The gastrointestinal tract is colonized by an abundant and diverse community of microorganisms which has a profound impact on the health of the host. The profiling of these microbial communities with traditional culture-based methods identifies only a fraction of microbes present with limited specificity, high labour costs and limited sample throughput. To overcome these limitations, a molecular hybridization assay was developed and characterized using the target gene chaperonin 60 (cpn60). The interspecies discriminatory ability of the hybridization assay was determined by hybridizing cpn60 gene fragments from a known species to a series of cpn60 gene fragments derived from related species with distinct but similar cpn60 sequences. Species with less than 85% cpn60 sequence identity to the probe DNA were effectively distinguished using the hybridization approach. To characterize complex microbial communities, universal PCR primers were used to amplify a fragment of 549-567 nucleotides from cpn60 (the cpn60 universal target (UT)) using template DNA extracted from the ileal contents of pigs fed diets based on corn (C), barley (B), or wheat (W), or from plasmids containing the cpn60 UT selected from a clone library generated from these contents. The intensity of hybridization signals generated using labelled probes prepared from library clones designated B1 (Bacillales-related), S1 (Streptococcus-related), C1 (Clostridiales-related), and L10 (Lactobacillales-related) and targets prepared from ileal contents of C, W, or B-fed pigs correlated closely with the number of genomes of each bacterial group as determined by quantitative PCR. Universal PCR primers were also used to amplify genomic DNA extracted from jejeunal contents of pre- and post-weaning piglets. Labelled probe DNA was prepared from S1, L10, LV (Lactobacillus vaginalis-related) and EC (E.coli) library clones. The resulting signal intensities correlated with quantitative polymerase chain reaction (qPCR) data for L10 and LV, but minimal correlation was observed for the S1 and EC groups. A cpn60- based macroarray has potential as a tool for identification and semi-quantification of shifts in colonization abundance of bacteria in complex communities, providing a similar amount of data as techniques such as denaturation gradient gel electrophoresis or terminal restriction fragment length polymorphism analysis.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-08272007-133445 |
| Date | 17 September 2007 |
| Creators | Goldfinch, Angela Dawn |
| Contributors | Hemmingsen, Sean M., Dumonceaux, Tim, Buchanan, Fiona C., Van Kessel, Andrew G. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-08272007-133445/ |
| Rights | unrestricted, 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 University of Saskatchewan 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. |
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