Development of an mRNA based method for molecular detection of E. coli O157:H7 in carcass material

Sharon De Wet (2010). Development of an mRNA based method for molecular detection of E. coli O157:H7 in carcass material PhD Thesis, School of Chemistry & Molecular Bioscience, The University of Queensland.

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Author Sharon De Wet
Thesis Title Development of an mRNA based method for molecular detection of E. coli O157:H7 in carcass material
School, Centre or Institute School of Chemistry & Molecular Bioscience
Institution The University of Queensland
Publication date 2010-04
Thesis type PhD Thesis
Supervisor Prof. Lindsay Sly
Dr. Chris McSweeney
Dr. Stuart Denman
Total pages 146
Total colour pages 1
Total black and white pages 145
Formatted abstract
Since its emergence in 1981, E. coli O157:H7 has been identified by the United States
Food Safety and Inspection Service (FSIS) as a pathogen and a zero-tolerance policy has
been adopted with respect to the presence of the organism in food products. Australia is
the second largest exporter of red meat in the world and the United States constitutes
its largest customer. The responsibility lies with the exporter, that is, with Australia, to
prove that its product is free of E. coli O157:H7. The industry standard for detection of
viable E. coli O157:H7 relies on overnight enrichment of sampled carcass material
followed by concentration of cells by immuno-magnetic-separation (IMS) for isolation of
the pathogen prior to biochemical, serological and/or molecular identification of the
isolates. Detection of small numbers of E. coli O157:H7 in the presence of other faecal
flora can be difficult due to the high sensitivity required of the test. In addition, a false
positive result has very serious implications for the export trade and the methods thus
have to be highly specific as well. Standard culture and IMS procedures can take up to 72
hours to perform. Furthermore should the need to detect other unwanted pathogens
arise, a different culture conditions need to be implemented for each target organism.
The need for a sample processing procedure able to detect small numbers of viable E.
coli O157:H7 while omitting lengthy and species-specific culture enrichment and IMS was
identified. RNA extraction procedures for small numbers of cells from an environment
with low levels of inhibitors, such as carcass surfaces, was optimised, as well as
procedures for environments with high levels of cells and inhibitors, such as rumen fluid
and bovine faecal material. Investigations into limits of detection and non-biased
amplification of bacterial mRNA (cDNA) from lowest possible bacterial numbers were
also carried out. Following optimised RNA extraction procedures, a method for mRNA
enrichment was developed and compared to a commercial procedure. mRNA
amplification of the total mRNA enriched material was introduced as a means of
increasing sensitivity from small populations of cells, such as low levels of introduced E.
coli O157:H7. The sample processing procedure was developed and efficiency
demonstrated by agarose gel electrophoresis. The sample processing procedure was
compared to the industry standard method under laboratory conditions. The level of
detection was similar (19 CFU ml-1) but the time needed from sampling to detection and
identification of the desired target using the introduced sample processing procedure
was reduced to 30 hours, compared with 72 hours for the industry standard method.
Moreover, the single sample processing procedure has the potential to be utilised for the
detection of multiple pathogens.
Keyword E. coli O157:H7, RT-PCR, detection, mRNA enrichment, mRNA amplification
Additional Notes Landscape printing: Page 14-17 of thesis which are in factpage 32-35 of the final PDF document. Colour page: 54 of thesis which is page 89 of the PDF document. "Attachment" is a publication, which needs to be added to the back of the printed thesis.

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Created: Mon, 18 Oct 2010, 10:27:46 EST by Ms Sharon De Wet on behalf of Library - Information Access Service