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Identification of Bacterial DNA Markers for the Detection of Human and Cattle Fecal Pollution - SLIDES
Citation:
SHANKS, O. C. Identification of Bacterial DNA Markers for the Detection of Human and Cattle Fecal Pollution - SLIDES. Presented at Great Lakes WATER Institute, University of Wisconsin-Milwaukee, Anchor Watch , Milwaukee, WI, November 05 - 07, 2008.
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Description:
Technological advances in DNA sequencing and computational biology allow scientists to compare entire microbial genomes. However, the use of these approaches to discern key genomic differences between natural microbial communities remains prohibitively expensive for most laboratories. We report the application of a genome fragment enrichment method that identifies genomic regions that differ between the total DNA of different fecal microbial communities. In two separate experiments, either human or cow fecal microbial community DNA was hybridized against a pig fecal DNA background. A total of 819 individual clones were sequenced and screened for redundancy. Dot blot analysis of 677 non-redundant sequences confirmed that 97.7% of the sequences were specific for respective human or cow fecal microbial communities. Bioinformatic analyses of non-redundant sequences indicated a preponderance of Bacteroidetes-like regions (41.3%). Oligonucleotide primers capable of annealing to 29 of these sequences did not amplify pig fecal DNA and exhibited different levels of specificity with fecal DNA from other animal sources. Eight PCR assays exhibited high levels of specificity (> 99%) including four human-specific and three cow-specific assays. These assays also demonstrated a broad distribution of genetic markers among respective host source populations. Two cow-specific and two human-specific assays were modified for real-time quantitative PCR (qPCR) applications. Using respective host-specific qPCR assays, the abundance of genetic marker was measured in either untreated sewage samples or fecal samples collected from different cattle populations and compared to quantities determined by qPCR assays specific for ribosomal RNA gene sequences from total Bacteroidetes and enterococci fecal microorganisms.