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Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution
SHANKS, O. C., E. ATIKOVIC, A. D. BLACKWOOD, J. LU, R. T. NOBLE, J. W. SANTO-DOMINGO, S. SEIFRING, M. SIVAGANESAN, AND R. A. HAUGLAND. Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution. L. N. Ornston (ed.), APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 74(3):745-752, (2008).
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Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described cow feces-specific genetic markers and a method for the enumeration of these markers using a Markov Chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2x106 copies of target DNA with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the cow-specific genetic target and presence of amplification inhibitors. The assays only detected cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual cow samples (98-100%) collected from five geographically distinct locations. The abundance of each cow-specific genetic marker was measured in 48 individual samples and compared to qPCR enumerated quantities of rRNA gene sequences representing total Bacteroidetes, Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring cow fecal pollution in ambient waters.
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Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL RISK MANAGEMENT RESEARCH LABORATORY
WATER SUPPLY AND WATER RESOURCES DIVISION
MICROBIAL CONTAMINANTS CONTROL BRANCH