Quantitative PCR for genetic markers of bovine fecal pollution. Export

@article{citeulike:2137485, abstract = {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 2x10(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 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 bovine samples (98-100\%) collected from five geographically distinct locations. The abundance of each bovine-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 bovine fecal pollution in ambient waters.}, 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, USA; University of North Carolina at Chapel Hill, Institute of Marine Sciences, 3431 Arendell St., Morehead City, NC 28557, USA; U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, OH. 45268, USA.}, author = {Shanks, Orin C C. and Atikovic, Emina and Blackwood, A Denene D. and Lu, Jingrang and Noble, Rachel T T. and Domingo, Jorge Santo S. and Seifring, Shawn and Sivaganesan, Mano and Haugland, Richard A A.}, citeulike-article-id = {2137485}, citeulike-linkout-0 = {http://dx.doi.org/10.1128/AEM.01843-07}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/18065617}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=18065617}, day = {7}, doi = {10.1128/AEM.01843-07}, issn = {0099-2240}, journal = {Appl Environ Microbiol}, keywords = {bovin, marker}, month = {December}, posted-at = {2007-12-17 16:38:07}, priority = {2}, title = {Quantitative PCR for genetic markers of bovine fecal pollution.}, url = {http://dx.doi.org/10.1128/AEM.01843-07}, year = {2007} }

TY - JOUR ID - citeulike:2137485 L3 - citeulike-article-id:2137485 N2 - 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 2x10(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 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 bovine samples (98-100%) collected from five geographically distinct locations. The abundance of each bovine-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 bovine fecal pollution in ambient waters. JF - Appl Environ Microbiol SN - 0099-2240 AD - 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; University of North Carolina at Chapel Hill, Institute of Marine Sciences, 3431 Arendell St., Morehead City, NC 28557, USA; U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, OH. 45268, USA. TI - Quantitative PCR for genetic markers of bovine fecal pollution. KW - bovin KW - marker AU - Shanks, Orin C AU - Atikovic, Emina AU - Blackwood, A Denene AU - Lu, Jingrang AU - Noble, Rachel T AU - Domingo, Jorge Santo AU - Seifring, Shawn AU - Sivaganesan, Mano AU - Haugland, Richard A PY - 2007/12/07/ UR - http://dx.doi.org/10.1128/AEM.01843-07 ER -