Genus and species-specific identification of Legionella

Details Genus and species-specific identification of Legionella Genus and species-specific identification of Legionella

1996-04-25

2001-02-27

Jones, W. Gary (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100, C536S024300, C435S091200

Reexamination Certificate

active

06194145

ABSTRACT:

Subject matter of the invention is a method for amplifying nucleic acids of the genus legionella, and a method for the genus and species-specific detection of bacteria of the genus legionella and reagents suitable therefore.
Legionella are aquatic, ubiquitous gramnegative aerobic, facultatively intracellular rod-like bacteria. The legionellaceae family consists of a genus (legionella) and 48 currently known species and 51 sero groups. There exist 16 known serovars from the most important species L. pneumcphila. Their most important habitats include water pipes, air conditioning systems, and cooling towers. Infection of humans usually occurs via Legionella-containing aerosols. Legionellosis frequently occurs as an epidemic, e.g. via shower heads of warm-water systems, contaminated cooling water in air-conditioning system or sporadic. Person-to-person transmission has not yet been described.
Legionella infections can be divided in two groups: The legionnaires' disease, an acute severe pneumonia that is often accompanied by high fever, abdominal pain, headaches, myalgias, and confusion and other neurological symptoms; further, the Pontiac fever which is a self-limiting variant of legionellosis is accompanied by influenza-like symptoms.
The most important sero group of L. pneumophila is the L. pneumophila sero group 1 (L. pn. Sero. 1) which is the most frequently cause of the legionnaires' disease (approx. 80%). However, there exist great regional differences, especially when dealing- with nosocomially acquired legionellosis. As opposed thereto, L. micdadei and other non-pneumophila species have been described as the causative agent for Pontiac fever.
The diagnostical treatment of legionella is difficult. Legionella are difficult to dye with fuchsin, rendering a gram stain of the causative agent practically impossible. Legionella do not grow on commonly used culturing media, but only on special agars and at an atmosphere that contains 2.5-5% CO
2
. The sensitivity of the culture is only approx. 20% for
L. pneumophila
and approx. 5% for other species.
For the detection of legionella, one has employed, inter alia, DNA-DNA hybridisation, Pulsfeld electrophoresis, ribotyping, restriction fragment length polymorphism, fatty acid and ubiquinone analysis, rRNA sequencing, RT-PCR and Southern blot, latex agglutination, Fourier-transformed infrared spectroscopy, indirect immunofluorescence and carbohydrate utilisation (BIOLOG system).
Med. Microbiol. Lett. 1994; 3: 279-290 and Clin. Lab. 1994; 40: 211-216 describe the sequencing of 5S-rDNA of legionella.
A kit for the detection of legionella in water samples is commercially available. The detection with the aid of this kit includes a first step where a fragment of the 5S-rDNA is genus-specifically amplified. A fragment of the MIP gene of the
L. pneumophila
species is amplified in the same vessel. The kit contains a total of seven primers for carrying out a multiplex PCR wherein two PCR amplificates are generated. Subsequently, the amplificates are detected via reverse dot blot. The necessity of great numbers of primers renders the method that is implemented with the aid of said kid rather complex as is its manufacture. Moreover, said method is unsatisfactory with respect to sensitivity.
It was, hence, an object of the present invention to provide reagents that facilitate the detection of legionella while comprising a smaller number of components. It was another object of invention to provide a more specific and potentially more variable legionella detection method.


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