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Detection and Quantification of Human Fecal Pollution with Real-Time PCR
Citation:
SHANKS, O. C., C. A. KELTY, K. M. WHITE, M. SIVAGANESAN, J. W. SANTO-DOMINGO, M. VARMA, AND R. A. HAUGLAND. Detection and Quantification of Human Fecal Pollution with Real-Time PCR. Presented at American Society of Microbiology 108th General Meeting, Boston, MA, June 01 - 05, 2008.
Impact/Purpose:
Poster Presentation
Description:
ABSTRACT Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for enumeration of two recently described human-specific genetic markers. Both assays exhibited a range of quantification from 10 to 1x106 copies of target DNA with a coefficient of variation of < 2.5%. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominately detected human fecal specimens when tested against 267 fecal DNA extracts from 20 different animal species. The abundance of each human-specific genetic marker was measured in raw sewage samples collected from 20 geographically distinct populations and compared to quantities determined by real-time PCR assays specific for 16S rRNA gene sequences from total Bacteroidetes and enterococci fecal microorganisms. A sample preparation control was also included for all raw sewage samples to monitor extraction efficiency. Significant differences in extraction efficiency were observed between two different extraction protocols. Assay performances combined with the prevalence of DNA targets across different human populations provide experimental evidence supporting the future application of these quantitative methods in monitoring fecal pollution in ambient environmental waters.