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Standardization of enterococci density estimates by EPA qPCR methods and comparison of beach action value exceedances in river waters with culture methods
Haugland, Rich, S. Siefring, M. Varma, A. Dufour, K. Brenner, Tim Wade, E. Sams, S. Cochran, S. Braun, AND Mano Sivaganesan. Standardization of enterococci density estimates by EPA qPCR methods and comparison of beach action value exceedances in river waters with culture methods. JOURNAL OF MICROBIOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 105(7):59-66, (2014).
Provide technical information on the performance of the EPA Enterococcus qPCR methods for monitoring recreational beach water quality to potential decision makers, stake-holders and end-users
The U.S.EPA has published recommendations for calibrator cell equivalent (CCE) densities of enterococci in recreational waters determined by a qPCR method in its 2012 Recreational Water Quality Criteria (RWQC). The CCE quantification unit stems from the calibration model used to estimate enterococci densities in recreational beach waters in multiple epidemiological studies under the EPA National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study and directly informed the derivation of the RWQC recommendations. This calibration model assumes that recovery efficiencies of DNA fragments containing the targeted gene region (target sequences) of the qPCR assay in extracts of filters containing cultured Enterococcus cells (calibrator samples) correspond to the recovery efficiencies from cells collected by filtration of ambient water samples. Comparability of CCE estimates to each other and to RWQC values also requires that the number of recoverable target sequences per calibrator cell be consistent when different cultured Enterococcus cell preparations are used in the method by different laboratories or studies. Assessments from recent studies have suggested that this requirement is not being met. To address this issue, recoveries of target sequences and the mean target sequence/cell ratio for the NEEAR study calibrator samples were retrospectively estimated with a corroborated standard curve from the study. The resulting overall mean ratio of 15 sequences/cell can be used to facilitate comparisons of future CCE estimates with the RWQC qPCR recommendations. To illustrate this process, a modification of the method calibration model was used to directly estimate enterococci target sequence densities from qPCR analysis results of 234 water samples from eight midwestern U.S. rivers. CCE estimates were obtained by dividing these target sequence estimates by the NEEAR study sequences/cell ratio. This process resulted in a high degree of agreement in beach action decisions (determinations of whether bacterial fecal indicator densities are above or below RWQC-recommended values) from results of the qPCR method and from culture dependent enumeration of both enterococci and Eschericia coli in the corresponding water samples.