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WO2003066894A2 - Sonde geniques pour detecter des membres de l'espece oenococcus - Google Patents

Sonde geniques pour detecter des membres de l'espece oenococcus Download PDF

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WO2003066894A2
WO2003066894A2 PCT/EP2003/001198 EP0301198W WO03066894A2 WO 2003066894 A2 WO2003066894 A2 WO 2003066894A2 EP 0301198 W EP0301198 W EP 0301198W WO 03066894 A2 WO03066894 A2 WO 03066894A2
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seq
oenococcus
detection
oligonucleotide
species
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PCT/EP2003/001198
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German (de)
English (en)
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WO2003066894A8 (fr
WO2003066894A3 (fr
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Jürgen FRÖHLICH
Helmut König
Steffen HIRSCHHÄUSER
Bruno Bandenburg
Original Assignee
Froehlich Juergen
Bruno Bandenburg
Koenig Helmut
Hirschhaeuser Steffen
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Application filed by Froehlich Juergen, Bruno Bandenburg, Koenig Helmut, Hirschhaeuser Steffen filed Critical Froehlich Juergen
Priority to AU2003205743A priority Critical patent/AU2003205743A1/en
Publication of WO2003066894A2 publication Critical patent/WO2003066894A2/fr
Publication of WO2003066894A3 publication Critical patent/WO2003066894A3/fr
Publication of WO2003066894A8 publication Critical patent/WO2003066894A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • 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).
  • BSA biological acid degradation
  • Malic acid is converted into lactic acid, which is perceived as less acidic.
  • 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).
  • 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.
  • 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.
  • 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.
  • probes or probe mixtures which enable reliable and simple identification of species of the genus Oenococcus.
  • the probes should be constructed so that they provide a signal suitable for detection, such as a good fluorescence yield.
  • the process should be fast, cheap and reliable and should also be applicable on an industrial scale.
  • 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.
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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 Oe ⁇ ococcw.s specific nucleic acids which are present in the sample in the form of an rRNA or cDNA.
  • 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.
  • a solid phase e.g. a chip that is bound.
  • the hybridization is detected by means of fluorescence measurement.
  • 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.
  • 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.
  • probes lying opposite or partially overlapping on the rRNA secondary structure can be used.
  • 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.
  • 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.
  • 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.
  • a laboratory culture such as a starter or enrichment culture.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 Oo ⁇ ” 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
  • 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.
  • 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.
  • 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.
  • oligonucleotide probes 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.
  • Cy3 indocarbocyanine
  • 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).
  • sequences of the 5S rDNA according to the invention differ within the lactic acid bacteria examined here.
  • 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.
  • the consensus 16S rRNA secondary structure of Oenococcus oeni 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.
  • 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; ⁇ 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).
  • the 23S rRNA secondary structure of Oenococcus oeni 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.
  • 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.
  • 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.
  • subtilis 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 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 3 Sequences and characteristics of the constructed 5S oligonucleotides.
  • 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.
  • 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.
  • the hybridization buffer (calculated for 1 ml hybridization solution) consisted of 100 ⁇ l 200 mM Tris / HCl buffer (pH 7.2), 333.4 ⁇ l 2.7 M NaCl solution, 1 ⁇ l 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 ⁇ l (2 ⁇ g / ml) DAPI solution. layered and incubated for 10 min at room temperature. After a short washing step with deionized water, add 50 ⁇ l 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).
  • 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.
  • 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.
  • probe Oo_2 and probe Oo_4 should give stronger signals or signals at all.
  • 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.
  • 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.
  • 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).
  • probe Ool CY3 - TTC TCT GAA TTC AGT TAT TC
  • Probe Oo3 CY3 - GTA CCG TCA AGC TGA
  • the three 5S rRNA oligonucleotide probes were also examined for the specific detection of oenococci.
  • Mixtures of O. oeni strain B139 ⁇ , the isolates strain B356 and strain 7 3 were prepared with the reference organisms (see above) and hybridized with the individual probes.
  • 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.

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Abstract

La présente invention concerne des sondes ou mélanges de sondes qui permettent une identification simple et fiable de membres de l'espèce Oenococcus. Les sondes doivent être mises au point de sorte qu'elles produisent un signal pouvant être utilisé pour la détection, par exemple un bon rendement en fluorescence. L'invention a également pour objet un procédé pour détecter et/ou différentier un type de bactéries appartenant à l'espèce Oenococcus et son utilisation par ex. dans la cadre de la production de vin.
PCT/EP2003/001198 2002-02-06 2003-02-06 Sonde geniques pour detecter des membres de l'espece oenococcus WO2003066894A2 (fr)

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AU2003205743A AU2003205743A1 (en) 2002-02-06 2003-02-06 Gene probes for the detection of the oenococcus species

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DE2002104858 DE10204858B4 (de) 2002-02-06 2002-02-06 Gensonden zum Nachweis von Spezies der Gattung Oenococcus
DE10204858.4 2002-02-06

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