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Comparison of Enterococcus qPCR analysis results from fresh and marine water samples on two real-time instruments -
Citation:
HAUGLAND, R. A., M. VARMA, R. OSHIRO, J. Parr, AND M. Doolittle. Comparison of Enterococcus qPCR analysis results from fresh and marine water samples on two real-time instruments - . Presented at National Beaches Conference 2009, Huntington Beach, CA, April 20 - 22, 2009.
Impact/Purpose:
The first objective of this study is to develop and evaluate qPCR assays and test methods for the detection and quantification of genetic markers from indicator bacteria that are associated with human fecal waste and to determine the occurrence and densities of these markers in waste waters and fecal material from different animal sources. The second objective is to similarly develop and/or evaluate qPCR assays and test methods for genetic markers from two other groups of general fecal indicator bacteria that historically have been widely used or are favored in specific regions. These bacterial groups include E. coli which has been accepted and widely used for freshwater monitoring and C. perfringens which has been favored in tropical and subtropical regions. The final objective is to determine the densities of these human and general fecal indicator markers, as well as previously identified markers for enterococci and total Bacterioidales,in a wide range of surface and recreational waters including archived samples from the previous NEEAR studies. Results from these studies will be used to determine correlations between indicator marker densities obtained by the best available new qPCR assays and test methods and swimming associated health risks by the National Health and Environmental Effects Laboratory and for the development of new national water quality criteria by the Office of Water.
Description:
EPA is currently considering a quantitative polymerase chain reaction (qPCR) method, targeting Enterococcus spp., for beach monitoring. Improvements in the method’s cost-effectiveness may be realized by the use of newer instrumentation such as the Applied Biosystems StepOneTM and StepOnePlusTM series instruments that can retail for under $20 K and provide 48 or 96 sample analysis capacity. In this study we compared the results obtained on a StepOnePlusTM 96 well instrument with those obtained on the Cepheid Smart Cycler® which has been the primary source of the method’s results to date. Analyses were performed simultaneously on DNA extracts from multiple, replicate filter retentates of 12 marine and 12 freshwater samples from diverse locations using study and data analysis designs from EPA's microbial alternate test procedure protocol. Precision among log10 target sequence copy (TSC) estimates in the samples from the two instruments were compared with no significant difference (p > .05) based on the one-way ANOVA of Levene's Test for Homogeneity of Variance. Three–way ANOVA with fixed factors: instrument, matrix, instrument*matrix; and random factors: sample (nested in matrix) and inst*sample (nested in matrix) was used to compare the mean log10 TSC estimates with no significant difference seen between the instruments (p > .05). Given the wide variety of qPCR instruments that are already available and the likelihood that additional advances will occur in instrument technology, this study may provide a useful model for the design and implementation of additional comparative studies in the future.