You are here:
COMPARISON OF REAL-TIME PCR FECAL BACTERIA MEASUREMENTS IN RECREATIONAL WATERS USING DIFFERENT INSTRUMENTS AND REAGENT SYSTEMS
Citation:
HAUGLAND, R. A., S. SIEFRING, M. VARMA, E. ATIKOVIC, AND L. J. WYMER. COMPARISON OF REAL-TIME PCR FECAL BACTERIA MEASUREMENTS IN RECREATIONAL WATERS USING DIFFERENT INSTRUMENTS AND REAGENT SYSTEMS. Presented at 106th General Meeting of the American Society for Microbiology, Orlando, FL, May 21 - 25, 2006.
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:
U.S. EPA guidance on the safety of surface waters for recreational use is currently based on concentrations of culturable fecal indicator bacteria. Attention is now shifting to more rapid molecular monitoring methods. A multi-year epidemiological study is in progress to determine the relationship between illness rates in bathers and concentrations of fecal bacteria in recreational water as determined by real-time PCR analysis. Analyses in the first 2 years of the study were limited to one PCR reagent and type of instrument. Acceptance of this technology will be aided by the availability of choices in instruments and newer PCR reagents that offer even shorter analysis times. DNA extracts of 50 ml beach water filtrates (N = 396) collected from Biloxi, MS during the 2005 study were analyzed on 3 instruments: Applied Biosystems model 7700 (analysis time ~ 2 hr); Cepheid Smart Cycler (analysis time ~ 30 min with TaqMan probe & ~45 min with Scorpion probe); and Applied Biosystems fast block model 7900 (analysis time ~ 35 min), using customized PCR reagents and primer/probe sets for each instrument. Mean Enterococcus and Bacteroidetes calibrator cell equivalents/filtrate determined from all analyses were 37 and 388 (CV between instrument means: 0.26 and 0.24), based on an amplification efficiency of 0.92 for both assays. Significant differences (P < 0.05) were found in the means for both groups of organisms between each of the systems with exception of the Bacteroidetes results on the models 7700 and 7900. Normalization of results using reference control analyses made these differences non-significant in all comparisons except those involving the model 7700. Failure to show comparability between model 7700 and other systems results may be related to the use of a different reference assay. Variance in replicate analyses was highest on the model 7700; this system gave a significantly lower percentage of non-detects. Our results suggest that different instrument/reagent systems can give comparable results but further analyses are needed to demonstrate relationships between epidemiological results and fecal bacteria measurements with the newer systems.