WO2001016363A2 - Rapid, highly specific detection method for pseudomonas aeruginosa using multi-probe detection - Google Patents

Abstract

The invention describes a rapid, highly specific, molecular detection of Pseudomonas aeruginosa.

Description

 FASTER, HIGH-SPECIFIC DETECTION OF PSEUDOMONAS AERUGINOSA BY MULTIPLE PROBE DETECTION

DESCRIPTION

The invention relates to agents for the detection of Pseudomonas ae ruginosa, methods for the specific detection of Ps. aeruginosa and the use of the agent disclosed according to the invention.

Representatives of the bacterial species Pseudomonas aeruginosa are widespread germs in the environment (e.g. in surface waters). They are gram negative, need oxygen for growth (aerobic) and are oxidase positive. As a rule, they grow at 41 ° C in the presence of cetyltrimethylammonium bromide (cetrimide) in the nutrient medium, form the pigments pyocyanin and pyoverdin (fluoresce weakly blue-green under UV excitation), form nitrate and nitrite and are sensitive towards polymyxin. They are able to utilize a wide range of nutrients and can multiply even with a very limited supply of usable carbon sources. Only a small part of all germs of the Pseudomonas aeruginosa bacterial species is pathogenic. This ubiquitous type of bacteria, however, has considerable secondary pathogenic significance as an opportunistic germ in connection with hospitalism.

In the context of so-called nosocomial (= acquired in hospital) infections, Pseudomonas aeruginosa can cause wound infections, sepsis, endocarditis, inflammation of the genitourinary tract, but in particular infections of the respiratory tract, including pneumonia. Immun-suppressed patients in particular are often affected. Patients with neutropenia or cystic fibrosis usually have very poor prognoses when they are infected with Pseudomonas aeruginosa. A quick and specific early detection of this germ is therefore of great importance in medicine, especially in medical diagnostics.

In the beverage industry, this germ is also a valuable indicator of contamination with surface water and unhygienic production. Therefore, according to legal regulations in Germany and Europe, e.g. the regulation on mineral, spring and table water, bottled products containing mineral, spring or table water are checked for the presence of this germ.

Conventional procedures based on the cultivation of these germs in pure culture and subsequent identification of the phanotype are lengthy and not always clear. This is due to the property of this germ and closely related organisms (pseudomonas) to absorb genetic material from other germs and also to develop the associated phenotype. Examples are also known which include some strains of Ps. aeruginosa no longer show typical properties (such as the formation of the pigment pyocyanin), and occasionally have completely lost them. An object of the invention is the description of a quickly executable, highly specific method for the detection of Pseudomonas aeruginosa and the naming of the materials required for this.

This object is achieved according to the invention by a means for the detection of Ps. aeruginosa, which is characterized in that it contains at least two probes with e at least ten successive nucleotides of the following two sequences:

PAE1: 5 - tca ctc cgt ggt aac cgt ccc cct tgc ggt tag act agc tac ttc tg -3 ^x

PAE2: 5 - tct cct tag agt gcc cac ccg agg tgc tgg taa cta ag - 3

and / or at least one or two derivatives of these sequences, each of which is a combination of at least two of these sequences for the specific detection of Ps. aeruginosa are suitable.

Furthermore, a method for the specific detection of Ps. aeruginosa disclosed with the following process steps:

Contact of the DNA or RNA of one of the presence of Ps. aeruginosa sample to be examined with at least the two probes PAE1 and PAE2 and / or their derivatives and detection of the hybridization of the probes with the RNA or the DNA of the sample to check for the presence of Ps. to show aeruginosa specific DNA and / or RNA sequences Preferred embodiments of the invention result from the subclaims and the following description and the exemplary embodiments. The invention is not limited to the preferred configurations mentioned below, but encompasses all of the embodiments covered by the claims, even if they are not specifically mentioned below.

The detection method according to the invention is based on the detection of defined nuclear acid sections of Ps. aeruginosa, with highly specific sequences of the 16S πbosomal ribonucleic acid (16S rRNA) from Pseudomonas aeruginosa and strains derived therefrom being recognized selectively. The agent provided according to the invention can be used for the rapid identification of Ps. Aeruginosa, since neither selective cultivation of Ps. aeruginosa to pure cultures still the formation of a defined phanotype are necessary. It differentiates Ps reliably and reproducibly. aeruginosa from other closely related Pseudomonas species, for example Pseudomonas alcaligenes, Pseudomonas mendocma and Pseudomonas fluorescens.

The agent according to the invention is characterized in that it comprises a combination of two probes which are characterized by the nucleotide sequences PAE1 and PAE2. Only the combined use of both probes leads to a reliably discriminatory detection of Ps. aeruginosa and its strains from other Pseudomonas species and their strains.

The probes disclosed here specifically bind to sequences of the 16S rRNA on Ps. aeruginosa. A germ that reacts positively with both probes is called Ps. aeruginosa identified. A single identification probe can identify Ps. Do not guarantee aeruginosa. Only the combination according to the invention tion from both probes allows the specific identification of Ps. aeruginosa.

Various approaches to solving Ps. identify aeruginosa. In comparison to the present invention, however, the known methods have serious disadvantages which are avoided according to the invention. The state of the art is discussed below:

1. Standard detection in the beverage industry: Incubation of the sample on cetrimides (US Pat. No. 4072573, Aldπdge et al., 1978) - agar at 41 ° C. for 48 h. Illuminate the agar plates with ultraviolet light. Identification of bluish green fluorescent colonies as Pseudomonas aeruginosa.

2. Probe for the diagnosis of Pseudomonas aeruginosa (US Patent No. 5798211, Appl. No. 921177, Ohno et al., 1998)

3. Isolation of suspect germs and pure culture. Identification of Pseudomonas aeruginosa by Fourier transformation infrared red spectroscopy (Helm et al., 1991; Naumann et al., 1991).

4. Nucleic acid probes and methods for the detection of group I pseudomonas (US Pat. No. 5,677,127, Hogan et al., 1997; Gen-Probe Incorporated, Appl. No. 453439).

The disadvantages of the detection methods mentioned above are as follows:

Re 1. The proof takes too long (2 days). In the case of doubtful results (only about 75% of all Pseudomonas aeruginosa strains form the pigment Pyocyanm), lengthy confirmation tests (for physiological properties) must be carried out for the test. Damaged and starved germs grow up to the selective medium only poorly or no longer.

Re 2. The binding sites of these ^λ probes are not known since they are HINDIII fragments of the DNA that were cloned in E. coli. As a result, the production of the probe is complex and expensive compared to oligonucleotides. Detection at the individual cell level is not possible because the probe is directed against the DNA and there are therefore too few molecular target sites per cell. The probe can presumably only be used for detection of nuclear acid extracts. Furthermore, it is not evident whether the probe between Ps. aeruginosa and closely related pseudomonas can be distinguished.

For 3. Mixed cultures cannot be analyzed with FT-IR spectroscopy. Therefore, the extraction of pure cultures and their cultivation on standardized media is the basic requirement for their FT-IR analysis. Accordingly, the entire detection procedure takes at least 48 hours too long.

Re 4. The developed probe is directed against the 23S rRNA. It detects Ps. aeruginosa, but also other fluorescent pseudomonas, e.g. Ps. trim, Ps. mendocma, Ps fluorescens, Ps putida and many more. A distinction between Ps. aeruginosa and other Group I pseudomonas is not possible. Furthermore, this probe cannot be used for the detection of Pseudomonas aeruginosa cells using FISH, but only for the detection of m nuclear acid extracts.

In the tests carried out in accordance with the invention, the probe designated PAE3 in FIGS. 1 and 2 was found as an additional probe. This is one of the 23S rRNA derived probe with the following sequence: 5 -GACCAGCCAGAGCTTACG-3. However, as Figure 2 shows, this PAE3 probe does not succeed in detecting all Pseudomonas aeruginosa isolates from mineral waters. Isolates NE54 and NE55 are not detected by the probe.

The way proposed according to the invention, by using at least two probes Ps. To discriminate aeruginosa safely from closely related species and to select probes that specifically recognize the 16S rRNA is new and also has an inventive performance compared to the prior art mentioned.

The agent proposed according to the invention for the detection of Ps. aeruginosa comprises at least two probes with the sequences PAE1 and PAE2 mentioned in claim 1 and derivatives thereof. In the detection method according to the invention, at least 10 consecutive nucleotides from PAE1 and PAE2 are used in combination. According to the invention, “combination ^* ” means both the sequential use of PAEl and PAE2 and the use of PAEl and PAE2 in one step.

In preferred embodiments of the invention, the probes comprise more than 10 consecutive nucleotides, preferably 12 to 26 nucleotides, further preferably 14 to 22 nucleotides, more preferably 16 to 20 nucleotides and particularly preferably 18 nucleotides. It is obvious to the person skilled in the art that probes with lengths other than those specifically described here can also be used, provided they are in the range from 12 to 26 successive nucleotides of the sequences PAE1 and PAE2. According to the invention, probes with the following sequences are particularly preferably used in combination: gt aac cgt ccc cct tgc g

gt gcc cac ccg agg tgc t.

It is also obvious to a person skilled in the art that, based on the sequences disclosed here, also modifications of these sequences for the detection of Ps. use aeruginosa. According to the invention, “modifications *” are to be understood as meaning derivatives of the sequences in which one, two or three nucleotides on one or both edges of the probe have been replaced by other nucleotides. The prerequisite here is that a specific binding to the 16 rRNA on Ps. aeruginosa remains, so that the object of the invention is achieved. According to the invention, “derivatives *” are also to be understood to mean those probes which are derived from the probes PAE1 and PAE2 and in which one or two nucleotides have been deleted or replaced by another nucleotide in the interior of the probe, but of course only such ones Variations are to be considered in which the specific binding to Ps. aeruginosa nucleotide sequences are retained. On the basis of the nucleotide sequences of the probes disclosed here, the person skilled in the art can easily determine which modifications of the sequences for the highly specific detection of Ps. aeruginosa are suitable and which are not.

The invention also encompasses the sequences and reverse-complementary sequences which are reverse to PAE1 and PAE2 and their derivatives.

The probes provided in the present invention are used in methods for the specific detection of Ps. aeruginosa used. Methods in which DNA probes are specific Detection of a microorganism are known per se. The person skilled in the art can use these methods known per se with the present probes. Here, the DNA or the RNA is based on the presence of Ps. aeruginosa sample to be examined with at least the two probes PAEl and PAE2 or derivatives of these probes, as described in more detail above, brought into contact. The hybridization of the probes with the RNA or the DNA of the sample is then detected in order to determine the presence of Ps. to show aeruginosa specific DNA and / or RNA sequences.

Various modifications are possible according to the invention in order to use the probes. Thus, the probes can be provided with a label, for example a radioactive label, digoxigenin, peroxidase-biotin label, or a fluorescent label in order to carry out a specific hybridization with Ps. to detect aeruginosa specific DNA or RNA.

Further modifications or derivatizations of the probes are e.g. PNAs in which the bases are bound to PNA and not to a sugar-phosphate backbone.

In a further embodiment of the invention, the probes are bound to a matrix (reverse hybridizations) and hybridization is carried out with fluorescence-labeled DNA or a PCR amplificate. Examples of such matrices are microchips and microtiter plates.

Certain derivatizations of the probes have been described above. However, the invention also encompasses derivatives of the probes or the individual nucleotides of the probes that are not described separately here, for example chemical changes. improvements to the probes that facilitate the detection procedure. Such derivatives are known to the person skilled in the art and can be applied to the probes provided according to the invention.

The present invention serves for the highly specific detection of Pseudomonas aeruginosa with the aid of a combination of at least two probes. It is known that a species like Ps. aeruginosa is naturally inconsistent, i.e. split up into individual trunks. In terms of taxonomy, a "strain" is usually understood to mean a bacterial clone defined by a strain number and its descendants. There is a so-called type strain that represents the species and thus the properties of the species. Since all strains of a species, such as Ps. aeruginosa, closely related to each other, the detection methods and probes provided according to the invention can of course also be applied to all strains of Ps. aeruginosa.

The DNA or RNA of the sample to be examined is either isolated from the sample organisms or the organisms are disrupted in a suitable manner so that direct contact of the probes with the DNA and / or the RNA of the sample organisms is made possible without extensive and time-consuming cleaning procedures beforehand must be carried out.

The probes are hybridized with the DNA and / or the RNA of the sample organisms under stringent conditions, preferably highly stringent conditions. These conditions are explained in more detail below.

In hybridization, the reaction buffer and wash buffer are composed of the following functional components: a) Buffer system for adjusting and stabilizing the pH between 7 and 8 (eg Tris / HCl) b) Water as ^λ solvent 'c) Possibly chelating agents, which at low concentrations of monovalent cations have the influence of divalent cations

(e.g. Ca ²⁺ ), which can be present as a contaminant, chemicals. Especially used in the wash buffer. d) Detergent for lowering the surface tension of aqueous solutions e) Nonionic, aprotic detergents (eg formamide) weaken the binding energy of duplex molecules of nuclear acid. f) Salt (functional units are cations that neutralize the negative charges of the nuclear acid phosphate groups and thereby facilitate the duplex formation of two single-stranded nucleic acids (eg Na ⁺ m NaCl).

Four factors influence the formation of specific hybrids from probe and target molecule:

Components e) and f) influence the binding strengths of duplex nuclear acid. Increasing the monovalent cations in the reaction or wash solution stabilizes the duplex molecules formed, while with an increasing content of e.g. Formamide the duplex formulations are weakened.

A suitable probe concentration must be used. The hybridization must take place at a suitable temperature {the higher the temperature, the weaker the binding of the hybrids. Strict hybridization and washing conditions are the reaction conditions (the correct choice of the four factors) under which only duplex molecules between the probe and the desired target molecules (perfect hybrids) are formed or only the desired target organism is detected.

Strict reaction conditions mean, for example, a hybridization temperature of approximately 5-10 ° C. below the respective primer melting point. The stability of the DNA / DNA or RNA / DNA hybrids must be ensured even at low salt concentrations corresponding to 0.1 x SSC / 0.5% SDS. In this way undesirable cross-reactions with other species can be prevented. However, the respective temperature conditions can differ depending on the chosen test conditions and depending on the DNA sample to be examined and must then be adapted accordingly. The hybridization product can be detected, for example, by autoradiography in the case of radioactively labeled primer molecules or by fluorimetry when using fluorescence-labeled oligonucleotides.

Further examples of strict conditions are listed in the examples below. The person skilled in the art can adapt the conditions to the selected examination method in a manner known per se, in order to actually achieve strict conditions and to enable a specific detection method. Suitable strain requirements can be determined, for example, using reference hybridizations in which DNA or RNA from Ps. aeruginosa with samples of other Pseudomonas species is examined and compared with the samples provided according to the invention. In a further embodiment of the invention, the probes are used as forward and backward polymer for a PCR reaction.

The above probes are complementary to areas of rRNA. Therefore these probes can bind to the rRNA. However, you can also bind to the anti-Smn DNA strand of the rRNA gene (which is the same sequence as the rRNA). In order to amplify the rRNA gene region between the two probe regions by means of PCR, a primer must bind to the anti-Smn strand, the second primer to the sense strand.

The area complementary to PAE2 (5'-ctt agt tac cag cac ctc ggg tgg gca Ctc taa gga ga -3 ^λ ) offers itself as a forward primer '(PAE _{vorwaι: tΞ} ), as a' backward primer '(PAE _ruckwar r _s ) Area PAEl (5'- tca ctc cgt ggt aac cgt ccc cct tgc ggt tag act agc tac ttc tg -3 ').

The derivatives and preferred modifications described above can be applied analogously.

When using these primers in PCR, at a suitable annealing temperature, only strains of Ps. aeruginosa em specific amplicon of 337 bp can be generated. Extracted DNA from pure cultures or enrichments, or whole cells from pure cultures or enrichments, or water samples before and after enrichments, or clinical material, can be used as the sample to be examined.

The probes according to the invention thus enable highly specific and rapid detection of representatives of the bacterial species Ps. Aeruginosa. For example, by marking of probes with different fluorescent dyes and their use at FISH in fixed samples Ps. aeruginosa can be identified at the single cell level when a cell fluoresces in two colors. In this way Ps. aeruginosa can also be specifically detected in mixed cultures. Growing on selective nutrient media and obtaining pure cultures is no longer necessary. Since it is based on the optical perception or measurement of two different fluorescent dyes, the identification can also be carried out by laypersons.

The PCR method has the advantage that very small amounts of DNA can be detected. Depending on the material to be detected, the temperature conditions and the cycle numbers of the PCR have to be modified. The optimal reaction conditions can be determined by hand tests in a manner known per se. An example of a PCR is as follows:

- Denaturation of the DNA sample to be examined at 94 ° C for at least 3 minutes;

Binding of the probe pairs for 1 minute at 60 ° C. to the two complementary single strands of the DNA to be examined; two-minute extension reaction of the individual primers along the DNA template at 72 ° C. by linking deoxy-ribonucleoside triphosphates using a thermostable DNA polymerase;

30 to 40 reaction cycles each consisting of: DNA denaturation at 94 ° C (30 seconds) followed by primer binding at 60 ° C (1 minute) and primer extension at 72 ° C (2 minutes);

- Final reaction to fill up the two 3 ends of the reaction product at 72 ° C (for about 5-8 minutes). The characteristic, species-specific DNA marker fragments formed in the course of the PCR amplification by the extension of the primer sequences can be detected, for example, by gel electrophoresis or fluorimetry using fluorescence-labeled oligonucleotides. Of course, other detection methods known to the person skilled in the art can also be used.

A particular advantage of the probe combination provided according to the invention is its use in situ detection methods. Often there are problems with hybridization, and the proteins present in the cell can jeopardize the success of m situ hybridization by masking important nucleotides. The possibility of using the present probe combination for hybridizations, in particular for in situ hybridizations, and not only PCR methods was therefore also not predictable. Hybridization methods and PCR methods are known per se to the person skilled in the art, and reference is only made here, for example, to the publication by C. Mulhardt, The Experimentator: Molecular Biology, G. Fischer, Stuttgart, Jena, Luebeck, Ulm, 1999. Reference is hereby made in full to this publication.

The invention is illustrated in more detail below with the aid of non-restrictive exemplary embodiments and illustrations. The pictures show:

Fig. 1: Identification of Pseudomonas aeruginosa reference strains and kimic isolates with rRNA-directed probes.

Fig. 2: 25 isolates from bottled non-carbonated natural mineral water. Example 1 :

Fluorescence microscopic detection of fixed Ps. aeruginosa m water samples (bottled mineral water, spring or table water) or river enrichments.

250 ml of mineral water or bacterial enrichment from 250 ml of liquid medium is filtered through bacteria-proof, white membrane filters (e.g. polycarbonate filter, Mill pore GTTP02500, Eschborn) with standard filtration systems. The filters are then incubated with 5 ml of 3% paraformaldehyde solution (3% PFA) for 15 minutes (= fιxιert). After the fixation solution has been aspirated, 5 ml of sterile water is added to the filter and aspirated. The filters are placed on glass objects and dripped with 50 μl of hybridization solution (= 62.5 ng PAE1-CY3 and 250 ng PAE2 fluorescence with hybridization buffer (0.9M NaCl, 0.01 M Tris / HCl (pH 7.2 - 8.0), 0.01% SDS , 60% formamide) and for 90 minutes in a closed chamber under an aquimolar atmosphere (eg chamber contains tissue soaked in 2 ml of the hybridization buffer) at 46 ° C. The filter is then m 50 ml wash solution (14 mM NaCl, To remove unbound probe, incubate 0.01 M Tris / HCl, 0.01% SDS, 5mM EDTA (pH8.0) for 15 minutes at 48 ° C. After drying, the filter is placed on a carrier surface (eg glass object carrier) and with an anti-thread g - Dripped medium (eg Citifluor AF1, Citifluor Ltd., London) and covered with a cover glass To detect the signals, the filter is placed in the epifluorescence microscope under excitation with blue and green light (equipped with fluorescence filters for fluorescem and rhodam) at 400- bis 1000x Magnification examined. Pseudomonas aeruginosa cells fluoresce green when excited blue and red when excited green.

Example 2: Identification of Pseudomonas aeruginosa m liquid enrichments from clinical samples m selective or non-selective nutrient medium on slides.

1 ml of slightly cloudy liquid enrichment is mixed with 1 ml of absolute ethanol and incubated at 4 ° C for at least 15 minutes. Centrifuge at 5000g for 3 minutes. Discard the supernatant. Resuspend the pellet in 500 μl phosphate-buffered NaCl solution (PBS buffer), add 500 μl 100% ethanol. Mix well. Distribute 10 μl of the enrichment thus prepared in a reaction field of the hybridization slide (e.g. Paul Marienfeld KG, Bad Mergentheim, Germany; Art. No. 1215130 - slide with 6 reaction fields) and allow it to dry 10 mm at 46 ° C. 6 different samples can be placed on one of the slides mentioned above and examined at the same time.

The slide must be incubated for 3 minutes with 50%, 70% and 100% ethanol. After drying, each reaction field provided with a sample is mixed with 10 μl of hybridization solution (= 12.5 ng PAE1-CY3 and 50 ng PAE2 fluorescence in in hybridization buffer (0.9M NaCl, 0.01M Tris / HCl (pH 7.2 - 8.0 ), 0.01% SDS, 60% formamide)) and coated for 90 minutes in a closed chamber under an aquimolar atmosphere (eg chamber contains tissue soaked in 2 ml of the hybridization buffer) at 46 ° C. The hybridization solution is then rinsed from the slide with a wash solution preheated to 48 ° C. (14 mM NaCl, 0.01 M Tris / HCl, 0.01% SDS, 5 mM EDTA (pHδ.O)) and this 50 ml wash solution for 15 minutes at 48 ° C. mcubated to remove unbound probe. Rinse the slides carefully with distilled water and let them dry. Drip the slide with antifadmg medium (e.g. Citifluor AF1, Citifluor Ltd., London) and cover with cover glass. To detect the signals, the individual reaction fields are examined in the epifluorescence microscope under excitation with blue and green light (equipped with fluorescent filters for fluorescem and Rhodamm) at a magnification of 400 to 1000 times. Pseudomonas aeruginosa cells fluoresce green when excited blue and red when excited green.

Example 3:

Rapid screening of colonies grown on selective or non-selective near agar plates from clinical samples, environmental samples or mineral, spring and table water samples or their products for the identification of Pseudomonas aeruginosa.

Wells of a sterile microtiter plate are filled with 50 μl PBS buffer each. Samples of the bacterial colonies are now suspended from the agar plate in the wells using sterile toothpicks. Add 50 μl of 100% ethanol each to the wells that contain suspended colonial samples. Mix. Distribute 10 μl of the samples fixed in this way per reaction field of the hybridization slide (e.g. Paul Marienfeld KG, Bad Mergentheim, Germany; Art. No. 1215130 - slide with 6 reaction fields) and allow it to dry 10 mm at 46 ° C. 6 different fixed colonies can be placed on one of the slides mentioned above and examined at the same time.

After the samples have dried, the slide must be incubated with 50%, 70% and 100% ethanol for 3 minutes. After drying, each of the reaction fields provided with a sample of a fixed colony is each provided with 10 μl hybridization solution (= 12.5 ng PAE1-CY3 and 50 ng PAE2-Fluorescem m Hybπdisierungsbuffer (0.9M NaCl, 0.01 M Tris / HCl (pH 7.2 - 8.0;, 0.01% SDS, 60-formamide)) overcoated and for 90 minutes in a closed chamber under an aquimolar atmosphere (eg chamber contains tissue that with 2 ml of Hybridization buffer is soaked) at 46 ° C mcubiert. The hybridization solution is then rinsed from the microscope slide with a wash solution preheated to 48 ° C. (14 mM NaCl, 0.01 M Tris / HCl, 0.01% SDS, 5 mM EDTA (pH 8.0)) and this 50 ml wash solution for 15 minutes at 48 ° C. mcubated to remove unbound probe. Rinse the slides carefully with distilled water and let them dry. Drip slides with anti-dmg medium (eg Citifluor AF1, Citifluor Ltd., London) and cover with cover glass. To detect the signals, the individual reaction fields are examined in the epifluorescence microscope under excitation with blue and green light (equipped with fluorescence filters for Fluoresce and Rhodamm) at a magnification of 400 to 1000 times. Pseudomonas aeruginosa cells fluoresce green when excited blue and red when excited green.

Example 4:

Possible application: Direct in situ identification of Pseudomonas aeruginosa in the mucus of patients with cystic fibrosis (= Mucovιszιdose patients).

Mucus sample is placed in 100-1000 ul PBS buffer (pH 7.2-7.4) and mixed with 100-1000 ul absolute ethanol and incubated at 4 ° C for at least 15 minutes. Spread the fixed mucus sample over the reaction field of the hybridization slide (eg Paul Marienfeld KG, Bad Mergentheim, Germany; Art. No. 1215130 - slide with 6 reaction fields) and let it dry 10 mm at 46 ° C. 6 different samples can be placed on one of the slides mentioned above brought and examined at the same time.

The slide must be incubated in 50%, 70% and 100% ethanol for 3 minutes each. After drying, each of the reaction fields provided with a sample is mixed with 10 μl hybridization solution (= 12.5 ng PAE1-CY3 and 50 ng PAE2 fluorescence in hybridization buffer (0.9M NaCl, 0.01 M Tris / HCl (pH 7.2 - 8.0), 0.01% SDS, 60% formamide)) and for 90 minutes in a closed chamber under an aquimolar atmosphere (eg chamber contains tissue soaked with 2 ml of the hybridization buffer) at 46 ° C. The hybridization solution is then rinsed from the slide with a wash solution preheated to 48 ° C. (14 mM NaCl, 0.01 M Tris / HCl, 0.01% SDS, 5 mM EDTA (pH 8.0)) and this 50 ml wash solution for 15 minutes at 48 ° C to remove unbound probe. Rinse the slides carefully with distilled water and let them dry. Drip slides with anti-fading medium (e.g. Citifluor AF1, Citifluor Ltd., London) and cover with cover glass. To detect the signals, the individual reaction fields are examined in the epifluorescence microscope under excitation with blue and green light (equipped with fluorescence filters for fluorescence and Rhodamm) at a magnification of 400 to 1000 times. Pseudomonas aeruginosa cells fluoresce green when excited blue and red when excited green.

Claims

claims 1. Means for the detection of Pseudomonas aeruginosa, characterized in that it contains at least two probes, each with at least ten successive nucleotides of the following two sequences: PAEl: 5'- tca ctc cgt ggt aac cgt ccc cct tgc ggt tag act agc tac ttc tg -3 PAE2: 5'- tct cct tag agt gcc cac ccg agg tgc tgg taa cta ag -3 ^λ which each in combination of at least two of these sequences for the specific detection of Ps. aeruginosa are suitable and / or at least one or two derivatives of these sequences in which one, two or three of the nucleotides at one or both edges of the probe and / or one or two nucleotides inside the probe are deleted or replaced by another nucleotide , the specific binding to the DNA or RNA of Ps. aeruginosa remains. 2. Composition according to claim 1, characterized in that the derivative is a probe with at least 12 - 26 consecutive nucleotides. 3. Agent according to one of claims 1 or 2, characterized in that the derivative is a probe with 14-22, preferably 18 consecutive nucleotides. 4. Composition according to one or more of the preceding claims, characterized in that a combination of the following two nucleotide sequences is used as the probe: gt aac cgt ccc cct tgc g gt gcc cac ccg agg tgc t 5. Composition according to one or more of the preceding claims, characterized in that it comprises the complementary sequence of the probes. 6. Composition according to one or more of the preceding claims, characterized in that it is specific to sequences of the 16S rRNA of Ps. aeruginosa is complementary. 7. Composition according to one or more of the preceding claims, characterized in that the probes have a label. 8. Composition according to claim 7, characterized in that the label is a radioactive label, fluorescent label, digoxigenm label, peroxidase label or biotm label. 9. Composition according to one or more of the preceding claims, characterized in that it is present bound to a solid phase matrix. 10.Means according to one or more of the preceding claims, characterized in that the probes are chemically modified. 11.Means according to claim 10, characterized in that the bases are bound to PNA. 12.Use of an agent according to one or more of the preceding claims, which contains at least the two probes PAEl and PAE2 or derivatives thereof according to one or more of the preceding claims, for the specific detection of Ps. aeruginosa. 3. Procedure for the specific detection of Ps. aeruginosa with the following process steps: Contacting the DNA or RNA of one for the presence of Ps. aeruginosa sample to be examined with at least the two probes PAEl and PAE2 and / or their derivatives according to one of the preceding claims and detection of the hybridization of the probes with the RNA or the DNA of the sample to determine the presence of Ps. to show aeruginosa specific DNA and / or RNA sequences. 14. The method according to claim 13, characterized in that the contacting takes place under stringent conditions. 15. The method according to claim 13 or 14, characterized in that the method is designed as a PCR method. 16. The method according to one or more of the preceding claims, characterized in that the probes are used as forward and reverse primers for a PCR reaction in order to multiply the area between the probes on the gene for the rRNA, and subsequent detection of the reproduced, for Ps. aeruginosa specific region of the rRNA. 17. The method according to claim 13 or 14, characterized in that the method is designed as an in situ hybridization method. 18th Kit, characterized in that it has at least one agent according to one or more of the preceding claims. 19. Solid phase matrix, characterized in that it has at least one of the probes as defined in one of claims 1-8 bound to the matrix.