Standardization of enterococci density estimates by EPA qPCR methods and comparison of beach action value exceedances in river waters with culture methods.

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 the EPA National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study and directly informed the derivation of the RWQC recommendations. Recent studies have demonstrated that CCE estimates from the method can vary when using different cultured Enterococcus cell preparations in calibrator samples. These differences have been attributed to differences in the quantities of targeted gene copies (target sequences) that are recovered per nominal calibrator cell by DNA extraction. Standardization of results from the calibration model will require the estimation of target sequence recoveries from the calibrator and water samples. In addition, comparisons of water sample results with the RWQC values will require a knowledge of target sequence recoveries from the NEEAR study calibrator samples. In this study 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. A modification of the calibration model was then used to estimate enterococci target sequence quantities in water samples from eight midwestern U.S. rivers. CCE estimates were obtained by dividing these target sequence quantities by the mean NEEAR study target sequence/cell ratio. This target sequence-based quantification approach 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 CCE results of the qPCR method and from culture dependent enumeration of both enterococci and Eschericia coli in the corresponding water samples.

Comparison of Enterococcus quantitative polymerase chain reaction analysis results from Midwest U.S. river samples using EPA Method 1611 and Method 1609 PCR reagents.

Enterococci target sequence density estimates from analyses of diluted river water DNA extracts by EPA Methods 1611 and 1609 and estimates with lower detection limits from undiluted DNA extracts by Method 1609 were indistinguishable. These methods should be equally suitable for comparison with U.S. EPA 2012 Recreational Water Quality Criteria values.

Comparison of Enterococcus quantitative polymerase chain reaction analysis results from fresh and marine waters on two real-time instruments.

The U.S. Environmental Protection Agency will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for implementation of this and other qPCR methods is whether the results are comparable on different PCR instruments. In this study, quantitative estimates of Enterococcus densities from marine and freshwater samples were determined by the qPCR method from cycle threshold (Ct) measurements obtained on Applied Biosystems StepOnePlus and Cepheid SmartCycler instruments. Three variations of a comparative Ct model, differing in their sources of calibration data, were used in the estimations. Both traditional and Bayesian statistical modeling approaches were examined in the instrument comparisons. The traditional analysis of variance (ANOVA) approach indicated no significant differences (p>0.05) between mean density estimates from the instruments in two of the three model variations. The Bayesian approach indicated that the 95% Bayesian credible intervals of density estimates from the instruments overlapped in all models; however, the uncertainty of the estimates varied depending on the model. These results support the interchangeable use of the two instruments in the method and also illustrate the importance of defining the source of calibration data used in the comparative Ct model.