Application of the fluorogenic probe technique (TaqMan PCR) to the detection of Enterococcus spp. and Escherichia coli in water samples.
A recent PCR detection technique (TaqMan) based on the 5'-3'-exonuclease activity of the Taq DNA polymerase was applied to the detection of indicator organisms in water samples. In this technique, an increasing fluorescence signal is measured online which enables direct assessment of results after PCR without additional detection steps. The test is completed within about 5 h. Two sets of primers and probes were designed and tested: a genus-specific assay for the detection of Enterococcus spp. based on 23S rRNA sequence and an Escherichia coli-specific assay based on the uidA gene sequence. Specificity of the assays was confirmed by testing strains of target bacteria and potential interfering microorganisms. Application of the tests to 55 natural water samples showed the need of an overnight enrichment step to achieve compliance with detection limits of existing regulations. Compared with a parallel microbiological examination of the samples, agreement was 96% with the Enterococcus assay and 98% with the E. coli assay. The rapidity and feasibility of the method point to benefits in drinking water analysis, particularly in emergency situations and, thus, to improved public health management.
Frahm
Edith
E
Forschungszentrum Karlsruhe GmbH, ITC-WGT, Abt. Umweltmikrobiologie, PF 3640, D-76021 Karlsruhe, Germany. frahm@t-online.de
Obst
Ursula
U
D016983:Enterococcus / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D005456:Fluorescent Dyes / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011634:Public Health; D012680:Sensitivity and Specificity; D012984:Software; D019914:Taq Polymerase / Q000378:metabolism; D014871:Water Microbiology
D005456:Fluorescent Dyes; D019914:Taq Polymerase