β-Glucuronidase assay for the detection of Escherichia coli in foods
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.
"β-Glucuronidase assay for the detection of Escherichia coli in foods"
Dissertations and Master's Theses (Campus Access).