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Single Laboratory Comparison of Quantitative Real-time PCR Assays for the Detection of Fecal Pollution
Citation:
KELTY, C. A., K. M. WHITE, J. C. BLANNON, M. C. MECKES, O. C. SHANKS, M. SIVAGANESAN, M. VARMA, AND R. A. HAUGLAND. Single Laboratory Comparison of Quantitative Real-time PCR Assays for the Detection of Fecal Pollution. Presented at American Society for Microbiology 110th General Meeting, San Diego, CA, May 23 - 27, 2010.
Impact/Purpose:
To inform the public.
Description:
There are numerous quantitative real-time PCR (qPCR) assays available to detect and enumerate fecal pollution in ambient waters. Each assay employs distinct primers and probes that target different rRNA genes and microorganisms leading to potential variations in concentration estimates of fecal pollution in impacted waters. Laboratory comparisons are needed to determine which assays are best suited for characterization of fecal pollution originating from different animal sources. We report the performance assessment of four previously published general fecal indicator qPCR assays that detect rRNA genes from Clostridales, Escherichia coli, enterococci, and Bacteroidales microorganisms. All assays exhibited a range of quantification from 10 to 4x104 copies of target DNA with a mean coefficient of variation across the range of quantification less than 2.1%. Each assay was tested against a reference collection of 54 untreated wastewater samples collected from different geographical locations across the United States and 169 fecal DNA extracts from 21 different animal species. Genetic markers were broadly distributed among untreated wastewater samples (100%) and specific animal sources (57% to 100%). In addition, the abundance of each general fecal indicator genetic marker was determined in both untreated sewage and specific animal fecal samples. Performance experiments indicate that all assays offer precise estimates of respective in DNA targets, but show substantial differences in genetic marker concentration between different animal sources. Notice - Although this work was approved by EPA for publication, it may not reflect official Agency policy.
