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Evaluation of Commercial Cell Preparations as Sources of Calibration Standards for Real-Time qPCR Analysis of Enterococci in Recreational Waters
Citation:
SIEFRING, S., R. A. HAUGLAND, AND L. J. WYMER. Evaluation of Commercial Cell Preparations as Sources of Calibration Standards for Real-Time qPCR Analysis of Enterococci in Recreational Waters. Presented at Great Lakes Beaches Association Annual Meeting, Porter, IN, September 15 - 17, 2008.
Impact/Purpose:
1. Determine effects of storage by freezing of NEEAR study samples on QPCR measurements of enterococci. 2. Identify, from an expanded pool of candidates, the general fecal indicator microorganism(s) (i.e. different taxonomic or phylogenetically-related groups from all animal sources) whose densities in NEEAR study water samples, as measured by QPCR analysis, best correlate with illness rates monitored in the NEEAR study. 3. Determine whether illnesses rates in the NEEAR studies are better correlated with QPCR measurements of fecal indicator organisms from human as opposed to general sources (subject to the availability or development of suitable QPCR assays for human fecal indicators).
Description:
In response to the Beach Act, the U.S. EPA has developed a quantitative PCR (qPCR) method for enterococci that meets requirements for rapid, risk-based water quality assessments of recreational waters. Widespread implementation of this method will require that different laboratories obtain consistent quantitative results. An important prerequisite will be the availability of reproducible, stable and sustainable standards. In this study two commercial sources of enumerated and lyophilized Enterococcus cells, BioBallTM (BTF, Ltd.) and EpowerTM (MicroBioLogics, Inc.) were used to prepare calibration samples for the qPCR method. The precision and recovery of target sequences from multiple samples prepared from each these cell sources and from currently used laboratory cultured cells were determined from qPCR cycle threshold (CT) results and by comparisons with a standard curve from known target sequence quantities. Unlike cultured cells, samples prepared from both of these products required filtration to remove PCR-inhibitory materials before DNA extraction. The ratios of variances in target sequence recoveries from replicate Epower and BioBallTM samples were 2.48 (p = 0.068) and 1.10 (p = 0.433), respectively, compared to the cultured cell samples. Lower precision obtained from the BioBallTM samples may be related to the lower cell quantities in these samples (~550 CFU vs. ~10^5 CFU in the EpowerTM and cultured cell samples). Absolute recoveries of target sequences per CFU from the three cell sources were similar. Each of these products has potential advantages in either cost (EpowerTM) or ease of use and more appropriate cell numbers for quantifying recreational water sample results (BioBallTM).