Citation:

VARMA, M., L. J. WYMER, AND R. A. HAUGLAND. FECAL INDICATOR BACTERIA MEASUREMENTS BY QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ANALYSIS IN FRESH ARCHIVED DNA EXTRACT OF WATER SAMPLE FILTRATES. Presented at American Society for Microbiology 105th General Meeting, Atlanta, GA, June 05 - 09, 2005.

Impact/Purpose:

The primary objective of this task is to identify, from a statistically significant pool of candidates, the fecal indicator microorganism(s) whose densities in recreational waters best correlate with the rates of illnesses monitored in the current health studies.

A second objective (pending the availability or development of suitable assays) will be to determine whether the rates of these illnesses are correlated with fecal pollution from humans or from various specific animal sources.

Description:

The U.S. EPA has initiated a new recreational water study to evaluate the correlation between illness rates in swimmers and Enterococcus concentrations determined by the mEI agar membrane filter (MF) method and several new technologies including QPCR analysis. Results of this study in 2003 showed a significant positive correlation between QPCR and MF method measurements of Enterococcus concentrations (Haugland et al. Water Research, 2005). A potential benefit of molecular methods such as QPCR is that they may be able to provide occurrence data for new indicator organisms by analyses of freezer-archived DNA extracts with new assays. To test whether QPCR analyses of archived DNA extracts provide comparable measurements to those of the fresh extracts, DNA extracts from the 2003 study were reanalyzed in 2004 and the results of the two data sets compared. The 2004 analyses were also performed with a newly developed QPCR primer and probe assay for enterococci (Entero2) that provides greater selectivity against related non-Enterococcus species. Fresh DNA extracts of cultured Enterococcus cells were used as calibration standards for measuring Enterococcus cell equivalents in the test samples in both sets of analyses and gave similar results. The overall mean of Enterococcus calibrator cell equivalents per sampling visit (N = 47) from the 2004 analyses was approximately five-fold lower than that from the initial 2003 analyses. The overall mean from the Entero2 assay was 28% lower than that from the original assay. In contrast, correlations between log10-transformed Enterococcus colony forming units (CFU), determined in 2003 by the MF method, and the 2003 and 2004 QPCR measurements were the same (r = 0.76). Correlations between CFUs and the 2004 Entero2 assay results were similar (r = 0.77). These results indicate that archived DNA extracts may undergo some deterioration that will bias absolute measurements of indicator bacteria, however, the changes in different samples are relatively consistent. Measurements of new indicators in archived DNA extracts by QPCR may therefore be useful for comparisons with epidemiological health data.

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