The diagnostic methods implemented at the Canadian Food Inspection Agency for the detection of Shiga toxin producing E. coli (STEC) are time consuming and tedious, taking up to 5 days before a positive sample can be confirmed. The goal of this project was to streamline the detection procedure for serogroup O157 and 6 important non-O157 serogroups of STEC. Following a short enrichment step (4-6 hrs), two approaches were considered: (1) the filtration of enrichment culture through a gradient of filtration membranes (decreasing pore sizes), followed
by capture using specific monoclonal antibody (mAb)-coated Dynabeads, and detection via fluorescence microscopy, (2) the addition of enrichment culture into a flow through system consisting of a column packed with large polystyrene beads (≥ 100 μm) coated with specific mAbs for capture. The results indicate that the filtration approach can only be applied to simpler food matrices. However, at least 100 CFU of the target STEC could be recovered using the filtration system following 4 hrs of enrichment of these matrices spiked with ≤ 15CFU of the target STEC. Similar capture results were obtained in the second approach using specific mAbs immobilized on covalently coupled protein G polystyrene beads and diluted enrichment media. A combination of these strategies together with immunofluorescence microscopy (IMS) and polymerase chain reaction (PCR) could provide diagnostic laboratories with a means to confirm a positive sample within 2 days of testing.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/34284 |
| Date | January 2016 |
| Creators | Kumaran, Dilini |
| Contributors | Wang, Lisheng, Lin, Min |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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