Multi-laboratory evaluations of the performance of Catellicoccus marimammalium PCR assays developed to target gull fecal sources |
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| Authors: | Christopher D. Sinigalliano,Jared S. Ervin,Laurie C. Van De Werfhorst,Brian D. Badgley,Elisenda Ballesté ,Jakob Bartkowiak,Alexandria B. Boehm,Muruleedhara Byappanahalli,Kelly D. Goodwin,Michè le Gourmelon,John Griffith,Patricia A. Holden,Jenny Jay,Blythe Layton,Cheonghoon Lee,Jiyoung Lee,Wim G. Meijer,Rachel Noble,Meredith Raith,Hodon Ryu,Michael J. Sadowsky,Alexander Schriewer,Dan Wang,David Wanless,Richard Whitman,Stefan Wuertz,Jorge W. Santo Domingo |
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| Affiliation: | 1. National Oceanic and Atmospheric Administration, Atlantic Oceanographic and Meteorological Laboratory, 4301 Rickenbacker Causeway, Miami, FL 33149, USA;2. Bren School of Environmental Science & Management, University of California, Santa Barbara, CA, USA;3. Earth Research Institute, University of California, Santa Barbara, CA, USA;4. BioTechnology Institute, University of Minnesota, St. Paul, MN, USA;5. Conway Institute, UCD School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland;6. Cooperative Institute for Marine and Atmospheric Studies, University of Miami, Miami, FL, USA;g Environmental and Water Studies, Department of Civil and Environmental Engineering, Stanford University, Stanford, CA, USA;h U.S. Geological Survey Great Lakes Science Center, Lake Michigan Ecological Research Station, Porter, IN, USA;i Laboratoire de Microbiologie, MIC/LNR, Département Ressources Biologiques et Environnement, Unité Environnement, Microbiologie et Phycotoxines, Ifremer, ZI Pointe du diable, Plouzané, France;j Southern California Coastal Water Research Project, Costa Mesa, CA, USA;k Department of Civil and Environmental Engineering, University of California Los Angeles, Los Angeles, CA, USA;l Division of Environmental Health Sciences, College of Public Health, Ohio State University, Columbus, OH, USA;m Department of Food Science & Technology, Ohio State University, Columbus, OH, USA;n Institute of Marine Sciences, University of North Carolina at Chapel Hill, Morehead City, NC, USA;o U.S Environmental Protection Agency, Microbial Contaminants Control Branch, National Risk Management Research Laboratory, Cincinnati, OH, USA;p Department of Civil and Environmental Engineering, University of California Davis, Davis, CA, USA;q Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore;r Department of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, VA, USA |
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| Abstract: | Here we report results from a multi-laboratory (n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR® Green qPCR method, and two TaqMan® qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions. |
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