Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay
| Autor: | Anu Tilsala-Timisjärvi, Tarja T. Leskelä, Antje Breitenstein, Peter Neubauer, Jaana Kusnetsov |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Microbiology (medical)
Detection limit DNA Bacterial Chromatography biology Legionella Oligonucleotide Hybridization probe Nucleic Acid Hybridization Ribosomal RNA 16S ribosomal RNA biology.organism_classification Microbiology Molecular biology Polymerase Chain Reaction Sensitivity and Specificity Spectrometry Fluorescence Water Supply RNA Ribosomal 16S Nucleic acid Oligomer restriction DNA Probes Water Microbiology Molecular Biology |
| Zdroj: | Journal of microbiological methods. 62(2) |
| ISSN: | 0167-7012 |
| Popis: | The aim of this study was to develop a sensitive, cultivation-independent analytical method for Legionella in man-made water systems which can be performed within one day in crude sample extracts. The new assay for the genus Legionella is a paramagnetic bead based fluorescence sandwich hybridization assay (SHA) for the 16S rRNA based on two oligonucleotide probes which makes the method highly specific. An advantage over RT-PCR is the exclusive detection of viable cells and, due to the high number of 16S RNA molecules, the possibility to apply the method directly in crude cell extracts without prior purification of the nucleic acids. A high sensitivity was obtained by modifying the probe chemistry and hybridization conditions. The most sensitive assay uses a 3'-end biotin-labelled capture probe and a 3'-end DIG tailed detection probe and has a detection limit of 20 amol target molecules corresponding to 1.2x10(7) molecules of 16S rRNA and approximately 1800 Legionella cells. Using this assay type the number of Legionella cells was determined in Legionella contaminated water samples. The results show that the developed SHA can be applied for estimation of the approximate number of Legionella cells based on the number of 16S rRNA molecules in a water sample. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect