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Comparison of Enterococcus quantitative polymerase chain reaction analysis results from midwest U.S. river samples using EPA Method 1611 and Method 1609 PCR reagents
Citation:
Sivaganesan, Mano, S. Siefring, M. Varma, AND Rich Haugland. Comparison of Enterococcus quantitative polymerase chain reaction analysis results from midwest U.S. river samples using EPA Method 1611 and Method 1609 PCR reagents. JOURNAL OF MICROBIOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 101(6):9-17, (2014).
Impact/Purpose:
Impact: 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
Description:
The U.S. Environmental Protection Agency (EPA) has provided recommended beach advisory values in its 2012 recreational water quality criteria (RWQC) for states wishing to use quantitative polymerase chain reaction (qPCR) for the monitoring of Enterococcus fecal indicator bacteria in their waters. Two alternative qPCR methods (currently available EPA Method 1611 and soon to be released EPA Method 1609) will be available for these water quality analyses. These EPA-validated methods are similar in most respects with the primary difference being in the PCR reagents that they feature. Method 1611 uses an older reagent formulation (Universal Master Mix) while Method 1609 will use a more recently introduced reagent (Environmental Master Mix). Analysis results with both reagents of DNA extracts from 221water samples from seven major midwestern U.S. rivers were used to compare frequencies of samples that failed EPA acceptance criteria for two different PCR interference control assays and to compare Enterococcus target sequence density estimates obtained from the EPA calibration model. Differences in control assay results were most pronounced in analyses of undiluted DNA extracts with Universal Mix analyses showing >50% acceptance criteria failures as opposed to <5% for Environmental Mix. Conversely, control assay failure rates were similar for the two reagents (<5%) in analyses of five-fold dilutions of the extracts. Enterococcus target sequence density estimates from analyses of five-fold diluted DNA extracts of the samples with the two reagents were not significantly different as determined using a traditional statistical analysis model and showed highly overlapping 95% credible intervals using a Bayesian statistical model. Similar results were observed in comparisons of estimates from analyses of undiluted and diluted DNA extracts using Environmental Master Mix only. These findings suggest that Enterococcus density estimates from analyses of five-fold diluted DNA extracts by Methods 1611 and 1609 may be equally compared to the EPA RWQC beach advisory values. Moreover, analysis results from undiluted DNA extracts by Methods 1609 also may be applicable for this purpose.
