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Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study |
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| Authors: | Alexander Schriewer Kelly D. Goodwin Christopher D. Sinigalliano Annie M. Cox David Wanless Jakob Bartkowiak Darcy L. Ebentier Kaitlyn T. Hanley Jared Ervin Louise A. Deering Orin C. Shanks Lindsay A. Peed Wim G. Meijer John F. Griffith Jorge SantoDomingo Jennifer A. Jay Patricia A. Holden Stefan Wuertz |
| Affiliation: | 1. Department of Civil and Environmental Engineering, University of California Davis, One Shields Ave, Davis, CA 95616, USA;2. National Oceanic and Atmospheric Administration, Atlantic Oceanographic and Meteorological Laboratory, Miami, FL, USA;3. National Oceanic and Atmospheric Administration, Northwest Fisheries Science Center Laboratory, Seattle, WA, USA;4. Cooperative Institute for Marine and Atmospheric Studies, University of Miami, Miami, FL, USA;5. Department of Civil and Environmental Engineering, University of California Los Angeles, 5732 Boelter Hall, Los Angeles, CA, USA;6. Bren School of Environmental Science & Management and Earth Research Institute, University of California, Santa Barbara, CA, USA;g UCD School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland;h U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, Cincinnati, OH, USA;i Southern California Coastal Water Research Project, Costa Mesa, CA, USA;j U.S Environmental Protection Agency, Microbial Contaminants Control Branch, National Risk Management Research Laboratory, Cincinnati, OH, USA;k Singapore Centre on Environmental Life Sciences Engineering (SCELSE), School of Biological Sciences and School of Civil and Environmental Engineering, Nanyang Technological University, Singapore |
| Abstract: | The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment. |
| Keywords: | Quantitative PCR Microbial source tracking Bacteroidales Canine fecal pollution |
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