Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Pathology

Supervisor

Dr. Michael J. Rieder

Abstract

Escherichia coli O157 is a persistent pathogen linked to food and waterborne infectious outbreaks with severe health consequences such as hemorrhagic colitis and hemolyticuremic syndrome (HUS). Because it is considered one of the major pathogens that contributes to the global burden of foodborne disease, its early detection within the food chain is an important milestone towards reducing foodborne diseases and economic losses due to contaminated food. Herein, the development and validation of a lateral flow immunoassay (LFIA) point-of-care (POC) device is described. Application of the LFIA test kit was focused on detection of E. coli O157 in raw meat products due to the fact that ground beef has been one of the major food items implicated in E. coli outbreaks and recalls within Canada. Moreover, the LFIA Test Kit was subjected to an independent validation study based upon Health Canada’s guidelines for the validation of alternative microbiological methods as established in the Compendium of Analytical Methods. The protocol comprised a pre-collaborative study, where the LFIA Test Kit was compared against the reference culture method, MFHPB-10, using eight different raw meat products following an unpaired samples experimental layout. The results demonstrated that the newly developed LFIA Test Kit exceeds the performance parameters criteria established by the Microbiological Methods Committee (MMC), thus suggesting that the LFIA Test Kit represents a reliable alternative for meat producers in order to obtain presumptive presence/absence results in less than one day. The design and expression of a single-chain variable fragment (scFv) targeting E. coli O157 is also presented. Recombinant antibody fragments such as scFv have not been extensively exploited within food safety diagnostics, especially for pathogen detection. Thus, in this project the anti-O157 mouse monoclonal antibody (mAb) used as the detection reagent in the LFIA Test Kit was genetically sequenced prior to bioengineering a scFv that could potentially be used to improve the performance of the LFIA Test Kit.

Available for download on Saturday, September 29, 2018


Share

COinS