Specific detection of Legionella pneumophila: construction of a new 16S rRNA-targeted oligonucleotide probe - PubMed (original) (raw)

Specific detection of Legionella pneumophila: construction of a new 16S rRNA-targeted oligonucleotide probe

D Grimm et al. Appl Environ Microbiol. 1998 Jul.

Abstract

Based on comparative sequence analysis, we have designed an oligonucleotide probe complementary to a region of 16S rRNA of Legionella pneumophila which allows the differentiation of L. pneumophila from other Legionella species without cultivation. The specificity of the new probe, LEGPNE1, was tested by in situ hybridization to a total of four serogroups of six strains of L. pneumophila, five different Legionella spp. and three nonlegionella species as reference strains. Furthermore, L. pneumophila cells could be easily distinguished from Legionella micdadei and Pseudomonas aeruginosa cells by using in situ hybridization with probes LEGPNE1, LEG705, and EUB338 after infection of the protozoan Acanthamoeba castellanii.

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Figures

FIG. 1

FIG. 1

Phase-contrast (a) and epifluorescence (b) micrographs of L. pneumophila Corby-infected A. castellanii cells 16 h postinfection. The micrograph (b) represents double exposure of the sample after hybridization with the CY3-labeled probe LEGPNE1 (red) and the fluorescein-labeled probe LEG705 (green). Magnifications, ×955. Either amoebae are filled with bacteria localized in particular areas of the cells which may be the phagosomes, or bacteria are present as single cells.

FIG. 2

FIG. 2

FISH of A. castellanii cells infected simultaneously with L. pneumophila Corby and L. micdadei (6 h postinfection) with a mixture of CY3-labeled probe LEGPNE1 (red) and fluorescein-labeled probe LEG705 (green). Phase-contrast (a) and epifluorescence (b and c) micrographs of identical microscopic fields are shown. All micrographs were done at the same magnification, i.e., ×744. Arrows indicate amoeba cells infected with L. micdadei.

FIG. 3

FIG. 3

FISH of A. castellanii cells infected simultaneously with L. pneumophila Corby and P. aeruginosa (16 h postinfection) with a mixture of CY3-labeled probe LEGPNE1 (red) and fluorescein-labeled probe EUB338 (green). The epifluorescence micrograph was done by image processing. Magnification, ×1,206.

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