Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources |
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| Authors: | Meredith R. Raith Catherine A. Kelty John F. Griffith Alexander Schriewer Stefan Wuertz Sophie Mieszkin Michele Gourmelon Georg H. Reischer Andreas H. Farnleitner Jared S. Ervin Patricia A. Holden Darcy L. Ebentier Jennifer A. Jay Dan Wang Alexandria B. Boehm Tiong Gim Aw Joan B. Rose E. Balleste W.G. Meijer Mano Sivaganesan Orin C. Shanks |
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| Affiliation: | 1. 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;2. Southern California Coastal Water Research Project, 3535 Harbor Blvd STE 110, Costa Mesa, CA 92626, USA;3. University California, Davis, Department of Civil & Environmental Engineering, One Shields Avenue Davis, CA 95616, USA;4. IFREMER, Laboratoire de Microbiologie – EMP/MIC, ZI Pointe du Diable BP 70, 29280 Plouzané, France;5. Research Group Environmental Microbiology and Molecular Ecology, Institute of Chemical Engineering, Vienna University of Technology, and Interuniversity Cooperation Center Water and Health, Gumpendorfer Straße 1a/166, A-1060 Vienna, Austria;6. University of California, Santa Barbara, Earth Research Institute and Bren School of Environmental Science & Management, 3508 Bren Hall, Santa Barbara, CA 93106, USA;g University of California, Los Angeles, Civil and Environmental Engineering, 7678 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA 90095, USA;h Stanford University, Department of Civil & Environmental Engineering, Jerry Yang & Akiko Yamazaki Environment & Energy Building, 473 Via Ortega, Stanford, CA 94205, USA;i Department of Fisheries and Wildlife, 13 Natural Resources Building, Michigan State University, East Lansing, MI 48824, USA;j UCD School of Biomolecular and Biomedical Science, University of College Dublin, Dublin 4, Ireland |
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| Abstract: | The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation. |
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| Keywords: | Microbial source tracking Fecal pollution Ruminants Cattle |
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