Use of PNA oligonucleotides for the in situ detection of Escherichia coli in water

Abstract

A species-specific Peptide Nucleic Acid (PNA) oligonucleotide probe directed against the V1region of the 16S rRNA molecule was synthesized for the detection of Escherichia coli in water. The specificity of the probe was tested in dot blot hybridizations against a number of environmental isolates including those from the genera Escherichia , Klebsiella , Enterobacter and Citrobacter . In situ hybridization experiments were performed with biotinylated PNA oligonucleotide probes with subsequent detection of the biotin label using a combination of Streptavidin-Horseradish Peroxidase and a tyramide signal amplification system. The results obtained enabled the specific detection of E. coli in under 3 h. Hybridizations were also performed on cells which were treated with chlorine (1·5 mg l−1) for up to 30 min. Escherichia coli cells were still detected after storage for 14 days at room temperature. No cells were detected by conventional plate count or the «Colilert» assay, a method currently used for the routine detection of E. coli and coliforms in the water industry. Cell viability was assessed by the ability of cells to reduce 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to highly fluorescent formazan crystals through bacterial respiration. Only cells that had not been chlorinated were detected. These results confirm that ribosomal RNA exists within the cell long after cell death has occurred and that rRNA cannot be used to assess the viability of individual cells. However rRNA probes in combination with viability markers should enable the specific detection of viable cells in situ . Hybridization experiments were also performed successfully on seeded tap water samples. The number of fluorescent cells detected correlated well with those obtained by plate count analysis. This represents the first reported use of PNA oligonucleotides forin situ detection of micro-organisms and offers a fast efficient alternative to conventional DNA approaches.