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Development of rapid methodology utilizing immunomagnetic separation for the detection of Listeria monocytogenes and Escherichia coli O157:H7 in foods
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The use of immunomagnetic separation (IMS) to supplant lengthy traditional enrichment procedures currently required by many rapid foodborne pathogen detection methods was investigated. IMS was combined with enzyme-amplified electrochemical immunoassays for both Listeria monocytogenes and Escherichia coli 0157:H7 and the resulting procedures evaluated using pure culture and inoculated food samples. An assay combining IMS with an enzyme-linked immunosorbent assay (ELISA) for E. coli 0157:H7 was also developed and evaluated against the current USDA-FSIS protocol for the detection of E. coli 0 1 57: H7 in ground beef. The procedures developed through incorporation of IMS and an enzyme-amplified electrochemical immunoassay produced inconsistent and unreliable data, making the dependable detection of either microorganism evaluated impossible. These findings led to the development of a new assay combining IMS with an ELISA procedure for E. coli 0157:H7. The IMS-ELISA procedure provided calorimetric detection of E. coli 0157:H7 in pure culture at levels as low as-1 log10 cells/ml, prompting further investigation evaluating inoculated ground beef samples. Subsequent analysis of the procedure indicated the possibility of detecting E. coli 0157:H7 at a lower limit of 100 cells/g in ground beef samples, but subjective visual scoring was believed to be causing false positive and negative results. In order to eliminate subjective visual scoring, an ELISA plate reader was used to measure sample absorbance for quantification and subsequent determination of test results. A centrifugation step was also incorporated into the procedure to further enhance assay sensitivity. Using the modified procedure, a lower limit of detection for E. coli 0157:H7 was determined to lie in the range of 2-4 log10 cells/g sample. In an effort to increase assay sensitivity sample size was increased. The sample size increase provided no significant increase in assay sensitivity, but did suggest that further evaluation of the procedure is needed. The USDA-FSIS protocol for detecting E. coli 0157:H7 in raw ground beef proved to be more sensitive than the IMS-ELISA. Recovery of E. coli 0157:H7 was most efficiently accomplished at cell numbers ranging from 1-4 log10 cells/g, but was also observed at a lower limit of-1 log10 cells/g.
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Cunningham, James Clark (1996). Development of rapid methodology utilizing immunomagnetic separation for the detection of Listeria monocytogenes and Escherichia coli O157:H7 in foods. Master's thesis, Texas A&M University. Available electronically from
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