EP1397519A2 - Procede pour la mise en evidence rapide specifique de bacteries a effet nefaste sur la biere - Google Patents

Procede pour la mise en evidence rapide specifique de bacteries a effet nefaste sur la biere

Info

Publication number
EP1397519A2
EP1397519A2 EP02762296A EP02762296A EP1397519A2 EP 1397519 A2 EP1397519 A2 EP 1397519A2 EP 02762296 A EP02762296 A EP 02762296A EP 02762296 A EP02762296 A EP 02762296A EP 1397519 A2 EP1397519 A2 EP 1397519A2
Authority
EP
European Patent Office
Prior art keywords
seq
lactobacillus
bacteria
beer
seqidno
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02762296A
Other languages
German (de)
English (en)
Inventor
Claudia Beimfohr
Jiri Snaidr
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vermicon AG
Original Assignee
Vermicon AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vermicon AG filed Critical Vermicon AG
Publication of EP1397519A2 publication Critical patent/EP1397519A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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

  • the invention relates to a method for the specific rapid detection of beer-damaging bacteria by in situ hybridization, oligonucleotide probes suitable for the method, and kits with which the detection method can be carried out.
  • the EU's annual beer production is around 313 million hectoliters, of which 112 million hectoliters are produced in Germany alone. With approximately 1270 breweries located here and an annual turnover of about DM 18 billion, the brewing process is one of the most important industrially used biotechnological processes in Germany ("Data from the European brewing industry 1999", Deutscher Brauer Bund, 2001, Bonn, http: //www.brauer -bund.de).
  • microbiological quality control is also of great importance.
  • microorganisms Due to the extremely selective and partly bacteriocidal effect of beer, only a very narrow spectrum of microorganisms is found in brewing systems. For persistence in the beer habitat, microorganisms must tolerate a low pH value, an anaerobic atmosphere, bitter hop substances, alcohol and a very small amount and variety of nutrients and growth agents. (W. Back, color atlas and manual of beverage biology, 1994, publisher Hans Carl, Nuremberg). Accordingly, predominantly lactic acid bacteria of the genera Lactobacillus and Pediococcus as well as representatives of the genera Pectinatus and Megasphaera are known as beer-damaging microorganisms.
  • Lactic acid bacteria are all gram-positive, non-spore-forming, catalase-negative rods and cocci. To date, nine different genera (Lactobacillus, Lactococcus, Leuconostoc, Carnobacterium, Bifidobacterium, Enterococcus, Pediococcus, Weissella and Streptococcus) are summarized under the term lactic acid bacteria. Phylogenetically, all representatives of the lactic acid bacteria are classified as members of the gram-positive bacteria with a low GC content in the DNA. (Brock, microbiology, 2001, Spektrum Akademischer Verlag GmbH Heidelb erg-B er lin).
  • Lactobacillus is used for the fermentation of cheese, yoghurt, buttermilk, sour cream, sauerkraut, meat and sausage products and other foods.
  • lactic acid bacteria are by no means only relevant in a positive sense for the food and beverage industry. Rather, some representatives of different genres play an important role as food spoilers.
  • Lactobacillus The representatives of the very heterogeneous genus Lactobacillus are described as gram-positive, non-spore-forming, homo- or heterofermentative, catalase-negative and usually immobile rods. There are currently over 50 different species in this genus, of which only a very small number are identified as harmful to beer.
  • Pediococci are characterized as gram-positive, non-spore-forming, homofermentative, catalase-negative and mainly found in tetrads. Also in this genus there are only a few species to grow in Beer and the resulting spoilage of the beer. (Brock, microbiology, 2001, Spektrum Akademischer Verlag GmbH Heidelberg-Berlin; general microbiology, H. Schlegel, 1992, Georg Thieme Verlag Stuttgart).
  • Lactobacillus brevis Lactobacillus buchneri, Lactobacillus lindneri, Lactobacillus coryniformis, Lactobacillus plantarum, Lactobacillus pseudoplantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus fructivorans, Lactobacillus per olens, Lactobacillus rhamnosus, Lactobacillus frigidus, Pediococcus damnosus, Pediococcus inopinatus (W. Back, color atlas and manual of beverage biology, 1994, publisher Hans Carl, Nuremberg). Lactobacillus brevis, Lactobacillus lindneri and Pediococcus damnosus are of particular importance in practice.
  • some gram-negative bacteria of the genera Pectinatus and Megasphaera are also known as beer spoilers.
  • the rod-shaped cells of the genus Pectinatus are described as strictly anaerobic, motile and slightly curved bacteria.
  • the spherical or slightly oval coccoid cells of the genus Megasphaera are also strictly anaerobic microorganisms. (W. Back, Color Atlas and
  • PCR the polymerase chain reaction
  • a characteristic piece of the respective bacterial genome is amplified with specific primers. If the primer finds its destination, a piece of the genetic material multiplies millions of times.
  • a qualitative evaluation can take place by means of an agarose gel that separates DNA fragments. In the simplest case, this leads to the statement that the target sites for the primers used were present in the examined sample. No further statements are possible; these target sites can originate from a living bacterium as well as from a dead bacterium or from naked DNA.
  • FISH fluorescence in situ hybridization method
  • the FISH technique is based on the fact that there are certain molecules in bacterial cells that, due to their vital function, have undergone only little mutation in the course of evolution: the 16S and the 23S ribosomal ribonucleic acid (rRNA). Both are components of the ribosomes, the sites of protein biosynthesis, and due to their ubiquitous distribution, their size, and their structural and non-constancy, they can serve as specific markers (Woese, CR, 1987. Bacterial evolution. Microbiol. Rev. 51, p. 221 -271). Based on a comparative sequence analysis, phylogenetic relationships can be established based solely on this data. To do this, this sequence data must be aligned.
  • rRNA ribosomal ribonucleic acid
  • these gene probes which are complementary to a specific region on the ribosomal target sequence, are introduced into the cell.
  • the gene probes are usually small, 16-20 base long, single-stranded deoxyribonucleic acid pieces and are directed against a target region, which is typical for a type or group of bacteria. If the fluorescence-labeled gene probe finds its target sequence in a bacterial cell, it binds to it and the cells can be detected in the fluorescence microscope due to their fluorescence.
  • the FISH analysis is always carried out on a slide, since during the evaluation the bacteria are visualized, ie made visible, by irradiation with high-energy light.
  • This is one of the disadvantages of the classic FISH analysis: since only relatively small volumes can naturally be analyzed on a slide, the sensitivity of the method can be unsatisfactory and not sufficient for a reliable analysis.
  • the present invention therefore combines the advantages of classic FISH analysis with those of cultivation. A comparatively short cultivation step ensures that the bacteria to be detected in sufficient numbers are available before the bacteria are detected using specific FISH.
  • the implementation of the method according to the invention for the specific rapid detection of beer-damaging bacteria thus comprises the following steps:
  • “cultivation” means the multiplication of the bacteria contained in the sample in a suitable cultivation medium.
  • the methods suitable for this are well known to the person skilled in the art.
  • fixing the bacteria is understood to mean a treatment with which the bacterial envelope is made permeable to nucleic acid probes. Ethanol is usually used for fixing. If the cell wall cannot be penetrated by the nucleic acid probes with these measures, the person skilled in the art will know Sufficient additional measures are known which lead to the same result, such as, for example, methanol, mixtures of alcohols, a low-percentage paraformaldehyde solution or a dilute formaldehyde solution, enzymatic treatments or the like
  • the enzymes include, for example, lysozyme, proteinase K and mutanolysin, and adequately suitable processes are known to the person skilled in the art and he will be able to easily determine which agent is particularly suitable for cell disruption depending on the bacterium.
  • the “fixed” bacteria are incubated with fluorescence-labeled nucleic acid probes for the “hybridization”.
  • These nucleic acid probes which consist of an oligonucleotide and a marker attached to it, can then penetrate the cell envelope and bind to the target sequence corresponding to the nucleic acid probe inside the cell.
  • the bond is to be understood as the formation of hydrogen bonds between complementary pieces of nucleic acid.
  • the nucleic acid probe can be complementary to a chromosomal or episomal DNA, but also to an mRNA or rRNA of the microorganism to be detected. It is advantageous to choose a nucleic acid probe that is complementary to an area that is present in the number of copies of more than 1 in the microorganism to be detected.
  • the sequence to be detected is preferably 500-100,000 times per cell, particularly preferably 1,000-50,000 times.
  • the rRNA is preferably used as the target site, since the ribosomes in the cell as sites of protein biosynthesis are present thousands of times in each active cell.
  • the nucleic acid probe in the sense of the invention can be a DNA or RNA probe which will generally comprise between 12 and 1000 nucleotides, preferably between 12 and 500, more preferably between 12 and 200, particularly preferably between 17 and 50 and between 15 and 40 and most preferably between 17 and 25 nucleotides.
  • the nucleic acid probes are selected on the basis of whether a complementary sequence is present in the microorganism to be detected. By selecting a defined sequence, a bacterial species, a bacterial genus or an entire bacterial group can be recorded. Complementarity should be at a probe of 15 Nucleotides must be given over 100% of the sequence. In the case of oligonucleotides with more than 15 nucleotides, one to more, preferably one, two or three mismatching sites are permitted.
  • nucleic probe molecules of the lengths and sequences given below are noted in the 5 '-3' direction.
  • the nucleic acid probe molecules according to the invention are for the detection of beer-damaging lactic acid bacteria of the genera Lactobacillus and Pediococcus, in particular of the species Pediococcus damnosus, Lactobacillus coryniformis, Lactobacillus per olens, Lactobacillus lactner, Lactobacillivobillillacillus, Lactobacillivobillillacillus, Lactobacillivacillusacillus Lactobacillusacillus, Lactobacillivacillusacillus Lactobacillusacillus Lactobacillusacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lacto
  • sequences SEQ ID No. 1 to 3 are particularly suitable for the detection of Lactobacillus perolens.
  • SEQ ID No. 1 to 3 illustrate preferred embodiments of the invention.
  • SEQ ID No.4 illustrate preferred embodiments of the invention.
  • SEQ ID No. 4 is particularly suitable for the detection of Lactobacillus buchneri.
  • SEQ ID No. 4 illustrates a preferred embodiment of the invention.
  • SEQ ID No. 5 is particularly suitable for the detection of Lactobacillus plantarum.
  • SEQ ID No. 5 illustrates a preferred embodiment of the invention.
  • SEQ ID No. 6 is particularly suitable for the detection of Lactobacillus fructivorans.
  • SEQ ID No. 6 illustrates a preferred embodiment of the invention.
  • SEQ ID NO. 7 is particularly suitable for the detection of Lactobacillus casei.
  • SEQ ID No. 7 illustrates a preferred embodiment of the invention.
  • SEQ ID NO. 8 is particularly suitable for the detection of Lactobacillus coryniformis.
  • SEQ ID NO. 8 illustrates a preferred embodiment of the invention.
  • SEQ ID No. 9 and 10 illustrate preferred embodiments of the invention. These sequences are particularly suitable for the detection of Lactobacillus brevis.
  • SEQ ID No. 11 is particularly suitable for the detection of Lactobacillus lindneri.
  • SEQ ID No. 11 illustrates a preferred embodiment of the invention.
  • SEQ ID No. 12 is particularly suitable for the detection of Pediococcus damnosus.
  • SEQ ID No. 12 illustrates a preferred embodiment of the invention.
  • SEQ ID No. 13 is particularly suitable for the detection of Lactobacillus lindneri.
  • GCT ACC CAY GCT TTC GAG SEQ ID No. 12 is suitable for the detection of the genera Pediococcus and Lactobacillus.
  • SEQ ID No. 15 is particularly suitable for the detection of bacteri of the genus Pediococcus, in particular of P. acidilactici, P. pentosaceus, P. damnosus, P. parvulus.
  • SEQ ID No. 16 is suitable for the detection of L. casei.
  • SEQ ID No. 17 is particularly suitable for the detection of L. coryniformis.
  • SEQ ID NO. 18 is particularly suitable for the detection of L. fructivorans.
  • SEQ ID No. 20 TTA CAA GAC CAG ACA GCC SEQ ID No. 19 and 20 are suitable for the detection of P. damnosus.
  • SEQ ID NO. 21 is suitable for the detection of L. brevis.
  • SEQ ID NO. 22 and 23 are suitable for the detection of L. plantarum.
  • SEQ ID No. 24 and 25 are suitable for the detection of the genera Pediococcus and Lactobacillus.
  • SEQ ID No. 26 is suitable for the detection of L. lindneri.
  • SEQ ID No. 27 is suitable for the detection of L. lindneri.
  • SEQ ID No. 27 is suitable for the detection of L. brevis.
  • the SEQ ID No. 28 to 67 are particularly suitable for the detection of P. damnosus.
  • sequences SEQ ID No.68 to 107 are suitable for the detection of L. brevis.
  • SEQ ID NO. 108 to SEQ ID No. 146 are suitable for the detection of L. lindneri.
  • SEQ ID NO.147 to 187 are suitable for the detection of L. buchneri.
  • SEQ ID No. 188 to 226 are suitable for the detection of L. casei.
  • SEQ ID NO. 227 to 265 are suitable for the detection of L. coryniformis.
  • the SEQ ID No. 266 to 304 are suitable for the detection of L. fructivorans.
  • SEQIDNo.312 AGACCATGCGGTCTCCGT
  • the SEQ ID No. 305 to 343 are suitable for the detection of Lactobacillus perolens.
  • the SEQ ID No. 344 to 356 are suitable for the detection of Lactobacillus plantarum.
  • TCC GAC ACT CCA GTC CGG SEQ H) NO. 357 and SEQ ID No. 358 are particularly suitable for the detection of Megasphaera cerevisiae.
  • SEQ ID No. 358 illustrates a preferred embodiment of the invention.
  • SEQ ID No. 359 and 360 are particularly suitable for the detection of Pectinatus cerevisuphilus.
  • SEQ ID No. 361 to 400 are particularly suitable for the detection of bacteria of the genus Pectinatus.
  • SEQ ID No. 401 to 439 are particularly suitable for the detection of Megasphaera cerevisiae.
  • SEQ ID No. 440 is particularly suitable for the detection of the genus Pectinatus.
  • SEQ ID No. 441 and 442 are particularly suitable for the detection of Pectinatus cerevisuphilus.
  • K stands for "G + T”
  • M stands for "A + C”
  • R for "A + G”
  • W for "A + T”
  • Y for "C + T”.
  • the invention also relates to modifications of the above oligonucleotide sequences SEQ ID No. 1 to SEQ ID No. 442.
  • This includes in particular a) Nuclear acid molecules which (i) with one of the above oligonucleotide sequences (SEQ ID NO. 1 to SEQ ID No. 442) in at least 80%, 84%, 87% and preferably in at least 90%, 92% and most preferably match in at least 94, 96, 98% of the bases (where the
  • Sequence area of the nucleic acid molecule is to be considered, which corresponds to the sequence area of one of the above-mentioned oligonucleotides (SEQ ID No. 1 to 442), and not approximately the entire sequence of a possibly. compared to the oligonucleotides given above by one to numerous bases extended oligonucleotides) or (ii) differ from the above
  • oligonucleotide sequences by one or more deletions and / or additions and a specific hybridization with nucleic acid sequences of lactic acid bacteria of the genera Lactobacillus and Pediococcus which are harmful to beer, in particular of the species Pediococcus damnosus, Lactobacillus coryniformis, Lactobacillus perolens,
  • the degree of sequence identity of a nucleic acid molecule with the probes SEQ ID No. 1 to SEQ ID No. 442 can be determined using standard algorithms.
  • the program for determining sequence identity is suitable here, which is available at http://www.ncbi.nlm.nih.gov/BLAST (on this page e.g. the link "Standard nucleotide-nucleotide BLAST [blastn]").
  • hybridizing can be synonymous with complementary.
  • the scope of this invention also includes those oligonucleotides that hybridize with the (theoretical) counter-strand of an inventive ohgonucleotide, including the modifications according to the invention of SEQ ID No. 1 to 442.
  • the nucleic acid probe molecules according to the invention can be used with various hybridization solutions in the context of the detection method according to the invention.
  • Various organic solvents can be used in concentrations of 0 - 80%.
  • Compliance with stringent hybridization conditions ensures that the nucleic acid probe molecule actually hybridizes with the target sequence.
  • Moderate conditions in the sense of the invention are, for example, 0% formamide in a hybridization buffer as described below.
  • Stringent conditions For the purposes of the invention, for example, 20-80% formamide are in the hybridization buffer.
  • “specifically hybridize” means that a molecule binds preferentially to a certain nucleotide sequence under stringent conditions if this sequence is present in a complex mixture of (for example total) DNA or RNA.
  • stringent conditions stands for conditions under which a probe will preferentially hybridize to its target sequence and to a significantly lesser extent or not at all to other sequences. Stringent conditions are in part sequence-dependent and will be different under different circumstances. Longer Sequences hybridize specifically at higher temperatures.
  • stringent conditions are selected so that the temperature is about 5 ° C below the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probe molecules complementary to the target sequence hybridize to the target sequence in an equilibrium state (since the target sequences are usually in excess, 50% of the probes are occupied in equilibrium) .
  • Typical stringent e Conditions where the salt concentration is at least about 0.01 to 1.0 M sodium ion concentration (or other salt) at a pH between 7.0 and 8.3 and the temperature is at least about 30 ° C for short probes (e.g. 10-50 nucleotides).
  • a typical hybridization solution contains 0-80% formamide, preferably 20-60% formamide and particularly preferably 35% formamide and has a salt concentration of 0.1 mol / 1 - 1.5 mol / 1, preferably 0, 5 mol / 1 to 1.0 mol / 1, more preferably from 0.7 mol / 1 to 0.9 mol / 1, particularly preferably from 0.9 mol / 1, the salt preferably being sodium chloride.
  • the hybridization solution usually comprises a detergent, such as sodium dodecyl sulfate (SDS), in a concentration of 0.001% to 0.2%, preferably in a concentration of 0.005-0.05%, more preferably 0.01-0.03% and am most preferably 0.01%.
  • a detergent such as sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various compounds such as Tris-HCl, sodium citrate, PIPES or HEPES can be used to buffer the hybridization solution, which are usually used in concentrations of 0.01-0.1 mol 1, preferably 0.01 to 0.08 mol 1, in a pH range of 6.0-9.0, preferably 7.0-8.0.
  • the preferred embodiment of the hybridization solution according to the invention contains 0.02 mol / 1 Tris-HCl, pH 8.0.
  • nucleic acid molecule enables specific detection of nucleic acid sequences from target organisms and can therefore be used reliably in the context of the invention.
  • the concentration of the probe can vary greatly depending on the marking and the number of target structures to be expected. To enable fast and efficient hybridization, the amount of probes should exceed the number of target structures by several orders of magnitude. However, with fluorescence in situ hybridization (FISH) care must be taken to ensure that an excessive amount of fluorescence-labeled hybridization probe leads to increased background fluorescence leads.
  • the amount of probe should therefore be in a range between 0.5 ng / ⁇ l and 500 ng / ⁇ l, preferably between 1.0 ng / ⁇ l and 100 ng / ⁇ l and particularly preferably 1.0–50 ng / ⁇ l.
  • the preferred concentration in the context of the method according to the invention is 1-10 ng of each nucleic acid molecule used per ⁇ l hybridization solution.
  • the volume of the hybridization solution used should be between 8 ⁇ l and 100 ml, in a particularly preferred embodiment of the method according to the invention it is 40 ⁇ l.
  • the duration of the hybridization is usually between 10 minutes and 12 hours; hybridization is preferably carried out for about 1.5 hours.
  • the hybridization temperature is preferably between 44 ° C. and 48 ° C., particularly preferably 46 ° C., the parameter of the hybridization temperature and the concentration of salts and detergents in the hybridization solution in
  • nucleic acid probes in particular their lengths and the degree of complementarity to the target sequence in the cell to be detected can be optimized.
  • the person skilled in the art is familiar with the relevant calculations here.
  • this washing solution can contain 0.001-0.1% of a detergent such as SDS, a concentration of 0.01% being preferred, and Tris-HCl in a concentration of 0.001-0.1 mol / 1, preferably 0.01 0.05 mol / 1, particularly preferably 0.02 mol / 1, the pH of Tris-HCl being in the range from 6.0 to 9.0, preferably from 7.0 to 8.0, particularly is preferably 8.0.
  • a detergent may be included, but is not essential.
  • the washing solution also usually contains NaCl, the concentration depending on the stringency required being from 0.003 mol / 1 to 0.9 mol 1, preferably from 0.01 mol / 1 to 0.9 mol / 1. A NaCl is particularly preferred. Concentration of 0.07 mol / 1. Furthermore, the washing solution can contain EDTA in a concentration of up to 0.01 mol 1, the concentration preferably being 0.005 mol / 1. Furthermore, the washing solution can also contain preservatives known to the person skilled in the art in suitable amounts.
  • buffer solutions are used in the washing step, which in principle can look very similar to hybridization buffers (buffered sodium chloride solution), except that the washing step is carried out in a buffer with a lower salt concentration or at a higher temperature.
  • the formamide content (which should be as low as possible due to the toxicity of the formamide) of the wash buffer can be replaced by a correspondingly lower sodium chloride content.
  • the “washing off” of the unbound nucleic acid probe molecules usually takes place at a temperature in the range from 44 ° C. to 52 ° C., preferably from 44 ° C. to 50 ° C. and particularly preferably at 46 ° C. for a period of 10-40 minutes, preferably for 15 minutes.
  • the nucleic acid probe molecules according to the invention are used in the so-called Fast-FISH method for the specific detection of the specified target organisms.
  • the Fast FISH method is known to the person skilled in the art and e.g. in German patent application DE 199 36 875.9 and international application WO
  • the specifically hybridized nucleic acid probe molecules can then be detected in the respective cells.
  • the prerequisite for this is that the nucleic acid probe molecule is detectable, e.g. in that the nucleic acid probe molecule is linked to a marker by covalent binding.
  • detectable markers e.g. fluorescent groups such as CY2 (available from Amersham Life Sciences, Inc., Arlington Heights, USA), CY3
  • nucleic acid probe molecules in such a way that at their 5 'or 3' end there is another suitable for hybridization Nucleic acid sequence is present.
  • This nucleic acid sequence in turn comprises approximately 15 to 1,000, preferably 15-50 nucleotides.
  • This second nucleic acid region can in turn be recognized by a nucleic acid probe molecule which can be detected by one of the means mentioned above.
  • Another possibility is to couple the detectable nucleic acid probe molecules with a hapten, which can then be brought into contact with an antibody that recognizes the hapten.
  • Digoxigenin can be cited as an example of such a hapten.
  • those skilled in the art are also well known.
  • the final evaluation is possible depending on the type of marking of the probe used with a light microscope, epifluorescence microscope, chemiluminometer, fluorometer etc.
  • Another advantage is the simultaneous detection of all relevant beer-damaging lactic acid bacteria as well as the simultaneous possible detection of gram-negative beer pests.
  • nucleic acid probe molecules used can be used to specifically detect the genus Lactobacillus as well as highly specifically the species Lactobacillus coryniformis, Lactobacillus perolens, Lactobacillus buchneri, Lactobacillus plantarum, Lactobacillus fructivorans, Lactobacillus Lobiobisociacti, Lactilliobioci, Lactilli, Lactilli, Speci, Lactilli, Lactilli, Speci, Lacti, Lactilli, Lactilli, Speci Likewise, both the genus Pectinatus and the highly specific species Pectinatus fr isingensis and Pectinatus cerevisuphilus as well as additionally the species Megasphaera cerevisiae can
  • Time saving the hybridization generally takes 4 hours in the prior art, and only 1.5 hours in the method according to the invention.
  • Another advantage of the method according to the invention is that it is easy to handle. Thus, the method can easily test large quantities of samples for the presence of the bacteria mentioned.
  • the method according to the invention can be used in a variety of ways. Both clear and cloudy yeast beers can be analyzed, as well as, for example, yeast samples (pure breeding, harvesting or operating yeasts and yeast bottoms) and rinsing water.
  • Another area of application for the method according to the invention is the microbiological control of all foods, in which the detected bacteria play a role as food spoilers.
  • kits for carrying out the method according to the invention are also provided.
  • the included in these kits are also provided.
  • Hybridization arrangement is e.g. described in German patent application 100 61 655.0. The disclosure contained in this document regarding the in situ hybridization arrangement is hereby expressly incorporated by reference.
  • kits comprise, as the most important component, the respective hybridization solution with the nucleic acid probe molecules described above for the microorganisms to be detected (referred to as the VIT solution).
  • the corresponding hybridization buffer (Solution C) and a concentrate of the corresponding washing solution (Solution D) are also included.
  • Fixation solutions Solution A and Solution B
  • an additional cell disruption solution Breaker_2
  • an embedding solution finisher
  • Finishers are commercially available, among other things they prevent the rapid fading of fluorescent probes under the fluorescence microscope. If necessary, solutions for the parallel execution of a positive control and a negative control are included.
  • a sample is appropriately cultivated for 20-44 h (e.g. NBB medium, 48 h, 28 ° C).
  • a suitable aliquot of the fixed cells (preferably 40 ⁇ l) is then applied to a slide and dried (46 ° C., 30 min or until completely dry).
  • the dried cells are then completely dehydrated by adding a further fixation solution (Solution B, absolute ethanol, preferably 40 ⁇ l).
  • Solution B absolute ethanol, preferably 40 ⁇ l
  • the slide is dried again (room temperature, 3 min or until completely dry).
  • a suitable volume of the cell disruption solution (Breaker_2, preferably 40 ⁇ l) is applied to the slide and the slide is incubated (10 min, RT).
  • the cell disruption solution is washed off by immersing the slide in a vessel filled with distilled water, preferably the VIT reactor, and the slide is then dried in a lateral position.
  • the hybridization solution (VIT solution) with the nucleic acid probe molecules which are described above and which are specific for the microorganisms to be detected in each case is then applied to the fixed, dehydrated cells.
  • the preferred volume is 40 ul.
  • the slide is then incubated in a chamber moistened with hybridization buffer (Solution C, corresponds to the hybridization solution without oligonucleotides), preferably the VIT reactor (46 ° C., 90 min).
  • the slide is then removed from the chamber, the chamber is filled with washing solution (Solution D, 1:10 diluted in distilled water) and the slide is incubated in it (46 ° C, 15 min).
  • washing solution Solution D, 1:10 diluted in distilled water
  • the chamber is then filled with distilled water, the slide is briefly immersed and then air-dried in the lateral position (46 ° C, 30 min or until completely dry).
  • the slide is then embedded in a suitable medium (finisher).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour la mise en évidence rapide spécifique de bactéries à effet néfaste sur la bière par hybridation in situ. L'invention concerne également des sondes oligonucléotidiques convenant au procédé, ainsi que des nécessaires permettant de mettre en oeuvre le procédé de mise en évidence.
EP02762296A 2001-06-19 2002-06-19 Procede pour la mise en evidence rapide specifique de bacteries a effet nefaste sur la biere Withdrawn EP1397519A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10129410A DE10129410A1 (de) 2001-06-19 2001-06-19 Verfahren zum spezifischen Schnellnachweis bierschädlicher Bakterien
DE10129410 2001-06-19
PCT/EP2002/006808 WO2002103043A2 (fr) 2001-06-19 2002-06-19 Procede pour la mise en evidence rapide specifique de bacteries a effet nefaste sur la biere

Publications (1)

Publication Number Publication Date
EP1397519A2 true EP1397519A2 (fr) 2004-03-17

Family

ID=7688610

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02762296A Withdrawn EP1397519A2 (fr) 2001-06-19 2002-06-19 Procede pour la mise en evidence rapide specifique de bacteries a effet nefaste sur la biere

Country Status (6)

Country Link
US (1) US20040219574A1 (fr)
EP (1) EP1397519A2 (fr)
JP (1) JP2004529662A (fr)
CA (1) CA2451067A1 (fr)
DE (1) DE10129410A1 (fr)
WO (1) WO2002103043A2 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI116030B (fi) * 2002-11-06 2005-09-15 Kemira Oyj Paperi- ja kartonkikoneiden termofiilisten mikrobien biofilminmuodostuksen esto
DE10344057B3 (de) * 2003-09-23 2005-06-09 Vermicon Ag Verfahren zum spezifischen Schnellnachweis getränkeschädlicher Mikroorganismen
JP2005229838A (ja) * 2004-02-17 2005-09-02 Sapporo Breweries Ltd 偏性嫌気性グラム陰性菌の検出・識別方法
CN1926245A (zh) * 2004-02-25 2007-03-07 三得利株式会社 细菌检测器具、细菌检测方法及细菌检测试剂盒
AU2006252346B2 (en) * 2005-06-02 2012-06-28 Advandx, Inc. Peptide nucleic acid probes for analysis of microorganisms
US9303281B2 (en) * 2012-07-23 2016-04-05 Pall Corporation Compositions for detecting foodstuff spoilage microorganisms
EP3447146A1 (fr) 2017-08-22 2019-02-27 Vermicon AG Procédé de détection spécifique de micro-organismes
US10918690B2 (en) * 2018-09-06 2021-02-16 Louise Wilkie Apparatus and method for processing organic bamboo leaf extract products
WO2020210140A1 (fr) * 2019-04-06 2020-10-15 Banu Ip Llc Appareil et procédé de traitement de produits extraits de feuilles provenant de bambous d'une forêt certifiée biologique

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0497464A1 (fr) * 1991-01-31 1992-08-05 Amoco Corporation Diagnose rapide de microbes par hybridisation in situ en suspension aqueuse
US5426025A (en) * 1992-05-28 1995-06-20 Florida State University Species-specific DNA probes for vibrio vulnificus methods and kits
US5484909A (en) * 1993-09-10 1996-01-16 Amoco Corporation Nucleic acid probes for the detection of bacteria of the genera Pediococcus and Lactobacillus and methods for the detection of the bacterial agents causing spoilage of beer
WO2000065093A2 (fr) * 1999-04-22 2000-11-02 Science And Technology Corporation Procedes d'hybridation in situ permettant de reduire l'occurrence de faux resultats positifs et de cibler des micro-organismes multiples
DE19945964A1 (de) * 1999-09-24 2001-04-05 Biotecon Diagnostics Gmbh Verfahren und Nukleinsäuren zum Nachweis von brauereirelevanten Mikroorganismen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO02103043A3 *

Also Published As

Publication number Publication date
WO2002103043A2 (fr) 2002-12-27
CA2451067A1 (fr) 2002-12-27
WO2002103043A3 (fr) 2003-09-25
DE10129410A1 (de) 2003-01-02
US20040219574A1 (en) 2004-11-04
JP2004529662A (ja) 2004-09-30

Similar Documents

Publication Publication Date Title
DE60131284T2 (de) Methode zum nachweis von mikroorganismen
DE69534516T2 (de) Gleichzeitiger nachweis, identifizierung und differenzierung von eubakterien unter verwendung eines hybridisierungs-assays
DE69434255T2 (de) Nukleinsäuresonde zum nachweis von lactobacillus
Juvonen et al. Group-specific PCR-RFLP and real-time PCR methods for detection and tentative discrimination of strictly anaerobic beer-spoilage bacteria of the class Clostridia
EP0739988B1 (fr) Procédé pour l'identification du genre et spécifique de espèce de Legionella
WO2002103043A2 (fr) Procede pour la mise en evidence rapide specifique de bacteries a effet nefaste sur la biere
DE10344057B3 (de) Verfahren zum spezifischen Schnellnachweis getränkeschädlicher Mikroorganismen
EP1214450B1 (fr) Procede et acides nucleiques servant a determiner la presence de micro-organismes presentant un interet dans le domaine de la brasserie
Nebra et al. The effect of reducing agents on the recovery of injured Bifidobacterium cells
DE102015012691A1 (de) Verfahren zum quantitativen Nachweis von Vibrio parahaemolyticus, Vibrio vulnificus und Vibrio cholerae
EP1397518A2 (fr) Procedes pour la mise en evidence rapide specifique de bacteries concernant l'eau potable
EP1366189A2 (fr) Identification de bacteries pathogenes
WO2004009839A2 (fr) Oligonucleotides pour la detection de micro-organismes
DE69631413T2 (de) Nachweis einer bakterie der pectinatus gattung
DE60118392T2 (de) Verfahren zum nachweis von mikroorganismen
DE10204858B4 (de) Gensonden zum Nachweis von Spezies der Gattung Oenococcus
EP1335991B1 (fr) Procede pour detecter des protozoaires du genre naegleria
EP1490507A1 (fr) Procede de detection de micro-organismes par une hybridation in situ et une cytometrie de flux
WO2003066893A1 (fr) Procede de detection specifique rapide de bacteries pathogenes presentes dans des denrees alimentaires
DE10004147A1 (de) Oligonukleotide zur spezifischen Amplifikation und zum spezifischen Nachweis von 16S-rRNA-Genen von Bakterien
DE10001140C2 (de) Oligonukleotide zur Amplifikation und zum Nachweis von bakteriellen Genen für extrazelluläre alkalische Metallopeptidasen (Apr), neutrale Metallopeptidasen (Npr) und Serinpeptidasen (Spr)
WO2002101089A2 (fr) Procede pour la detection rapide specifique de bacteries filiformes
AT503401A1 (de) Microarray zur erkennung von pathogenen
DE10160666A1 (de) Verfahren zum spezifischen Schnellnachweis von Trinkwasser relevanten Bakterien
DE102006022569A1 (de) Spezies-unabhängiges Nachweisverfahren für biologisches Material

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20031126

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

RIN1 Information on inventor provided before grant (corrected)

Inventor name: SNAIDR, JIRI

Inventor name: BEIMFOHR, CLAUDIA

17Q First examination report despatched

Effective date: 20060901

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20120103