You are here:
Laboratory Evaluations of the Enterococcus qPCR Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements
Citation:
HAUGLAND, R. A., S. SIEFRING, M. VARMA, AND L. J. WYMER. Laboratory Evaluations of the Enterococcus qPCR Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements. Presented at 108th General Meeting of the American Society for Microbiology, Boston, MA, June 01 - 05, 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:
The BEACH Act of 2000 directed the U.S. EPA to establish more expeditious methods for the detection of pathogen indicators in coastal waters, as well as new water quality criteria based on these methods. Progress has been made in developing a quantitative PCR (qPCR) method for enterococci that meets the requirements for rapid, risk-based water quality assessments at beaches. However, questions remain over how this method should be used in water quality criteria. These questions relate in part to the lack of a clear understanding of the performance characteristics of the method such as detection limits and precision under the influence of mixed sources of variation. In the present study, single laboratory results are reported that further define these attributes. The 95% confidence method detection limit (95% MDL) was estimated at ~100 calibrator cell equivalents (CE)/sample from analysis results of replicate 100 ml buffer samples spiked with 10-1000 cultured E. faecalis cells. This CE value can be translated to a 95% MDL of 3230 target sequence copies (TSC)/sample based on the recovery efficiency of 32 TSC/cell determined for these samples or 5.4 TSC/reaction based on the use of 0.166% of the sample extracts for PCR analysis. Pure method precision, expressed as log10 variance, was estimated as 0.018, 0.054 and 0.114 from spikes of 1000, 333 and 100 cells, respectively. Analyses of diverse surface water samples indicated that the influence of non-interfering matrices on log10 variance was negligible. Based on analyses of different sets of beach water samples from 2003-2005 U.S. EPA epidemiological studies where the geometric means for ambient enterococci by qPCR were within 100 to 1000 CE, the 90% prediction interval was estimated as 170 to 650 for a true value of 333 CE. These analyses further indicated that the major source of the measurement uncertainty was associated with sample variability. Accurate characterization of beach water quality by this method is therefore highly dependent upon the analysis of multiple samples.