Citation:

SIEFRING, S., L. J. WYMER, AND R. A. HAUGLAND. NEW TARGET AND CONTROL ASSAYS FOR QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ANALYSIS OF ENTEROCOCCI IN WATER. Presented at American Society for Microbiology 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:

Enterococci are frequently monitored in water samples as indicators of fecal pollution. Attention is now shifting from culture based methods for enumerating these organisms to more rapid molecular methods such as QPCR. Accurate quantitative analyses by this method requires highly specific primer and probe assays and the inclusion of effective controls to detect and adjust for influences of the sample matrix on both DNA template recovery and PCR efficiency. A new primer and probe set targeting a conserved rDNA sequence in Enterococcus species and with high predicted selectivity against related non-Enterococcus species was developed. A second highly discriminatory primer and probe set, targeting exactly the same rDNA region of the related species Lactococcus lactis, was also developed to allow known additions of this organism to water sample filtrates to be used for assessing variability in DNA recovery. Finally, an easy to perform method was devised for the generation of an artificial PCR template with extensive nucleotide sequence similarity to the same rDNA region but again with specific primer and probe target sequences. This template was designed for addition as a positive control to DNA extracts to test for PCR inhibition. Preliminary tests confirmed that there was no cross-reactivity between the three assays and that their amplification efficiencies were similar (~0.9). Different extraction conditions, designed to alter the efficiency of cell lysis, resulted in DNA recoveries from Lactococcus and Enterococcus that were strongly correlated (r = 0.85) as determined by the respective QPCR assays. Extracts of different water sample filtrates were spiked with Enterococcus, Lactococcus and positive control template DNAs and analyzed to determine the relative sensitivities of the respective assays to PCR inhibition. The three assays showed highly correlated changes in results in response to varying amounts of PCR inhibitory substances in the different water extracts as evidenced by r values of 0.9 or higher. This system shows great promise for improving the accuracy of enterococci measurements in water samples by QPCR.

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