An Alternative Method to Concentrate E. coli O157:H7 from Meat Homogenates to Facilitate Detection by Cultural and Molecular Methods

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dc.contributor.advisor MaryAnne Drake, Committee Co-Chair en_US
dc.contributor.advisor Donn Ward, Committee Member en_US
dc.contributor.advisor LeeAnn Jaykus, Committee Co-Chair en_US Taylor, Thomas Matthew en_US 2010-04-02T17:58:09Z 2010-04-02T17:58:09Z 2003-09-15 en_US
dc.identifier.other etd-06122003-171935 en_US
dc.description.abstract Escherichia coli O157:H7, first identified in 1982, is linked to approximately 73,500 cases of foodborne illness per year. Sensitive and efficient methods to detect this organism are crucial. Current bacterial concentration methods from foods, such as centrifugation and filtration, are problematic, often co-precipitating or becoming clogged with food particulates. Metal hydroxide concentration of bacterial cells has gained increased recognition as a new alternative method to non-specifically concentrate bacteria from foods (Lucore et al., 2000; Cullison and Jaykus, 2002). The objective of this research was to develop and optimize the application of a metal hydroxide concentration method for recovery of viable E. coli O157:H7 from ground beef with subsequent polymerase chain reaction (PCR) detection. Hypotheses that metal hydroxide concentration would lead to increased cell recoveries compared to controls and that subsequent detection by PCR would gain higher detection sensitivity were tested for E. coli O157:H7 in autoclaved and raw ground beef samples. In the study, 10 g autoclaved and raw ground beef samples were artificially contaminated with E. coli O157:H7 cells at 102, 104, or 106 CFU/ml; autoclaved and raw ground beef samples were also seeded with a low level inoculum (1 CFU/g beef) and allowed to incubate to previously mentioned inoculum levels. Bacterial concentration consisted of two primary steps: centrifugation (primary concentration) and metal hydroxide immobilization (secondary concentration). PCR primers targeted a 254-bp portion of the slt-II gene. Southern hybridization was conducted on nylon membranes to confirm presence of DNA amplicons and gain higher detection limits. Recoveries exceeded 70% for cooked beef samples without growth and 57% for cooked beef samples with growth. Further, recoveries for raw ground beef samples exceeded 66% for samples concentrated without growth and 55% for samples concentrated after growth in a selective medium. In all cases, loss to supernatant did not exceed 20%. Overall, there were no statistically significant differences in samples concentrated with the metal hydroxide and samples concentrated with a saline control. The PCR detection limit effectively was 102 CFU/ml for all samples except raw, no growth cell concentrates. Southern hybridization afforded confirmation and an increase in detection limits for all samples except from growth, raw beef cell concentrates. Data indicate that model food systems may not always be accurate indicators of a bacterium's behavior in a food matrix. Future research is recommended to investigate the mechanisms by which bacterial cells are less readily recovered following growth in a food. en_US
dc.rights I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. en_US
dc.subject PCR detection en_US
dc.subject E. coli O157:H7 en_US
dc.subject ground beef en_US
dc.subject metal hydroxides en_US
dc.subject Bacterial concentration en_US
dc.title An Alternative Method to Concentrate E. coli O157:H7 from Meat Homogenates to Facilitate Detection by Cultural and Molecular Methods en_US MS en_US thesis en_US Food Science en_US

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