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SPECIFICITY AND SENSITIVITY OF FECAL BACTEROIDETES HUMAN-SPECIFIC PRIMERS WITH FECAL AND WASTEWATER SAMPLES FROM THE U.S. MIDWEST AND NORTHEAST REGIONS
Citation:
TANG, J., J. SANTO-DOMINGO, O. C. SHANKS, C. WALDRON, P. WEISKEL, R. F. BREAULT, AND O. C. PANCORBO. SPECIFICITY AND SENSITIVITY OF FECAL BACTEROIDETES HUMAN-SPECIFIC PRIMERS WITH FECAL AND WASTEWATER SAMPLES FROM THE U.S. MIDWEST AND NORTHEAST REGIONS. Presented at American Society for Microbiology Annual Meeting, Atlanta, GA, June 04 - 09, 2005.
Impact/Purpose:
To inform the public
Description:
Numerous watersheds throughout the United States are impaired due to fecal contamination. Fecal Bacteroidetes is a group of anaerobic bacteria present in high concentrations in animal feces that has shown promise as a microbial source tracking indicator of human and other animal fecal contamination. We evaluated the specificity of fecal Bacteroidetes primers in two separate laboratories using gradient thermal cyclers from different manufacturers and fecal samples from three states (DE, MA, & OH). Fecal DNA extractions and PCR amplifications were carried out using identical protocols. PCR annealing temperatures were evaluated using a gradient ranging from 55 to 65°C. Primers were tested with human, chicken, turkey, goose, seagull, horse, sheep, goat, deer, pig, and cow fecal DNA. With the fecal Bacteroidetes group primer, all fecal samples yielded positive results on all thermal cyclers. The human-specific primers (HF134 and HF183) identified all human fecal extracts, and did not cross-react with deer, pig, or cow fecal DNA on all thermal cyclers. However, human-specific primers cross-reacted with non-target animals including chicken, goose, seagull, turkey, goat, sheep, and horse in at least one but not all thermal cyclers used in this study. For chicken and sheep feces collected from different sites, different results were obtained using the same thermal cycler. Validation appears to be necessary when using a different thermal cycler or when testing samples from different geographic regions. We also tested the sensitivity of the fecal Bacteroidetes group and human-specific primers with raw municipal wastewater. The Bacteroidetes group primer was positive when the sewage sample was diluted up to 10-4. However, the human-specific assays were 104 -fold less sensitive than the group primers. Since other studies have reported a higher incidence of these Bacteroidetes human clusters in raw sewage, our results may reflect analytical system differences (e.g., different thermal cycler), differences in municipal wastewater treatment plant characteristics (e.g., population, size served) or geographic variance in host distribution of these clusters.