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USE OF TAQMAN TO ENUMERATE ENTEROCOCCUS FAECALIS IN WATER
Citation:
Brown, S, J Santo Domingo, S D. Siefring, AND R A. Haugland. USE OF TAQMAN TO ENUMERATE ENTEROCOCCUS FAECALIS IN WATER. 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:
The Polymerase Chain Reaction (PCR) has become a useful tool in the detection of microorganisms. However, conventional PCR is somewhat time-consuming considering that additional steps (e.g., gel electrophoresis and gene sequencing) are required to confirm the presence of the target organism. To circumvent these steps a real time PCR (TaqMan) approach was developed to monitor the presence of Enterococcus faecalis in water samples. The TaqMan primers and probes used in this study align to regions wihtin the 16S rDNA of E. faecalis. When the method was empirically tested against 14 different enterococci species only E. faecalis cells produced cycle threshhold (Ct) values that were significantly different than negative controls. No differences in Ct values were obtained regardless if E. faecalis cells were innoculated in sterile distilled water, in untreated river samples, or in a background of biofilm material from a water distribution system. In contrast, a 100 fold dilution was necessary to detect E. faecalis in human feces. The detection limit was determined to be approximately 60 dfu/ml. The method was successfully used to identify E. faecalis strains from a culture collection of environmental fecal enterococi isolates. Biochemical profiles and 16S rDNA sequencing analysis of presumptive E. faecalis strains confirmed the TaqMan identification. Since no nucleic acid extraction was required, the presence of E. faecalis was detected in less than three hours.