WO2003066894A2 - Gene probes for the detection of the oenococcus species - Google Patents

Abstract

The invention relates to probes or probe mixtures, which enable a safe and simple identification of the Oenococcus species. The probes are constructed in such a way that they provide a suitable signal for detection, e.g. a good fluorescent yield. The invention also relates to a method for detecting and/or differentiating a species of bacteria belonging to the Oenococcus genus and the use thereof, e.g. in wine production for example.

Description

 Gene probes for the detection of species of the genus Oenococcus

description

The use of lactic acid bacteria is an approved oenological process, which is defined in EU Regulation 1493/1999. The wine is refined by the treatment with lactic acid bacteria, in particular by Oenococcus oeni, and thus enhanced in quality. These Gram-positive bacterial cultures cause the so-called "biological acid degradation" (BSA). Malic acid is converted into lactic acid, which is perceived as less acidic. Furthermore, other metabolic products favor the aroma of the wine (Nielsen, JC; Richelieu, M. Control of Flavor in wine during and after malolactic fermentation by Oenococcus oeni. Appl. Environ. Microbiol., 2: 740-745 (1999). There are currently several companies that offer oenococci as starter cultures. The growing importance of this industry and the associated Profit margins characterize the economic benefit of these bacteria and cause distributors to isolate other strains of this species and to check their industrial applicability.

The strains of Oenococcus oeni (previously Leuconostoc oenos [Dicks LMT, Dellaglio F, Collins MD: Proposal to reclassify Leuconostoc oenos as Oenococcus oeni [corrig.] Gen. Nov., Comb. Nov. Int. J. Syst. Bacteriol. 45: 395-397 (1995)]) always showed a homology of over 70% ("golden rule") to each other in DNA-DNA hybridization (Dicks LMT, van Vuuren HIJ, Dellaglio F: Taxonomy of Leuconostoc species, particularly Leuconostoc oenos as revealed by numerical analysis of total soluble cell protein patterns, DNA base compositions, and DNA-DNA hybridizations, Int. J. Syst. Bacteriol. 40: 83-91 (1990), Garvie EI: Hybridization between the deoxyribonucleic acids of some strains of heterofermentative lactic acid bacteria. Int. J. Syst. Bacteriol. 26: 116-122 (1976)).

To date, the genus Oenococcus consists of only one species, from which several thousand strains from different regions of the world have so far been isolated. According to the "golden rule", this can also be seen in the analysis of rDNA customary for bacterial identification, in which almost no sequence differences occur at approximately 1500 bp. The eubacterial, ribosomal gene cluster comprises the 16S, 23S and 5S rDNA, each of which is separated by an intergenic sequence segment (ISA). No sequences are known downstream of the 5S rDNA for O. oeni, so that no specific primers for a PCR can be generated here. The bacterial 5S rDNA is usually 120 bp and contains a middle, conserved section in other eubacteria. Another characteristic is the secondary structure of the 5S rRNA. The 5 'end is complementary to the 3' end of the sequence.

Eubacteria can be identified using the 16S rDNA sequence. This has already been determined for O. oeni, as has the 23 S rDNA sequence (Martinez-Murcia, AJ; Collins MD. A phylogenetic analysis of the genus Leuconostoc based on reverse transcriptase sequencing of 16S rRNA. FEMS Microbiol, 61: 2139 -2144 (1990); Martinez- Murcia, AJ; Harland, NM; Collins, MD. Phylogenetic analysis of some leuconostocs and related organisms as determined from large-subunit rRNA gene sequences: assessment of congruence of small- and large-subunit rRNA derived trees. J. Appl. Bacteriol., 5: 532-541 (1993)) and from gene databases (Benson, DA; Boguski, MS; Lipman, DJ; Osteil, J; Ouellette, BF. GenBank. Nucleic Acids Res., 26 : 1-7 (1998)). The sequence of the 5S rDNA sequence was previously unknown. However, the identification via an amplification of the rDNA genes and the subsequent sequencing is time-consuming and costly. The examination of the region used for the determination and detection of many other bacteria between the region of the chromosomal DNA coding for the 16S and 23S rRNA or 5S and 23S rRNA also proved to be unsuitable for direct strain identification (Le Jeune C, Lonvaud-Funel A. Sequence of DNA 16S / 23S spacer region of Leuconostoc oenos (Oenococcus oeni): application to strain differentiation. Res Microbiol. 148: 79-86 (1997); (Hirschhäuser S. diploma thesis: Fluorescence in situ hybridization and single cell -Micromanipulation as new applications in the identification of Oenococcus oew ^' . Strains. (2002); University of Mainz, Mainz) as well as hybridization with genomic DNA (Sohier D., Lonvaud-Funel A. Rapid and sensitive in situ hybridization method for detecting and identifying lactic acid bacteria in wine. Food Microbiology 15: 391-397 (1998)).

Molecular biological methods, such as pulse field electrophoresis of restriction fragments (Kelly, WJ; Huang CM; Asmundson RV. Comparison of Leuconostoc oenos strains by pulse-field gel electrophoresis. Appl. Environ. Microbiol., 59: 3969-3972 (1993)) and Randomly Amplified Polymo hic DNA-PCR (RAPD-PCR) (Zavaleta, AI; Martinez-Murcia AJ; Rodriguez-Valera F. Intraspecific genetic diversity of Oenococcus oeni as derived from DNA fingerprinting and sequence analysis. Appl. Environments Microbiol. 63 : 1261-1267 (1997)) have also been used so far and the previous methods of choice in the strain identification of O. oeni.

DE 100 21 947 discloses a general in situ method for the detection of nucleic acids, in particular ribosomal RNA molecules, by means of a detection probe, hybridization and the use of an unlabeled helper probe. DE 100 21 947 describes no detection probes for Oenococcus or a method for its detection.

DE 100 04 147 describes a conventional method for the detection of 16S rDNA with oligonucleotide probes, which were obtained by means of a simple sequence comparison of known 16S sequences. DE 100 04 147 does not describe any detection probes for Oenococcus or a method for its detection.

The above-mentioned increasing importance of oenococcal starter cultures for the wine industry combined with legal approval as an acid-reducing measure places high demands on the purity of the cultures, since only the starter cultures are approved as ingredients and no contaminated products. In the case of random samples of lyophilized oenococci from various manufacturers already available on the market, two out of five samples were found to be a mixed culture due to contamination with previously unidentified germs.

It is therefore an object of the present invention to develop probes or probe mixtures which enable reliable and simple identification of species of the genus Oenococcus. At the same time, the probes should be constructed so that they provide a signal suitable for detection, such as a good fluorescence yield.

It is a further object of the present invention, a method for the specific detection and / or differentiation of a species of bacteria of the genus Oenococcus to provide. The process should be fast, cheap and reliable and should also be applicable on an industrial scale.

One of the stated objects of the present invention is achieved by an oligonucleotide, in particular DNA oligonucleotide, which hybridizes selectively to an rRNA-specific nucleic acid sequence of a species of bacteria of the genus Oenococcus, which can be selected from the 23S, 16S and 5S rRNA and a sequence or Partial sequence with at least 9 nucleotides in length, selected from the group of sequences according to Seq ID NO: 1-31 (see Tables 1-3) or sequences complementary thereto.

For this purpose, the sequences of the 5S rDNA and the intergenic sequence section between the 5S rDNA and the 23S rDNA of six different O. oem strains and four phylogenetically closely related reference organisms were first determined (Dicks et al, 1995). As with strains of the genus Leuconostoc (Nour, M. Studies on the large subunit rRNA genes and their flanking regions of leuconostocs. Can. J. Microbiol., 9: 807-818 (1998)), neither the intergenic sequence section nor the 5S- rDNA variability between different O. oeni strains. Then the species-specific, fluorescence-labeled oligonucleotides according to the invention were developed on the basis of the sequence of the 23 S rRNA and the 5S rRNA. The phylogeny of the 16S rRNA gene from Oenococcus oeni shows a highly exposed position within the lactic acid bacteria (Dicks et al. 1995, see above). This means that there are sequence differences of over 10% to the closest related genera or species. These differences made it possible to construct genus-specific detection probes after comparing the available rRNA sequences. This was similar for the genes for the 5S rDNA and 23S rDNA.

The detection probes according to the invention are preferably oligonucleotides which contain a region which is complementary to the target nucleic acid. This range encompasses the sequences or sections thereof given in Seq ID Nos. 1 to 31 (Tables 1-3) and permits selective hybridization to a 5S, 16S or 23 S rRNA-specific nucleic acid sequence of a species of bacteria of the genus Oenococcus. The oligonucleotide used can preferably be DNA, but also any other nucleic acid, such as PNA, RNA or derivatives thereof, as long as these nucleic acids can hybridize with the target nucleic acid sequence. In contrast to the hybridization of chromosomal DNA using genomic DNA probes (Sohier and Lonvaud-Funel, 1998), the probes according to the invention bind to the complementary sequence of the rRNA. The oligonucleotides have to penetrate the three-dimensional structure of the ribosomes. This can reduce the fluorescence yield, since the hybridization sequences are also located in regions of the ribosome that are difficult to access (Fuchs, BM; Syutsubo, K; Ludwig, W; Amann, R. In situ accessibility of Escherichia coli 23S rDNA to fluorescently labeled oligonucleotide probes. Appl Environ. Microbiol., 2: 961-968 (2001)). For this reason, labeled or unlabeled helper oligonucleotides (Fuchs, BM; Glöckner, FO; Wulf, J; Amann, R. Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl. Environments. Microbiol ., 8: 3603-3607 (2000)) can be included in the hybridization. In this way, the secondary structure of the ribosomes is resolved and the actual hybridization sequence is opened for the labeled probe. According to the invention, a higher fluorescence yield can thereby be achieved. The use of secondary structures thus enables the design of probes in a manner independent of the conventional selection criteria, which completely ignore the secondary structure of the RNA and only rely on simple alignments.

The oligonucleotide according to the invention preferably comprises at least one detectable marker. This marker can further preferably be a dye, enzyme, antibody, radionuclide or fluorescent marker which can be present at the 3 'and / or 5' end of the detection probe, but also as a modification within the sequence of the detection probe. Markers such as biotin, fluorescein, rhodamine, phycoerythrin, carbocyanines (e.g. Cy3 or Cy5), phosphorus, digoxigenin, peroxidase or Texasred or derivatives thereof are particularly preferred. The marker can also generate an indirect detection signal, for example via an enzymatic reaction. Of course, two different markers can also be used, which increases the flexibility of the system.

The oligonucleotides according to the invention usually have a length of 10 to 30 nucleotides, the region which is complementary to the target nucleic acid being at least 9 nucleotides long in order to permit selective hybridization. However, longer oligonucleotides can also be used, these oligonucleotides not further include specific nucleic acid segments, such as PNA or RNA segments. These sections can impart further functional properties to the detection probes according to the invention, which can be advantageous for industrial applicability, for example for selective binding to a nucleic acid chip. However, the advantageous properties of the oligonucleotides according to the invention are retained.

A further object of the present invention is achieved by a method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus, the method comprising the steps of: a) bringing at least one oligonucleotide according to the invention into contact with a sample which is suspected is that it contains Oenococcus-specific nucleic acids, under conditions which allow a specific hybridization of the at least one oligonucleotide to the Oeococcws-specific nucleic acids, and b) detection of the hybridization of the at least one oligonucleotide. With the method according to the invention, all Oenococcus strains can now be detected for the first time, including Oenococcus oeni strain JCM 6125, Oenococcus oeni strain DSM 20252 and / or Oenococcus oeni strain NCDO 1674.

The detection and / or differentiation of a species of bacteria of the genus Oenococcus is preferably carried out on the basis of Oeπococcw.s specific nucleic acids which are present in the sample in the form of an rRNA or cDNA.

In a preferred embodiment, the method according to the invention for the detection and / or differentiation of a species of bacteria of the genus Oenococcus is characterized in that at least one oligonucleotide is attached to a solid phase, e.g. a chip that is bound. The use of chip technology allows the method according to the invention to be used in a high-throughput technology which is expedient for industrial use, since large numbers of samples may have to be checked there. At the same time, chip technology allows the use of robots and other suitable machines, which minimizes the number of manual steps that have to be carried out.

According to a particularly preferred method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus of the invention, the hybridization is detected by means of fluorescence measurement. In this case too, this technique is for one Particularly suitable for use on a large scale and at the same time allows safe, fast and inexpensive implementation.

Another method according to the invention for the detection and / or differentiation of a species of bacteria of the genus Oenococcus is characterized in that the hybridization is detected by means of PCR, optionally using a second non-specific, part-specific or specific oligonucleotide. PCR methods are known to the person skilled in the art from the relevant specialist literature. In the context of the invention, the term “specific” oligonucleotide means an oligonucleotide which hybridizes selectively to a 5S, 16S or 23S rRNA-specific nucleic acid sequence of a species of bacteria of the genus Oenococcus and a sequence or partial sequence with a length of at least 9 nucleotides, selected from the group of sequences according to Seq ID NO: 1-31 or sequences complementary thereto. "Partially specific" means that an oligonucleotide is used which hybridizes specifically to the target sequence only under stringent hybridization conditions. "Non-specific" oligonucleotides are all oligonucleotides that can produce a PCR product with the specific oligonucleotides of the invention and are themselves not suitable for the specific detection of a species of bacteria of the genus Oenococcus. Such non-specific oligonucleotides are thus upstream or downstream of the gene regions localized for the 5S, 23 S and 16S rDNA and usually no further than about 10 kb from the probes according to the invention.

Another aspect of the present invention relates to a method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus, which is characterized in that at least two labeled oligonucleotides are used, which are optionally partially complementary to one another. This use of oligonucleotides as supporting probes eliminates the formation of secondary structures of the target nucleic acid during the hybridization and at the same time increases the signal strength by using a second label. In particular, probes lying opposite or partially overlapping on the rRNA secondary structure can be used.

Another aspect of the present invention relates to kits for the detection and / or differentiation of a species of bacteria of the genus Oenococcus, comprising at least an oligonucleotide according to the invention, optionally together with suitable auxiliaries and additives. Suitable additives are, for example, buffer solutions and / or enzymes for the detection method or for hybridizing the oligonucleotides. The compilation of kits facilitates the industrial application of the method according to the invention and also allows individual samples to be carried out.

According to the invention, at least one of the above-mentioned oligonucleotides or the above-mentioned kits can be used for the detection and / or differentiation of a species of bacteria of the genus Oenococcus. The use for the detection of all Oenococcus strains, in particular Oenococcus oeni strain JCM 6125, Oenococcus oeni strain DSM 20252 and / or Oenococcus oeni strain NCDO 1674, is further preferred.

A particularly preferred aspect of the present invention relates to a method according to the invention and its use, the detection and / or differentiation in a natural sample, in particular a food sample, such as fruit juice, must, mash and / or wine or a sample not obtained from nature , in particular a laboratory culture, such as a starter or enrichment culture. In particularly preferred cases, this is used to check the purity of parent and laboratory cultures, to isolate new strains of Oenococcus, to produce starter cultures, to monitor the vinification and / or to control and ensure quality. The probes according to the invention are therefore an important aid for quality control and process control.

In this way, starter cultures and their purity can be checked using the detection probes according to the invention. It is essential that the starter cultures are made available in sufficient purity.

Similarly, new Oenococcus strains can be isolated by means of the detection probes according to the invention, wherein the probes according to the invention have so far been able to be used successfully with all examined Oenococcus strains. This also allows an effective screening of many samples to be examined without the false positives being feared or without new strains being identified. Based on the present invention, probes or probe mixtures were developed which ensure reliable identification with a high fluorescence yield. The hybridization protocol was modified and shortened so that a result was available within four to five hours. This period ensures a quick check of the purity during cultivation as well as the control of the spontaneous or biological acid degradation by oenococci in the cellar industry. The conventional method, the plate casting process and the observation of colony-forming units is comparatively unsuitable, since visible colonies only develop after five to seven days.

The invention will now be described in more detail below with the aid of examples with reference to the attached sequence listing and the figure. It shows:

Seq ID NO 1: The nucleotide sequence of the probe "Sonde_Ool" according to the invention; Seq ID NO 2: The nucleotide sequence of the probe "Sonde_Oo2" according to the invention; Seq ID NO 3: The nucleotide sequence of the probe “Sonde_Oo3” according to the invention; Seq ID NO 4: The nucleotide sequence of the probe according to the invention “Sonde_Oo4”; Seq ID NO 5: The nucleotide sequence of the probe "Sonde_Oo5" according to the invention; Seq ID NO 6: The nucleotide sequence of the probe "Probe Ooό" according to the invention; Seq ID NO 7: The nucleotide sequence of the probe "Sonde_Oo7" according to the invention; Seq ID NO 8: The nucleotide sequence of the probe "OENOS 23/1" according to the invention; Seq ID NO 9: The nucleotide sequence of the “OENOS 23/2” probe according to the invention; Seq ID NO 10: The nucleotide sequence of the “OENOS 23/3” probe according to the invention; Seq ID NO 11: The nucleotide sequence of the “OENOS 23/4” probe according to the invention; Seq ID NO 12: The nucleotide sequence of the “OENOS 23/5” probe according to the invention; Seq ID NO 13: the nucleotide sequence of the “OENOS 23/6” probe according to the invention; Seq ID NO 14: the nucleotide sequence of the “OENOS 23/7” probe according to the invention; Seq ID NO 15: the nucleotide sequence of the probe "OENOS 23/8" according to the invention; Seq ID NO 16: the nucleotide sequence of the probe "OENOS 23/9" according to the invention; Seq ID NO 17: The nucleotide sequence of the “OENOS 23/10” probe according to the invention; Seq ID NO 18: The nucleotide sequence of the “OENOS 23/11” probe according to the invention; Seq ID NO 19: the nucleotide sequence of the “OENOS 23/12” probe according to the invention; Seq ID NO 20: the nucleotide sequence of the “OENOS 23/13” probe according to the invention; Seq ID NO 21: The nucleotide sequence of the probe "OENOS 23/14" according to the invention; Seq ID NO: 22: the nucleotide sequence of the probe "OENOS 23/15" according to the invention; Seq ID NO: 23: the nucleotide sequence of the probe "OENOS 23/16" according to the invention; Seq ID NO: 24: The nucleotide sequence of the “OENOS 23/17” probe according to the invention; Seq ID NO: 25: The nucleotide sequence of the “OENOS 23/18” probe according to the invention; Seq ID NO: 26: The nucleotide sequence of the “OENOS 23/19” probe according to the invention; Seq ID NO: 27: The nucleotide sequence of the “OENOS 23/20” probe according to the invention; Seq ID NO: 28: the nucleotide sequence of the probe "OENOS 23/21" according to the invention; Seq ID NO: 29: the nucleotide sequence of the probe "OENOS 5/1" according to the invention; Seq ID NO: 30: the nucleotide sequence of the "OENOS 5/2" probe according to the invention; Seq ID NO: 31: the nucleotide sequence of the "OENOS 5/3" probe according to the invention; and Seq ID NO: 32: The nucleotide sequence of the 5S rDNA from O. oeni.

FIG. 1 shows a FISH with the 16S rRNA oligonucleotide probes Ool and Oo3. The red fluorescent O. oe «/ - cells can be seen in the center of the picture due to the hybridization conditions and the choice of probes. The other microorganisms fluoresce light blue in the DAPI counterstain. The red autofluorescence of sample contamination can also be seen at the top of the picture.

Examples

Production of the media used (30 ° C)

For the preparation, 2% tryptone, 0.5% yeast extract, 0.5% peptone, 0.5% glucose, 0.005% Tween 80 were dissolved in 1: 4 diluted tomato juice, adjusted to pH 5.2-5.5 and autoclaved for 15 min. DSMZ 59 was used as an alternative medium.

For one enrichment culture, 7% ethanol (final concentration; addition after autoclaving), 4 mg cycloheximide / 1 (fungicide, to suppress yeast / fungal growth) was added to the medium and the pH was adjusted to 4 with 1 M HCl solution. 0 set.

Structure of the probes

The probes were designed according to the two-dimensional alignment technique (Fröhlich, J. Dissertation: Development and use of isolation techniques for the phylogenetic characterization of symbiotic microorganisms in the intestine of termites (1999); University of Ulm; Ulm). The 16S rRNA sequence to be examined is included a sequence of a possible related kind, for which a secondary structure is known. The secondary structure diagrams are available on the following website: http://www.rna.icmb.utexas.edu cgi-bin / update / seq info.pl

The secondary structure diagrams are sometimes processed using Microsoft Office and the programs ClustalX 1.8 (Thompson, JD; Gibson, TJ; Plewniak, F; Jeanmougin, F; Higgins, DG. The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res., 24: 4876- 4882 (1997)) and Ghostview 4.3. A two-dimensional alignment is created on the basis of an already known rRNA secondary structure. The investigated rRNA sequence is integrated into this secondary structure and displayed graphically. The sequence differences that result from the alignment can also be highlighted in color. With this representation it is possible to use sequence sections with a high variability for the development of fluorescence-labeled oligonucleotides. This view also facilitates the generation of multiple oligonucleotides that can be placed in the immediate vicinity. Two or more hybridization sequences in the secondary structure can be partially complementary to one another. This helper probe system also differs from the classic helper probes (Fuchs et al, 2000) by the fluorescent labeling and the partial complementarity.

Using the probe design technique, it is possible to develop fluorescence-labeled oligonucleotides based on O. oeni's 5S, 16S, and 23 S rRNA secondary structures, some of which were generated using the helper probe system.

5S rRNA oligonucleotide probes

The newly created secondary structure of the 5S rRNA is based on the known secondary structure of Bacillus subtilis (Acc: Dl 1460). Few sequences of the 5S rDNA and secondary structures of the 5S rRNA are known from the representatives of the lactic acid bacteria. W. viridescens offers itself as a reference organism because of its close phylogenetic relationship to O. oeni. The secondary structure of O. oeni was then adapted to the secondary structure of W. viridescens using the procedure described above. The 5S rDNA sequence from O. oeni strain B241 (Acc: AJ510153) was selected for generation because the most complete sequence could be identified from this strain. By comparing the 5S rDNA sequences and the respective secondary structures of the reference organisms to O. oeni, three fluorescence-labeled oligonucleotide probes were created. In addition, an NCBI database comparison of the determined probe sequences was carried out to check the specificity for O. oeni. All oligonucleotide probes are reversely complementary to the selected 5S rRNA sequence and, for example, labeled with indocarbocyanine (Cy3) at the 5 'end.

The sequences of the fluorescence-labeled oligonucleotides were created using an alignment of the O. oeni 5S rDNA sequence with the following reference sequences: Weissella viridescens strain B175, (Acc: unpublished), Leuconostoc mesenteroides strain ATCC 10830 (Acc: AJ510154), Leuconostoc fallax strain DSM 20189, (Acc: AJ510154), Enterococcus faecalis strain ATCC 11420 (Acc: unpublished).

The sequences of the 5S rDNA according to the invention differ within the lactic acid bacteria examined here. For the development of specific, fluorescence-labeled oligonucleotides, the base variability within a potential probe sequence of approximately 15-20 bp is also of interest. A variability can be seen in the individual alignments within the first 25 bp, which favors the design of an oligonucleotide probe. A similarly pronounced variability is evident from base position 60. This is where a variable section of the 5S rDNA sequence begins. This area offers further binding options for two to three oligonucleotide probes, which are specific for oenococci.

With the aid of the sequence alignments, no satisfactory information about the binding of possible probes in the secondary structure of the smallest ribosomal subunit could be made. Therefore, the secondary structures of the 5S rRNA were created for O. oeni strain B241 (Seq ID NO: 32) and the reference organisms.

16S rRNA oligonucleotide probes

The consensus 16S rRNA secondary structure of Oenococcus oeni (strain JCM 6125, Acc: AB022924; ^τ strain DSM 20252, Acc: M35820; ^τ strain NCDO 1674, Acc: X95980) was, according to the scheme described above for the 5S rRNA of the 16S rDNA sequences available in the NCBI database. The existing secondary structure of Lactococcus lactis subsp served as the basis for the construction of the 16S rRNA secondary structure. lactis (Acc: AE006456). 7 probes were generated, some of which were complementary (Probe Ool &Oo3; probe Oo2 & Oo3) to each other. In addition, the special probe areas were compared with an alignment consisting of 16S rDNA sequences from the following organisms: Oenococcus oeni (strain JCM 6125, Acc: AB022924; strain DSM 20252, Acc: M35820; ^τ strain NCDO 1674, Acc: X95980) Leuconostoc mesenteroides strain NCFB , (Acc: AB023247), Lactobacillus casei strain ATCC 393 (Acc: D16551), Pediococcus damnosus strain JCM 5886 (Acc: D87678).

23S rRNA oligonucleotide probes

The 23S rRNA secondary structure of Oenococcus oeni (Acc: S60377) was created as with the 5S or 16S rRNA. Since there is no secondary structure of a phylogenetically closely related lactic acid bacterium in this case, the structure of Leuconostoc carnosum (Acc: S60371, Martinez-Murcia et al., 1993) was based on the existing secondary structure of Enterococcus faecium (Acc: X79341; Naimi, A; Beck, G; Monique, M; Lefebvre, G; Branlanti, C. Determination of the nucleotide sequence of the 23 S ribosomal RNA and flanking spacers of an Enterococcus faecium strain, reveals insertion-deletion events in the ribosomal spacer 1 of enterococci. Syst Appl. Microbiol., 1: 9-21 (1999)).

This was followed by the final presentation of the secondary structure of O. oeni based on that of L. carnosum. As already described above, the sequence differences were highlighted by red coloring and base deletions or insertions were corrected by visual inspection of the graphics and programming in the text in Word 97.

The sequences of the fluorescence-labeled oligonucleotides were created using an alignment of the O. oeni 23 S rDNA sequence with the following reference sequences: Weissella paramesenteroides (Acc: S60373), Weissella confusa (Acc: S60375), Leuconostoc mesenteroides (Acc: S60370), Leuconostoc carnosum ( Acc: X68037), Leuconostoc lactis (Acc: Z75491), Oenococcus oeni (Acc: S60377).

The position of the 21 generated 23 S rRNA oligonucleotide probes was checked in a two-dimensional representation by comparing the secondary structures of L. carnosum and O. oeni. In addition, a database comparison of the probe sequences determined was carried out to check the specificity for oenococci. All oligonucleotide probes are reversely complementary to the selected 23 S rRNA sequence and optionally labeled with indocarbocyanine (Cy3) at the 5 'end.

The exact placement of the hybridization sequence of a probe has the advantage of generating further probes in the immediate vicinity, so that these oligonucleotides can increase the fluorescence yield according to the helper probe principle. These need not necessarily be fluorescence-labeled probes. Unlabeled probes have the advantage of also hybridizing in conserved and thus occurring sequences in different species. Binding to such a sequence, however, facilitates the access of a specific, labeled probe to a more difficult-to-reach sequence in the three-dimensional structure of the ribosome.

Another possibility is offered by fluorescence-labeled, i.e. specific probes that also act on the helper probe principle. These probes hybridize with a specific sequence and thus facilitate the access of a second, also specific, probe to another sequence. These probe mixtures intensify the fluorescence yield even more than mixtures with unlabeled helper probes.

The functioning of the probes and the fluorescence yield depend directly on the transferability of the method by Fuchs et al. (Flow cytometric analysis of the in situ accessability of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes; Appl Environ Microbiol 64: 4973-82 (1998)) on bacteria other than Escherichia coli. For this question, the binding studies of the SSU rRNA from E. coli and Bacillus subtilis were compared (SSU rRNA secondary structure from E. coli and B. subtilis) (see http://www.rna.icmb.utexas.edu/ANALYSIS/16S .FOLD / 16sfold.html, and Konings and Guteil, A comparison of thermodynamic foldings with comparatively derived structures of 16S and 16S-like rRNAs. RNA 1: 559-74 (1995)). The structure of the secondary structures is largely the same. However, all seven generated 16S probes are in more restricted areas.

The positions of the probes were chosen so that the best possible fluorescence yield according to, or in some cases contrary to, Fuchs et al. Was achieved in 2000. The probe sequences were compared for their specificity with the RDP database (http://rdp.cme.msu.edu/cgis/probe_match.cgi?su=SSU). The probes were then removed synthesized and the 5 'position optionally marked with CY3 (MWG-BIOTECH AG, Ebersberg). Tables 1 to 3 show the sequences and characteristics of the oligonucleotides so constructed.

Table 1: Sequences and characteristics of the constructed 16S oligonucleotides.

Table 2: Sequences and characteristics of the constructed 23 S oligonucleotides. (Continuation)

Table 3: Sequences and characteristics of the constructed 5S oligonucleotides.

Database Comparison

The probe sequences were compared to check the specificity with the RDP database (http://rdp.cme.msu.edu/cgis/probe_match.cgi?su=SSU). With the exception of probe Oo_7, all probes showed sufficient specificity (more than 3 mismatches) in which there was 100% agreement with the SSU rRNA from Xanthomonas sp. revealed. Probe Oo_7 was nevertheless used as an Oenococc ws-specific detection probe, since the common occurrence of Oenococcus and Xanthomonas sp. is unlikely due to the different habitats.

The probe sequences of the 5S rRNA oligonucleotides did not match the rRNA sequences of other organisms in the NCBI sequence comparison. From this it can be seen that until now they are specific to O. oeni.

With one exception, the probe sequences of the 23 S rRNA oligonucleotides are specific for O. oeni. Probe 23/19 showed complete agreement (16 bp / 16 bp) in the 23S rDNA gene from Enterococcus faecium (Acc: AF432914). Since the probe 23/19 interacts with the probe 23/18 in a helper and detection function, the detection reaction for O. oeni can be given despite this complete homology. Furthermore, the occurrence of O. oeni is limited to wine, while E. faecium is predominantly found in faeces. The application and the specificity of OENOS 23/19 therefore depends on the sample material examined. When hybridizing wine samples, the possible cross-reaction with the 23 S rRNA from E. faecium can be neglected.

Fluorescence in situ hybridization FISH (Leitch, AR; Schwarzacher, T; Jackson, D; Leitch, IJ (1994) In 57 ^' tw hybridization, spectrum academic publisher GmbH Heidelberg, Berlin, Oxford, Bettenhausen B (ed.) ISBN 3- 86025-225-9)

The cells were centrifuged off at 5000 rpm (centrifuge 5403, Eppendorf, Hamburg). The supernatant medium was discarded. The bacteria were then washed with deionized water and centrifuged again. The cells were then resuspended in water and epoxy-coated slides (Menzel glasses, Braunschweig) were applied. After drying at room temperature, the slides were incubated at 90 ° C for 10 min. Then the samples were included in ascending order 50% -70% ethanol / lxPBS buffer mixture and finally dewatered 2x with 96% ethanol for 5 min each.

After the ethanol had evaporated, 99 μl of the hybridization buffer was mixed with 1 μl 50 pmol / μl probe solution and applied to the slides in the dark. The buffer / probe mixtures were previously denatured for 10 min at 70 ° C. and then cooled on ice. The hybridization buffer (calculated for 1 ml hybridization solution) consisted of 100 μl 200 mM Tris / HCl buffer (pH 7.2), 333.4 μl 2.7 M NaCl solution, 1 μl 10% SDS solution ., 0.1 g dextran sulfate, 565.6 ul deionized water. The slides were covered with coverslips and placed in preheated hybridization chambers, which were moistened with 2 ml buffer (50 ml Falkontubes, Becton Dickinson Labware, NJ, USA) and in the hybridization oven (Hybaid, Heidelberg) at 36 ° C (16S rRNA probes) or 45 ° C (5S and 23 S rRNA probes) for 1 h. The wash solution had the same composition as the hybridization buffer except for the dextran sulfate. The slides were left in the wash solution at 38 ° C (16S rRNA probes) or 48 ° C (5S and 23S rRNA probes) for 10 min and a second time for a further 10 min at 40 ° C (16S rRNA probes) ) or 50 ° C (5S and 23S rRNA probes). The slides were washed with 100 μl (2 μg / ml) DAPI solution. layered and incubated for 10 min at room temperature. After a short washing step with deionized water, add 50 μl DABCO solution and cover with a coverslip.

The slides were examined with an Axiophot2 fluorescence microscope (Zeiss, Göttingen) at 1000x magnification using filter sets 01 (excitation: 365 nm, emission: 397 nm, and Cy3 (excitation: 550 nm, emission: 610-675 nm).

fluorescence signals

The hybridization experiments were carried out with the following organisms: Oenococcus oeni Bl, Oenococcus oeni B139, Leuconostoc mesenteroides, Lactobacillus casei and Pediococcus damnosus. The probes should be so specific that signals only result from the various oenococci. In order to accelerate the hybridization and to minimize the background fluorescence, fixation with formaldehyde was dispensed with and the hybridization accelerator dextran sulfate was used (Leitch et al, 1994; see above). The fluorescence signals of the Oo_l and Oo_7 probes were comparatively stronger than with the Eub338 probe, which generally binds to all Eubacteria. The other probes showed only a weak or no signal. Only probe Oo_2 and probe Oo_4 should give stronger signals or signals at all. For probe 2, the area that Fuchs et al. Appl Environ Microbiol. 1998 Dec; 64 (12): 4973-82 suggested that the probe was not strictly adhered to, but, to be specific, had to be moved slightly into an area with low signal strength. The case for probe 4 is different because this area was described as less strongly bound in E. coli, while this area is strongly bound in Bacillus and Oenoccocus.

The area of the SSU rRNA to which the probe Oo_l hybridizes is even less bound in Oenococcus than in Bacillus or Escherichia and is also significantly A / T-rich. Therefore, this probe shows a strong fluorescence signal. Since probe Oo_3 gave a moderate signal, but hybridizes with an area that is partially complementary to probe area 1, mixed hybridization with probe Oo_l and Oo_3 should result in a particularly strong fluorescence signal, since probe 1 exposes the area for probe 3 during hybridization. The results exceeded our expectations. Lactobacillus casei showed a higher self-fluorescence under UV light due to its light pigmentation. Therefore, the weaker probes should not be used alone.

FIG. 1 shows a FISH with the 16S rRNA oligonucleotide probes Ool and Oo3. The red fluorescent O. oew ^' cells can be seen in the center of the picture due to the hybridization conditions and the choice of probes. The other microorganisms fluoresce light blue in the DAPI counterstain. The red autofluorescence of sample contamination can also be seen at the top of the picture.

Application example of probe mixtures

Based on the results mentioned, the probes Ool and Oo3 were applied to 84 strains from our institute strain collection. The tribes come from different regions of Germany, Portugal (B281, B283, B289, B290, B302) and South Australia (B5). The results are shown in Table 4. New isolates from wine were also reliably identified (strains: B355-B359) Despite different fluorescence yields during in situ hybridization, the probes show that oenococcal strains can be identified with certainty. The strains from Portugal identified as oenococci on the basis of their protein spectrum gave no signal with the probes (see Table 4). After sequencing the PCR amplificates of the 16S rDNA sequences, these strains were identified as representatives of the species Lactobacillus casei. The probe sequence and 5 'labels were as follows: Probe Ool: CY3 - TTC TCT GAA TTC AGT TAT TC Probe Oo3: CY3 - GTA CCG TCA AGC TGA A

Table 4: Results of the hybridizations of the oenococci examined with the probes Ool and Oo3:

Signal strength: *** very strong positive; ** strongly positive; * moderately positive; - Negative series 10: Newly isolated strains from wine tasting (Hirschhäuser 2002).

Despite different fluorescence yields during in situ hybridization, the probes show that oenococcal strains can be identified with certainty. Other probes of the following compositions have been used successfully:

1. Single probes

OENOS 23/2; OENOS 23/3; OENOS 23/4; OENOS 23/7, OENOS 23/9 and OENOS 23/20

2. Probe pairs

OENOS 23/1 & OENOS 23/12

3. Probe triplets

OENOS 23/2 & OENOS 23/3 & OENOS 23/4; OENOS 23/11 & OENOS 23/13 & OENOS 23/14

Application examples of the 5S rRNA oligonucleotide probes

Following the probe design, the three 5S rRNA oligonucleotide probes were also examined for the specific detection of oenococci. Mixtures of O. oeni strain B139 ^τ , the isolates strain B356 and strain 7 ³ , were prepared with the reference organisms (see above) and hybridized with the individual probes. In further approaches, all possible probe mixtures were produced and used in the hybridization. This was used to examine their helper probe properties.

Hybridizations with only one of the three 5S rRNA probes showed no detectable fluorescence yield. It was not possible to differentiate between O. oeni and the reference organisms. L. fallax also showed background fluorescence that was stronger than that of O. oeni. Therefore, none of the three 5S rRNA probes is suitable for the specific detection of oenococci. Only the mixture of all three probes in a hybridization reaction showed the clear one Differentiation between Oenococcus oeni and Leuconostoc fallax. The reference organisms could also be distinguished from Oenococcus oeni using this probe mixture.

Claims

claims 1. oligonucleotide, in particular DNA oligonucleotide, which hybridizes selectively to a 5S or 16S or 23S rRNA-specific nucleic acid sequence of a species of bacteria of the genus Oenococcus and a sequence or partial sequence with at least 9 nucleotides in length, selected from the group of sequences TTC TCT GAA TTC AGT TAT TC (Seq ID NO: 1), ATT CAG TTA TTC TTC CCT TA (Seq ID NO: 2), GTA CCG TCA AGC TGA A (Seq ID NO: 3), AGA TCA CAT GTG TGA TTT G (Seq ID NO: 4), TCA CTA GGA GGC GGA A (Seq ID NO: 5), TTA CAA AAG CGA TCA TTG G (Seq ID NO: 6), TTA CCG TCG CCG GTT T (Seq ID NO: 7) , AAA AGA TGA TGA TCA TTA AGA TC (Seq ID NO: 8), CTT ATA CCT TTG TAA CTC AAC AT (Seq ID NO: 9), TGT CAC CCT CTT TGG AAC (Seq ID NO: 10), TTA GAG AGT ACA AGA TTT CGC (Seq ID NO: 11), TAA CAC CCA CTT GTT CGG T (Seq ID NO: 12), TCC ACA GTT CAT CAA AGG G (Seq ID NO: 13), CCT GCA CAT ACT TAG CTG (Seq ID NO: 14), ATC CTA CCA TCT TAC GCA T (Seq ID NO: 15), CCC TTC ATC GCG TTA ATT A (Seq ID NO: 16), TTC CTT CGG C AA TTA AAT ATC (Seq ID NO: 17), CAA GTA AAC TTG GTA CAT TCA (Seq ID NO: 18), GCT TCA TCT AAT CTA TGA ATT C (Seq ID NO: 19), GCG TGA AAT CAC ACA ATT TC (Seq ID NO: 20), AGA CTA ACT GTT TAA AAC CAC AT (Seq ID NO: 21), GCT CCA ACA TGT CTT ACG (Seq ID NO: 22), CTG CGG CTG GTA TAA AAC (Seq ID NO: 23 ), ACC AAT GCC TCC ACC G (Seq ID NO: 24), TTT ATT GGT GAG GGT TAG AG (Seq ID NO: 25), TGA CAC TGT CTC CCA C (Seq ID NO: 26), GTC TTA CTT CTG ACG AAT G (Seq ID NO: 27), CCC CCG GGT AAG CTT (Seq ID NO: 28), GAC CTC ATC GGA ATT AAC (Seq ID NO: 29), TAC TTT GGG CCC TGA CA (Seq ID NO: 30) and ACC TTG CAA CAG GCG TT (Seq ID NO: 31) or sequences complementary thereto. 2. Oligonucleotide according to claim 1, characterized in that it comprises at least one detectable marker. 3. Oligonucleotide according to claim 2, characterized in that the marker is an enzyme, antibody, radionuclide or fluorescent marker. 4. Oligonucleotide according to claim 2 or 3, characterized in that the marker is biotin, fluorescein, carbocyanines (e.g. Cy3 or Cy5), phosphorus, digoxigenin, peroxidase or rhodamine, Texasred or derivatives thereof. 5. Oligonucleotide according to one of the preceding claims, characterized in that the oligonucleotide has a length of 10 to 30 nucleotides. 6. Oligonucleotide according to one of the preceding claims, characterized in that the oligonucleotide comprises further non-specific nucleic acid sections, such as PNA or RNA sections. 7. A method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus, comprising bringing at least one oligonucleotide according to any one of claims 1 to 6 into contact with a sample suspected of containing Oenococcus-specific nucleic acids Conditions which allow a specific hybridization of the at least one oligonucleotide to the Oenococcus-specific nucleic acids and detection of the hybridization of the at least one oligonucleotide. 8. A method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus according to claim 7, characterized in that all Oenococcus strains are detected, in particular Oenococcus oeni strain JCM 6125, Oenococcus oeni strain DSM 20252 and / or Oenococcus oeni strain NCDO 1674. 9. A method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus according to claim 7 or 8, characterized in that the Oenococcws-specific nucleic acids are present in the sample in the form of an rRNA or cDNA. 10. A method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus according to one of claims 7 to 9, characterized in that at least one oligonucleotide is coupled to a solid phase, for example a chip. 11. A method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus according to one of claims 7 to 10, characterized in that the hybridization is detected by means of fluorescence measurement. 12. A method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus according to one of claims 7 to 11, characterized in that the detection of the hybridization by means of PCR, optionally using a second non-specific, part-specific or specific oligonucleotide. 13. A method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus according to one of claims 7 to 12, characterized in that at least one oligonucleotide is used which acts as a helper probe. 14. A method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus according to one of claims 7 to 13, characterized in that at least two labeled oligonucleotides are used, which are optionally partially complementary to one another. 15. A method for the detection and / or differentiation of a species of bacteria of the genus Oenococcus according to one of claims 7 to 14, characterized in that the sample is a natural sample, in particular a food sample, such as fruit juice, must, mash and / or wine or a sample not obtained from nature, especially a laboratory culture such as is a starter or enrichment culture. 16. Kits for the detection and / or differentiation of a species of bacteria of the genus Oenococcus, comprising at least one oligonucleotide according to one of claims 1 to 6, optionally together with suitable auxiliaries and additives. 17. Use of at least one oligonucleotide according to one of claims 1 to 6 or a kit according to claim 16 for the detection and / or differentiation of a species of bacteria of the genus Oenococcus. 18. Use according to claim 17 for the detection of all Oenococcus strains, in particular Oenococcus oeni strain JCM 6125, Oenococcus oeni strain DSM 20252 and / or Oenococcus oeni strain NCDO 1674. 19. Use according to claim 17 or 18, wherein the detection and / or differentiation in a natural sample, in particular a food sample, such as fruit juice, must, mash and / or wine or a sample not obtained from nature, in particular a laboratory culture, such as eg a starter or enrichment culture. 20. Use according to one of claims 17 to 19 for checking the purity of master and laboratory cultures, for isolating new strains of Oenococcus, the production of starter cultures, tracking the wine development and / or quality control and assurance.