Abstract
Rapid and simple dot hybridization was designed and developed for the identification of Legionella species.
DNAs from 18 type strains of Legionella species were quantitatively spotted on nitrocellulose filters and kept in a desiccation chamber. When an organism was suspected of belonging to the Genus Legionella, the DNA of the isolate was quickly extracted from a single colony, or 1ml of the bacterial suspension that matched the turbidity of the No. 2 McFarland standard. The DNA was labeled with photobiotin within 15min and a dot hybridization experiment was carried out between the labeled DNA of the unknown organism and the reference DNAs of 18 type strains of Legionella species. Hybridization was stopped within 4hr. Hybridized spots were visualized with alkaline-phosphatase color detection method. The reaction for the color detection was stopped within 30min and the color intensity of the hybridized spots was measured with a color graphic analyzer to determine the strongest spot.
Among about 500 environmental strains identified as legionellae at the genus level, 20 strains were serologically unidentified. They were hippurate-positive legionellae but did not react to any antisera of hippurate-positive Legionella species. These strains were finally identified as L. pneumophila by our simplified colorimetric dot-hybridization method.
