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Applied and Environmental Microbiology, February 2008, p. 745-752, Vol. 74, No. 3
0099-2240/08/$08.00+0     doi:10.1128/AEM.01843-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution{triangledown} ,{dagger}

Orin C. Shanks,1* Emina Atikovic,3 A. Denene Blackwood,2 Jingrang Lu,1 Rachel T. Noble,2 Jorge Santo Domingo,1 Shawn Seifring,3 Mano Sivaganesan,1 and Richard A. Haugland3

U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, Ohio 45268,1 University of North Carolina at Chapel Hill, Institute of Marine Sciences, 3431 Arendell St., Morehead City, North Carolina 28557,2 U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, Ohio 452683

Received 8 August 2007/ Accepted 26 November 2007

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 106 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.

* Corresponding author. Mailing address: U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, OH 45268. Phone: (513) 569-7314. Fax: (513) 569-7328. E-mail: shanks.orin{at}epa.gov

{triangledown} Published ahead of print on 7 December 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, February 2008, p. 745-752, Vol. 74, No. 3
0099-2240/08/$08.00+0     doi:10.1128/AEM.01843-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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