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DETECTION OF FECAL ENTEROCOCCI USING A REAL TIME PCR METHOD
Citation:
Siefring, S D., J Santo Domingo, N Brinkman, AND R A. Haugland. DETECTION OF FECAL ENTEROCOCCI USING A REAL TIME PCR METHOD. Presented at American Society of Microbiology, Orlando, FL, May 19-24, 2001.
Impact/Purpose:
1) Develop rapid TaqMan PCR methods for the detection and quantification of total enterococci and specific groups or species of Enterococcous in water and determine, in a laboratory setting, the efficacy of the methods. 2) Use TaqMan methods to determine the occurrence of total and specific enterococci groups in fresh and marine recreational water samples and compare results with those of standard culture based methods.
Description:
In spite of their importance in public health, the detection of fecal enterococci is performed via culturing methods that are time consuming and that are subject to inaccuracies that relate to their culturable status. In order to address these problems, a real time PCR (TaqMan) method was used to monitor the presence of several fecal enterococci species in water samples. Using 16S rDNA sequencing data, three different TaqMan assays were designed to detect Enterococus faecalis, E. faecium, and E. casseliflavus. The specificity of each assay was empirically tested using known cultures. The results from these studies indicated that assay I was specific to E. faecalis, assay II specific to E. facium, E. hirae, E. durans, and E. dispar, while assay III was specific to E. casseliflavus and E. gallinarum. These assays were then used in analyses of environmental isolates to determine the composition of fecal enterococci from a freshwater pond with history of fecal contamination. Isolates obtained during three different months in each case gave signals in only one of the three TaqMan assays, further suggesting the specificity of the assays. TaqMan studies with total biomass harvested from membrane filtration media specific for fecal enterococci indicated the presence of each of the above described enterococci groups in the pond. The relative abundance of the enterococci groups in these samples was different each month suggesting changes in fecal enterococci composition. This result suggests the possible presence of different sources of fecal pollution during each month. The results from this study indicate that these assays could be used to potentially detect the most relevant enterococci species associated with fecal pollution.