A chemiluminescent assay using a 1,2-dioxetene derivative as substrate was compared to the fluorescent assay, and was found to be 10x more sensitive.; Anti-E. coli GUS antibodies were covalently immobilized on magnetic beads and used in a 30 min immunocapture method. GUS was recovered from E. coli grown at 37°C in an induction medium containing p-nitrophenyl-β-D-glucuronide at 0.3 mM concentration. This method provided GUS measurement increases up to 81x compared to the chemiluminescent assay in culture filtrate, and permitted detection of 1 CFU/ml of E. coli within 9 h.; The effect of other microorganisms on growth and GUS production of E. coli was tested using Salmonella typhimurium, Staphylococcus aureus, and E. coli O157:H7 with initial inoculum ratios of 1:1 and 100:1 of each microorganism to E. coli. E. coli O157:H7 was the only one which caused 1.5 and 4 h delay in GUS detection time, compared to the control.; The specificity of the chemiluminescent enzyme capture was tested with GUS preparations from shellfish and raw liver. Immunomagnetic beads did not give cross-reaction with GUS from any of the samples.; The minimum detection time for GUS was determined when E. coli was inoculated into pasteurized milk, raw vegetable and ground beef samples at ∼2 CFU/ml. In milk samples with ∼102 and ∼10 3 CFU/ml background microflora, GUS was detectable after 9 h of incubation. In cut vegetables with background microflora of ∼107 CFU/ml, the detection time for GUS was 12 h. In meat samples having background flora of ∼104 and 105 CFU/ml, GUS was detectable with a 5 min centrifugation step after 10 h of incubation. These results for E. coli detection in food were equivalent to the standard procedure that required 24 to 48 h incubation for presumptive results. |
