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Quantitative PCR for detection and enumeration of genetic markers of bovine fecal pollution.

Shanks OC, Atikovic E, Blackwood AD, Lu J, Noble RT, Domingo JS, Seifring S, Sivaganesan M, Haugland RA

U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, OH 45268, USA. shanks.orin@epa.gov

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 bovine 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 2 x 10(6) 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 bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-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 bovine fecal pollution in ambient waters.

Published 4 February 2008 in Appl Environ Microbiol, 74(3): 745-52.
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