Citation:

SHANKS, O. C. IDENTIFICATION OF BACTERIAL DNA MARKERS FOR THE DETECTION OF HUMAN AND CATTLE FECAL POLLUTION. Presented at US EPA WORKSHOP ON INNOVATIVE APPROACHES FOR DETECTING MICROORGANISMS IN WATER, CINCINNATI, OH, June 18 - 20, 2007.

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. Here we report the application of a genome fragment enrichment (GFE) method that identifies genomic regions that differ between the metagenomes 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 Bacteroidales-like regions (41.3%) predicted to encode membrane-associated proteins (52.3%). 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 extremely high levels of host-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 ranging from 61 to 100% of samples tested. Host-specific assays were then challenged against water samples collected from fecal impacted streams to explore their potential for water quality monitoring in ambient waters.

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