EP2129798A2 - Molécule d'acide nucléique hybridable spécifique d'un organisme - Google Patents

Molécule d'acide nucléique hybridable spécifique d'un organisme

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Publication number
EP2129798A2
EP2129798A2 EP08715931A EP08715931A EP2129798A2 EP 2129798 A2 EP2129798 A2 EP 2129798A2 EP 08715931 A EP08715931 A EP 08715931A EP 08715931 A EP08715931 A EP 08715931A EP 2129798 A2 EP2129798 A2 EP 2129798A2
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EP
European Patent Office
Prior art keywords
organism
nucleic acid
acid molecule
nucleotide sequence
dna
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.)
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Application number
EP08715931A
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German (de)
English (en)
Inventor
Marco Thines
Frank Braendle
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IdentXX GmbH
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IdentXX GmbH
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Filing date
Publication date
Application filed by IdentXX GmbH filed Critical IdentXX GmbH
Priority to EP16181282.1A priority Critical patent/EP3109328A1/fr
Priority to EP12153295.6A priority patent/EP2471955B1/fr
Publication of EP2129798A2 publication Critical patent/EP2129798A2/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method for producing an organism-specific hybridizable nucleic acid molecule, a nucleic acid molecule produced by this method, a kit comprising at least one of the nucleic acid molecules, the use of a nucleotide sequence lying between two elements on the genomic information of an organism Preparation of an Organism-Specific Hybridizable Nucleic Acid Molecule, and a Method of Detecting an Organism in a Biological Sample.
  • Hybridization By hybridization, the assembly of two nucleic acid Einzelstrlindc that have complementary base sequences, to double strands understood. Hybridization can occur between two DNA strands, one DNA and one RNA strand, and between two RNA strands.
  • Hybridisable nucleic acid molecules are, for example, primers for the polymerase chain reaction (PCR primer) or for other amplification methods based on the activity of a nucleic acid polymerase, as well as hybridization probes.
  • the primers for example for the PCR, or hybridization probes can be DNA, RNA or synthetic oligonucleotide molecules which can be used for the detection, characterization or localization of those target nucleic acid molecules which have a nucleotide sequence which corresponds to that of PCR primer or hybridization probes is complementary.
  • Methods of making PCR primers or hybridization probes are well known in the art; see. Sambrook and Russel (2001), Molecular Cloning - A Laboratory Handbook, 3rd Ed., CoId Spring Harbor Laboratory Press, New York.
  • Detection of the target nucleic acid molecules allows for indirect detection of the organism carrying the target nucleic acid molecule. In this way, it is possible, for example, to detect pathogenic organisms which contain the target nucleic acid molecule as part of their genomic information. In turn, the identification of pathogenic organisms is the prerequisite for a targeted therapy of a disease caused by the organism.
  • Modern phytopathology deals with the disease of plants by abiotic causes or biotic pathogens. These pathogens include plant viruses, bacteria, fungi or parasitic flowering plants.
  • pathogens include plant viruses, bacteria, fungi or parasitic flowering plants.
  • the subject of phytopathological research is, for example, the development of detection methods with which pathogens can be detected in seed and plant matter, in soil samples, liquid substrates and any other media.
  • Conventional test methods of phytopathology are essentially limited to the planting of potentially diseased plants and the visual characterization of the phenotype with regard to characteristic disease characteristics (scoring). However, these visual scoring-based detection methods for plant diseases are very time-consuming and labor-intensive.
  • serological methods for the detection of phytopathogens are used in which antibodies directed against the pathogens are used.
  • ELISA enzyme linked immunosorbent assay
  • Serological methods are very costly, since the production of specific monoclonal antibodies is associated with a high technical workload, and also because of the large homologies of the antigenic structures of eukaryotic pathogens are often very difficult to develop highly specific antibodies to eukaryotes that have no cross-reaction with closely related species or with formae specialis, ie highly specialized subspecies of a species.
  • moderately variable genome segments of the pathogen such as the so-called “internal transcript spacer” (ITS)
  • ITS internal transcript spacer
  • these moderately variable genome segments of the Pathogens have very large sequence homologies across species boundaries, and as a result, very closely related species of pathogens are virtually indistinguishable with this method, and by means of these conventional PCR-based methods, a differentiation of such pathogens within the same species, the different hosts infested or colonized or by formae specialis, not possible.
  • WO 95/33075 and DE 698 27 934 describe methods for identifying the origin of DNA samples from DNA-containing organisms.
  • existing genetic fingerprints of the DNA samples from DNA amplificates to be analyzed are created and compared with genetic fingerprints of DNA samples from reference organisms.
  • pattern comparison is disadvantageous: the patterns to be compared are usually not exactly identical, so that a comparison is extremely difficult. The effort is large and not suitable for rapid identification, as required by agricultural practice.
  • highly selective, organism-specific nucleic acid molecules are to be produced which have a reduced cross-reactivity with related pathogenic species or formae specialis.
  • a method of producing an organism-specific hybridizable nucleic acid molecule comprising the steps of: (1) providing genomic information derived from an organism; (2) generating a genetic fingerprint of the genomic information to obtain a pattern of amplicon genomic sections by the steps of: (2.1) amplifying portions of genomic information of the organism to obtain a mixture of amplicons, and (2.2) separating the mixture contained amplificates to obtain a pattern of amplicons of genome segments; (3) isolating at least one amplicon from the sample; (4) sequencing the isolated amplificate to obtain a nucleotide sequence; (5) selecting a portion of the nucleotide sequence of 3 to 1000 nucleotides suitable for producing an organism-specific hybridizable nucleic acid molecule, and (6) preparing a nucleic acid molecule having the portion selected in step (5).
  • an "organism” is understood to mean any living organism, in particular the living organism that can colonize or infect a plant, animal or human host, possibly causing diseases in the host, in particular plant diseases thought to be caused by plant viruses, bacteria, fungi, oomycetes, parasitic plants and animals, etc.
  • genomic information is understood as meaning nucleic acid which is specific for the organism, in particular the genome in the form of DNA, but also the RNA of the organism which arises from a copy of a genome part Information not only information-bearing but also inter- and / or exogenous sections of the genome.
  • step 2 of the method according to the invention 2.1 amplification of sections of the genomic information of the organism to obtain a mixture of amplificates, and 2.2 separation of the amplificates contained in the mixture to obtain a pattern of amplificates of genome sections.
  • a “genetic fingerprint” is understood to mean the characteristic genetic characteristics of an organism that allow it to be identified.This identification option is based on the occurrence of specific sequences of nucleotides in the genomic information of the organism, including, for example, repetitive, tandemly arranged DNA sequences Satellite DNA consists of a large number of copies of nucleotide sequences that occur in direct succession, examples of short such sequences are A, AC, AAT or AAC, although other sequences are also observed Satellite DNA with AC dinucleotide sequences is present on average once per 30 kb genome section Tri- or tetranucleotide sequences are less common, even less than a few hundred thousand base pairs In the genomic information of the organism, longer repeating sections are also reported Found 5 to 2000 nucleotides in length.
  • DNA fingerprinting Methods of generating genetic fingerprints used in step 2 of the present invention are well described in the art and are referred to as "DNA fingerprinting,” “DNA profiling,” or “DNA typing,” which are described, for example in Weising et al. (1995), Fingerprinting in Plants and Fungi, CRS Press, Boca Raton, Fla., USA; the content of this publication is incorporated herein by reference.
  • PCR primers can be made which hybridize to the satellite DNA. It will be appreciated that the forward and reverse primers each correspond to the same satellite DNA or to different satellite DNA, i. different nucleotide sequences, can hybridize. It is only important that the sections lying between two such repetitive sections of the genomic information are amplified, which are referred to below as "amplicons.” This is done in the given example by means of the PCR reaction. amplification by means of phi DNA polymerases or the thermophilic helicase dependent amplification (tHDA).
  • tHDA thermophilic helicase dependent amplification
  • the respective amplificates can then be separated according to the invention by means of chromatographic or electrophoretic methods, for example, their size, and made visible via specific dyes which bind or associate with DNA.
  • a pattern of amplificates is obtained, which is characteristic of the particular organism analyzed on account of the polymorphism mentioned. This pattern is called a genetic fingerprint.
  • inventive according to the pattern consist of only one amplificate. It is understood that by "an" amplificate according to the invention it is meant that there are several nucleic acid molecules with identical nucleotide sequence, which consequently do not differ from each other.
  • an "amplificate" is isolated from the obtained genetic fingerprint or the pattern of amplicons, for example by using a band containing the nucleic acid of an amplificate from an electrophoresis gel a knife is cut out, and the nucleic acid contained therein is eluted from the gel by well-known methods.
  • Sequencing of the isolated amplicon in step 4 of the present invention is also carried out according to methods well known in the art, for example by the Maxam-Gilbert or Sanger method; see. Sambrook and Russell (supra).
  • step 5 such a portion is selected which is suitable for the preparation of a hybridizable nucleic acid molecule.
  • This section has 3 to 1000 nucleotides.
  • the selection criteria used here are generally known to the person skilled in the art. For example, if the purpose of preparing a PCR primer is to be selected, it is preferred to select portions that are 3 to 10 to 36 nucleotides in length, preferably 18 to 25 nucleotides in length, but may be shorter or longer.
  • the criteria laid down by the person skilled in the art for the preparation of a PCR primer can be found, for example, in Sambrook and Russel (loc. Cit.), Chapter 8, Table 8.3, the content of which is incorporated herein by reference.
  • the selection criteria can be represented as follows: The G + C content should be between 40% and 60%, preferably between 45% and 55%, with an even distribution of all four nucleotides along the length of the primer.
  • the primer has the nucleotide G or C at the 3 'end. It is optimal if the last two nucleotides at the 3 'end the variations GG; GC; CG or CC have.
  • the PCR primer should not be able to hybridize to itself. It is optimal if ⁇ 3 identical nucleotides follow each other.
  • both primers ⁇ 3 have complementary nucleotides.
  • the distance between the two primers should be selected so that the amplificate can be detected well in the detection system used in each case.
  • Optimum for polyacrylamide gels 100-1500 nucleotides
  • Optimum for agarose gels 250-750 nucleotides
  • Optimal for real-time PCR systems 100 - 250 nucleotides.
  • the primer should be designed so that a hybridization temperature of ⁇ 50 0 C is possible.
  • the hybridization temperatures that apply to a primer can be calculated using software provided for this purpose.
  • the object underlying the invention is hereby completely solved.
  • the inventors have recognized that amplicons obtained in the generation of a genetic fingerprint of the organism are suitable templates for the preparation of highly selective organism-specific and hybridizable nucleic acid molecules, such as probes and PCR primers.
  • the inventors have found that in the context of DNA fingerprinting fragments of the genomic information of the organism of interest can be amplified, which are usually between the genes of the organism, ie intergenic, arranged, ie do not carry protein-coding information. Due to the low or even missing selection pressure, these fragments have a very high variability between individual organisms and thus a high specificity for the respective organism.
  • nucleic acid molecules which can bind highly selectively to such fragments.
  • nucleic acid molecules are probes and PCR primers or DNA oligomers.
  • a molecular biologist or plant pathologist can determine whether such a nucleic acid molecule produced according to the invention can hybridize to genomic information in any biological sample. In the case of hybridization, it can be concluded that the organism in question is contained in the sample.
  • the genomic information of the organism is double-stranded DNA.
  • This measure has the advantage that the genomic, double-stranded DNA of the organism is particularly suitable for generating a genetic fingerprint.
  • the art has described a variety of methods that allow the generation of the genetic fingerprint from the base of double-stranded DNA; see. Weising et al. (Supra). These include the PCR-based method of DNA fingerprinting described in more detail above.
  • the amplification in step (2.1) is preferably carried out by means of a polymerase chain reaction (PCR), with sections of satellite DNA, in particular microsatellite DNA and simple sequence repeats (SSRs), being particularly suitable as primer attachment sites.
  • PCR polymerase chain reaction
  • Satellite DNA is found mainly in the genome of eukaryotes occurring many times, i. understood repetitive, DNA sequences that can account for up to 90% of the total genome depending on the organism. Satellite DNAs are referred to differently by their repetitive nucleotide sequence length and the number of these tandemly arranged copies. Smaller satellite DNA is often referred to as “microsatellite DNA,” meaning di-, tri-, or tetranucleotides that occur at 10 to 30 copies within a "clustered” repeating unit. Microsatellite DNA also includes repetitive sequences of 20 to 40 nucleotides and extends over a range of a few kilobases. They are also called “variable number of tandem repeats" (VNTRs). “Single sequence repeats” (SSRs), also referred to as “simple sequence repeats”, are short, repetitive sequences of 1 to 4 nucleotides in length.
  • SSRs single sequence repeats
  • nucleotide sequences of a variety of satellite DNAs are described in the prior art. It is thus easy for the skilled person to generate suitable PCR primers which hybridize to the satellite DNA.
  • nucleotide sequences of satellite DNAs can be found on the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) website (loc.
  • Nx any nucleotide
  • x is an integer of 2-6 for SSRs
  • x is an integer of 7-30 for microsatellite DNA
  • x is an integer of 31-10000 for macrosatellite DNA
  • n is generally an integer from 2 to 1000, with greater values being possible for both n and x.
  • the forward primer in the context of PCR, to a randomized nucleotide sequence having a length of 3 to 30, preferably from 4 to 20, most preferably from 6 to 10 nucleotides, on the the first DNA strand of the organism hybridized, and as the reverse primer, one to be selected, which to a nucleotide sequence having a length of 3 to 30, preferably from 4 to 20, most preferably from 6 to 10 nucleotides, on the first DNA Strand hybridized to complementary DNA strand of the organism.
  • This measure can also be used to create a genetic fingerprint in the form of a pattern of amplification codes of fragments of the genomic information of the organism of interest.
  • a forward PCR primer is made having from 3 to 10 arbitrarily selected nucleotides.
  • the reverse PCR primer is chosen accordingly.
  • the inventors have found that for statistical reasons in a large number of organisms, corresponding nucleotide sequences can be found distributed over the genome.
  • the intervening fragments of the genomic information of the organism are then amplified, which according to findings of the inventors for statistical reasons also have a very high organism specificity, since a large part of the genome is information-free, and therefore for the production of organism-specific hybridizable nucleic acid molecules suitable.
  • the forward primer in the context of the PCR, one which hybridizes to a traceable to a viral genome nucleotide sequence on the first DNA strand of the organism, and as the reverse primer is selected one which attaches to a a virus genome back feasible nucleotide sequence on the DNA strand complementary to the DNA strand of the organism hybridized.
  • Nucleotide sequences attributable to a virus genome can be identified by sequence homologies.
  • the invention refers to a nucleotide sequence attributable to a viral genome when portions of the genomic information of at least 10 nucleotides have homology with a corresponding portion of a virus genome of at least 30%, preferably at least 40%, 50%, 60%, 70%, 80% %, 90%, 95%, and most preferably at least 100%.
  • the homologies can be determined, for example, by means of the software MegAlign of the laser gene program of DNAStar Inc.
  • Nucleotide sequences attributable to a viral genome are described extensively in the art, cf. Cooper and Geoffrey (2000), The Cell - A Molecular Approach, 2nd Edition, Sunderland (MA): Sinauer Associates, Inc., Brown, TA (2002), Genomes 2nd Edition, New York and London: Garland Science; Pearson and Morrow (1981), "Discrete-length repeated sequences in eukaryotic genomes," Proceedings of the National Academy of Sciences of the United States of America, Vol. 78, pp. 4016-4020; Bernardi, G. (2005 ), “Structural and evolutionary genomics - natural selection in genome evolution", Elsevier Science Publications, and Baltimore, D.
  • the forward primer one is selected which hybridizes to a nucleotide sequence of a transposable element on the first DNA strand of the organism
  • the reverse primer one is selected which can be attached to a nucleotide sequence Nucleotide sequence of a transposable element on the DNA strand complementary to the DNA strand of the organism hybridized.
  • Transposable or transposable elements are mobile genetic elements, i. DNA regions that can change their position within the genome.
  • the inventors have recognized that such transposable elements are similar to, for example, satellite DNA distributed over the genome and intervening genome segments are usually intergenic and highly variable between organisms or individuals.
  • transposable elements that can simultaneously represent viral genome-traceable sequences are described in the prior art; see. see. Cooper and Geoffrey (supra); Brown, T.A. (supra); Pearson and Morrow (supra); Bernardi, G. (supra) and Baltimore, D. (supra).
  • the forward primer is preferably one which hybridizes to a repetitive nucleotide sequence of a telomer or centromere on the first DNA strand of the organism
  • the reverse primer is one selected from a repetitive nucleotide sequence of a telomere or centromere on the DNA strand complementary to the DNA strand of the organism hybridized.
  • this measure also succeeds in producing a genetic fingerprint of the genetic information of the organism in the form of a pattern of amplicons of genome fragments. So it is known that In the centromeric and telomeric regions of chromosomes, there are repeats of hundreds or thousands of sequenced DNA segments that correspond in structure to that of satellite DNA. These repetitive nucleotide sequences also exhibit a very high variability between individuals and are therefore equally suitable for the development of highly specific PCR probes and primers for the detection of these individuals or organisms. Repetitive nucleotide sequences in the centromeric and telomeric regions of chromosomes are well described in the art; see. Brown, TA (supra); Griffiths, et al.
  • fragments of genomic information which can be obtained by means of DNA fingerprinting of organisms have a very high variability not only between organisms of different species but also between forma specialis and even individuals and, therefore, for the production of organism-specific hybridisable Nucleinklakülen, for example.
  • PCR primers and / or hybridization probes are particularly suitable.
  • These fragments are usually located between repetitive DNA sequences randomly distributed throughout the genome of the microorganism, such as between satellite DNA or even between such nucleotide sequences attributable to a virus genome, between transposable elements or between repetitive nucleotide sequences of a telomere or centromere.
  • nucleotide sequences the between randomized short nucleotide sequences of 3 to 30 nucleotides can be used for this purpose.
  • the present invention also relates to the use of a nucleic acid molecule having a nucleotide sequence lying between two elements on the genetic information of an organism, for producing an organism-specific hybridizable nucleic acid molecule, preferably a PCR primer, wherein the two elements are each independently selected from the group consisting of: satellite DNA nucleotide sequence, viral genomic traceable nucleotide sequence, nucleotide sequence of a transposable element, repetitive nucleotide sequence of a telomere / centromere.
  • Between two elements on the genetic information of an organism means according to the invention that the nucleotide sequence in question is flanked 5'- and 3'-ward by the two elements, ie to the 3 'end of the nucleotide sequence closes the one element, and the 3 'end joins the other element.
  • nucleotide sequences of such nucleic acid molecules are derived from the genomes of the plant pathogens Peronospora arborescens, Peronospora swinglei s.l., Fusarium oxysporum, Plasmopara petroselini and Peronospora valerianellae and are flanked both 5'- and 3'-wards by microsatellites or single-sequence repeats (SSRs).
  • SSRs single-sequence repeats
  • the subject matter of the present invention is also a nucleic acid molecule which has one of the nucleotide sequences SEQ ID Nos. 1 to 8 from the attached sequence listing or a nucleotide sequence complementary thereto.
  • Said nucleic acid molecules have proven to be particularly suitable for acting as a template for the preparation of hybridisable nucleic acid molecules with which the organisms Peronospora arborescens, Peronospora swinglei sl, Fusarium ox ⁇ sporum, Plasmopara petroselini and Peronospora valerianellae can be detected in a biological sample.
  • the abovementioned nucleic acid molecules have a nucleotide sequence of 3 to 812, preferably 10 to 64, more preferably 12 to 48, more preferably 14 to 36, more preferably 16 to 27 consecutive nucleotides from one of the nucleotide sequences SEQ ID NO 1 to 8 or a nucleotide sequence complementary thereto.
  • nucleic acid molecules are already provided which have a portion of sufficient length to act as PCR primer for the detection of the organisms Peronospora arborescens, Peronospora swinglei s.l., Fusarium oxysporum, Plasmopara petroselini and Peronospora valerianellae.
  • These PCR primers are capable of hybridizing to those nucleic acid molecules having one of the nucleotide sequences SEQ ID Nos. 1 to 8 or a sequence complementary thereto.
  • a further subject matter of the present invention relates to a nucleic acid molecule which is at least 20%, preferably at least 30%, more preferably 40%, preferably 50%, more preferably 60%, more preferably 70%, further preferably 80%, further preferably 90%. , more preferably 95%, more preferably 99% sequence homology with the above nucleic acid molecules.
  • the three mentioned plant-pathogenic microorganisms can be detected highly selectively and specifically in a biological sample on account of the high homology.
  • the homologies can be easily determined by means of methods known to the person skilled in the art, for example a BLAST Analysis or by means of the MegAlign module of the laser gene program of DNAStar Inc. The identities indicated relate to the entire, above-mentioned, inventive nucleic acid molecule or to a section thereof with> 10 nucleotides.
  • a further subject relates to such a nucleic acid molecule which hybridizes under stringent conditions to one of the abovementioned nucleic acid molecules.
  • nucleic acid molecule also has the properties of the nucleic acid molecules described above and is therefore suitable for the specific detection of plant pathogens.
  • stringent conditions are meant those under which only nearly perfect, i. to 80%, 85%, 90%, 95%, 98%, 99% base-paired nucleic acid strands are formed and remain stable. Stringent conditions can be adjusted, for example, by changing the salt concentrations, the pH, the temperature, etc. in the medium in which the binding of the nucleic acid strands is to take place.
  • Another object of the invention relates to a method for producing an organism-specific hybridizable nucleic acid molecule, comprising the following steps: (1) providing a genomic information derived from an organism; (2) incubating the genomic information of the organism with at least one restriction endonuclease to obtain a mixture of restriction fragments; (3) Ligation of oligonucleotides of a defined nucleotide sequence (adapter sequence) to the free ends of the restriction fragments to obtain modified sections of genomic information; (4) generating a genetic fingerprint of the genomic information to obtain a pattern of amplicons of genomic sections by the steps of: (4.1) amplifying the fragments of the genomic information of the organism to obtain a mixture of amplicons, (4.2) separating the mixture contained in the mixture Amplificates to obtain a pattern of amplicons from genomic sections; (5) isolating at least one amplicon from the sample; (6) sequencing the isolated amplificate to obtain a nucleotide sequence; (7) selecting a portion of the nucleotide
  • AFLP amplified fragment length polymorphism
  • the genome fragment lying between the adapters is then obtained.
  • measures can be taken to reduce the number of genomic fragments, should this, because of the chosen restriction endonuclease particularly high and thus be difficult to handle.
  • further amplifications can be followed, resulting in a reduction of the genomic fragments.
  • the AFLP method is described, for example, in Weising et al. (Supra). This publication is incorporated herein by reference.
  • Another object of the invention relates to a method for producing an organism-specific hybridizable nucleic acid molecule, comprising the following steps: (1) providing a genomic information derived from an organism; (2) generating a genetic fingerprint from the genomic information to obtain a pattern of amplicons of genomic sections by the steps of: (2.1) incubating the genomic information with at least one restriction endonuclease to obtain a mixture of fragments of the genomic information, (2.2) ligation of the fragments (2.3) introduction of the vectors into host organisms, optional (2.3 ') plating of the host organisms on culturing plates, (2.4) amplification of the vectors in the host organisms, (2.5) isolation of the amplified vectors to obtain a mixture of amplificates of Genome sections, and (2.6) separating the amplicons contained in the mixture to obtain a pattern of amplicons of genome sections; (3) isolating at least one amplicon from the sample; (4) sequencing the isolated amplificate to obtain a nucleotide sequence
  • this method represents an alternative procedure for the generation of a genetic fingerprint, which can be carried out reliably by means of laboratory-standard reagents and methods.
  • the singulation of the amplificates contained in the mixture takes place, for example, by means of a chromatographic and / or electrophoretic method.
  • gel electrophoretic methods in which the amplificates are separated from each other by the migration in an electric field, for example, according to their size.
  • chromatographic processes such as high performance liquid chromatography (HPLC), as well as all processes which are suitable for the separation of nucleic acid molecules of different composition and / or length and / or charge present in a mixture.
  • HPLC high performance liquid chromatography
  • At least one such amplificate is isolated from the pattern of amplicons of genome sections, which has been amplified to an above-average extent in relation to the other amplificates.
  • Such amplicons which are present in high amounts, are particularly suitable as a template for the preparation of a species-specific hybridizable nucleic acid molecule.
  • the sections on which such amplicons are based are present in the genomic information or the genome of the organism of interest either in high copy number or form hardly any disturbing secondary structures during the amplification.
  • Such particularly readily amplifiable sections can be identified, for example, after gel electrophoretic separation of the mixture of amplicons and subsequent staining with a suitable substance, for example ethidium bromide, using a particularly pronounced or prominent gel band.
  • the gel bands can, for example, be measured densitometrically and an average of the measured values can be formed.
  • an "above-average" strong amplification of an amplificate is present if above-average values can be assigned to a gel band in densitometry, and it is particularly preferred if an above-average amplification Amplicon is amplified twice, three times, four times, five times, or ten times as much as the average of the amplicons.
  • the isolation of such an above-average amplified amplificate takes place, for example, in that the corresponding band is excised from the gel and the amplificate contained therein is eluted by methods known in the art.
  • the organisms preferably microorganisms such as Escherichia coli, "repair" the vector and thus make the amplificate particularly well sequenced.
  • the amplicon contained in the vector is thereby available for the subsequent process steps in optimal form.
  • the portion selected to produce an organism-specific hybridizable nucleic acid molecule preferably has a length of 10 to 64, more preferably 12 to 48, more preferably 14 to 36, more preferably 16 to 27 nucleotides, more preferably the nucleic acid molecule prepared in the subsequent step is designed as a PCR primer or alternatively as a hybridization probe.
  • hybridization probes or PCR primers have the abovementioned number of nucleotides which are complementary in their sequence to the target sequence to be detected.
  • Hybridization probes and PCR primers may be 5, 10, 15, 20, 30, 50, 100, 150, and 200 to 2000 nucleotides, respectively, whose sequence is complementary to the target sequence. Ideally the number of nucleotides at 20 ⁇ 10. Criteria considered by those skilled in the design of PCR primers can be found in Sambrook and Russel (supra), Chapter 8, Table 8.3, the contents of which are incorporated herein by reference ,
  • the inventors have been able to use the method according to the invention to produce a large number of organism-specific hybridizable nucleic acid molecules which can be used as PCR primers and are described in the attached sequence listing under SEQ ID NO. 9 to 35 are listed; see. also table 2.
  • the present invention therefore furthermore relates to a nucleic acid molecule which has one of the nucleotide sequences SEQ ID Nos. 9 to 35 from the attached sequence listing or a nucleotide sequence complementary thereto.
  • a further subject of the present invention relates to a nucleic acid molecule which is at least 20%, preferably at least 30%, more preferably 40%, preferably 50%, more preferably 60%, further preferably 70%, more preferably 80%, further preferably 90% preferably 95%, more preferably 99% sequence homology with the above nucleic acid molecules.
  • nucleic acid molecules can be detected highly selective and species-specific a phytopathogenic microorganism due to the high degree of homology.
  • the homologies can easily be determined by methods known to the person skilled in the art, for example a BLAST analysis or by means of the MegAlign module of the laser gene program of DNAStar Inc.
  • the identities indicated relate to the entire aforementioned nucleic acid molecules according to the invention or to a section thereof with> 10 nucleotides.
  • Another object of the present invention relates to a Nucleinklaremole- molecule which hybridizes under stringent conditions to one of the nucleic acid molecules described above.
  • nucleic acid molecule also has the properties of the nucleic acid molecules described above and is therefore suitable for the specific detection of said microorganisms.
  • determination of stringent conditions reference is made to the definition given above.
  • Another object of the present invention relates to a kit comprising at least one of the aforementioned nucleic acid molecules, a description of the use of the nucleic acid molecule, preferably in the context of the following inventive detection method, and optionally buffer and chemicals.
  • kits can be compiled, for example, for each microorganism to be detected as a diagnostic kit.
  • a diagnostic kit then contains, for example, the highly selective for the respective pathogenic organism to be detected PCR primer and a DNA sample of the corresponding organism as a positive control.
  • a diagnostic kit contains a description with detailed information on how to perform the subsequent detection method.
  • the kit may include suitable buffers, chemicals and other adjuvants to perform the diagnostic procedure. The provision of such a kit has the advantage that all the necessary chemicals and information are provided, so that a corresponding detection method can also be carried out without errors by personnel who may be less well-trained.
  • Another object of the invention relates to a method for detecting an organism in a biological sample, for example.
  • a seed sample comprising the following steps: 1. Provision of a nucleic acid-containing biological sample, 2. Isolation of the nucleic acid from the sample, 3. subjecting the isolated 4. Polymerase Chain Reaction Nucleic Acid (PCR), 4. Determine if in step 3 5. Amplification of the isolated nucleic acid, and 5. Correlation of a positive determination in Step 4 with the presence of the organism in the sample, and correlation of a negative determination in Step 4 with the absence of the organism in the sample, in Step 3 as PCR At least one nucleic acid molecule which has been produced by the process according to the invention is used.
  • the PCR reaction is preferably designed as a "nested” or nested PCR reaction.
  • This measure has the particular advantage that the specificity and efficiency of the PCR performed is further increased.
  • a first round of PCR within the framework of so-called pre-PCR, a first template is amplified over a few cycles.
  • the primers are chosen here so that they are relatively far apart, i.
  • the forward PCR primer hybridizes relatively close to the 3 'end, while the antisense PCR primer, for example, hybridizes near the 5' end of the intergenic organism-specific DNA segment.
  • the amplificate of this pre-PCR is then amplified in another PCR round, the secondary PCR or post-PCR, using a new PCR primer pair.
  • the new PCR primers are located further inside than the first, so that in this second step only the specific DNA segments from the pre-PCR are amplified.
  • the sensitivity and efficiency of the method according to the invention is significantly increased.
  • Fig. 1 shows a classical DNA fingerprint for performing a prior art patterning to identify the pathogen Xanthomonas campestris in kohlrabi field samples
  • FIG. 2 shows a schematic representation of the occurrence of organism-specific DNA sequences with flanking repetitive DNA sequences using the example of a eukaryotic chromosome
  • FIG. 3 shows a schematic representation of important steps of the method according to the invention for the production of organism-specific nucleic acid molecules.
  • FIG. 4 shows the result of the detection method according to the invention with reference to an agarose gel stained by ethidium bromide in order to detect the pathogen Peronospora swinglei s.l. in basil seed.
  • the bacterial pathogen Xanthomonas campestris pathovar campestris is one of the most important pests in cabbage cultivation. To identify the organism is in the The method and the problems associated therewith are demonstrated below with reference to Figure 9, isolated from the field crops of kohlrabi and various wild plants isolated pure cultures.
  • the standard protocol in the prior art essentially comprises the following 4 steps, namely (a) providing the organisms in the form of a pure culture, (b) the DNA extraction, (c) the production of a genetic fingerprint and (d) the electrophoretic separation of the genetic fingerprints
  • the macroscopically identified as diseased plant parts were transferred to a sterile test tube in which a liquid medium such as the NYG medium was introduced. After an incubation period of min. 12 h at 30 0 C were prepared on agar plates Verkowsausstriche with various concentrated solutions of the liquid medium and incubated overnight at 30 0 C. On the other day, isolated bacterial colonies were lifted off the agar plate and further dilution smears made. This process was repeated until it was certain that the smear was indeed a pure culture of the organism. Reinfections were used to verify pathogenicity with these pure cultures according to Koch's postulates. For further experiments, only those pure cultures were used, which had the typical infection symptoms of the pathogen.
  • a PCR reaction is carried out in a PCR-suitable reaction vessel, for example consisting of 2.5 .mu.l of 1Ox PCR buffer, 2.5 .mu.l of a 2 mM solution of the four deoxynucleotide triphosphates (dNTPs), 2.5 .mu.l magnesium chloride solution (20 mM), 1 U of a thermostable DNA polymerase, for example Taq DNA polymerase, 1 ⁇ l of, for example, a 25 mM solution of the PCR primer GTCs (5'GTCGTCGTCGTCGTC-S '; SEQ ID No. 39).
  • the primer is designed to bind to the microsatellite GTC.
  • the mixture is made up to 25 ⁇ l with water.
  • the PCR is carried out, for example with the following conditions:
  • the incubation in a thermocycler takes place in accordance with the device-specific modalities. First, for example, an incubation, for example. At 94 ° C for 2 min. Subsequently, an incubation at eg. 94 ° C, for 1 min. Thereafter, incubation, for example. At 56 ° C, for 1 min. Subsequently, an incubation, for example. At 72 ° C, for 2 min. The last three steps are then repeated, for example, 30 times. It may then, for example, a final incubation, for example. At 72 ° C for 4 min, performed.
  • the amplificates obtained in the PCR are separated, for example by means of gel electrophoresis, for example in an agarose gel.
  • This can be prepared, for example, by placing 1 g of agarose in a vessel, filling it to 100 ⁇ l with an ion-containing liquid, for example with Tris-borate-EDTA buffer (TBE).
  • TBE Tris-borate-EDTA buffer
  • the PCR reaction solution is placed in a preformed well of the agarose gels, for example.
  • the electropho- rese itself is carried out in a dedicated electrophoresis chamber filled with an isotonic ion-containing solution to the gel, preferably with the same, which has been used to prepare the agarose gel.
  • the migration of the DNA fragments is enforced by the agarose gel, which is separated by the selective retention capacity, which is based in particular on the size of the migrating fragments.
  • a voltage for example 140 V
  • the selective retention capacity which is based in particular on the size of the migrating fragments.
  • staining with a dye which associates or binds to nucleic acids for example ethidium bromide, for example by incubating the agarose gel for half an hour in a dyebath containing ethidium bromide.
  • FIG. 1 agarose gel, 1.5%, stained with ethidium bromide; Primers used: GTCs lane 1: Xanthomonas campestris field sample 1; Lane 2: Xanthomonas campestris reference sample; Lane 3: Xanthomonas campestris field sample 2; Lane 4: Xanthomonas campestris field sample 3; Lane 5: Xanthomonas campestris field sample 4; Lane 6: Xanthomonas campestris field sample 5; Lane 7: Xanthomonas campestris field sample 6; Lane 8: Xanthomonas campestris field sample 7; Lane 9: Xanthomonas campestris field sample 8; Lane 10: Xanthomonas campestris field sample 9; Lane 11: DNA size marker (standard) 1
  • the presented example shows the typical disadvantages of identification on the basis of a "pattern comparison" of genetic fingerprints: the patterns to be compared are usually not exactly identical, individual amplificates are weaker or stronger, amplicons are missing completely or new ones appear
  • different fingerprints based on different PCR primers are made in practice with the same DNA solution.
  • the individual comparative investigations are then compared with each other in a complex procedure. The effort is large and not suitable for rapid identification, as required by agricultural practice.
  • the result is highly dependent on the separation matrix used or the conditions of the separation as a whole, which makes comparability of test results between individual laboratories very difficult.
  • FIG. 2 schematically shows part of the genome of a phytopathogen.
  • organization level I a chromosome is shown schematically; the organization stage II shows the DNA double strand on which coding gene segments and regions of intergenic DNA sequences are shown;
  • Organizational step IV represents a sequence example of repetitive (e.g., microsatellites, solid line) and organism-specific (dotted line) DNA sequences.
  • intergenic sequences including organism-specific DNA sequences lying between or flanking repetitive sequences, are eminently suited as templates for the preparation of organism-specific hybridizable nucleic acid molecules, such as PCR primers, for the following reasons: Intergenic sequences are not protein coding. (Fig. 2 (H)), this results in a low to absent selection pressure. They have a very high variability between individual organisms or species, but have a high specificity for the respective organism or the respective species. They can be quickly identified by the flanking repetitive DNA sequences, since they are largely statistically distributed in the genome (FIG. 2 (H)), but are frequently found in intergenic sections (FIG. 2 (HI)). B eisp. 1..3.:. one .
  • 5 mg of the sporangia are in a reaction vessel of. 2 ml, by means of a device suitable for this purpose, for example a shaking mill, with the addition of grinding media, for example small iron balls, comminuted.
  • the comminuted sporangia are mixed with 500 ⁇ l of a detergent solution in order to destroy the membranes of the organism and thus release the genomic DNA.
  • the solution may, for example, consist of 0.1 M Tris pH 8, 0.05 M EDTA, 2% SDS, proteinase K.
  • the comminuted sporangia are incubated in the reaction vessel with the abovementioned solution for several minutes, for example 30 minutes, at 65 ° C, incubated.
  • the reaction vessel is centrifuged, for example, for 5 min at 10,000 s / t 2 * 9.81 to sediment cell debris.
  • the liquid phase is removed and transferred to a new reaction vessel.
  • a protein-precipitating preferably a phase-separating solution, for example. Chloroform and isoamyl alcohol in a ratio of 24 to 1, for example, added in the same amount as the liquid already in the reaction vessel.
  • the phases are mixed, for example by repeatedly pivoting the reaction vessel along the longitudinal axis.
  • the reaction vessel is mixed, for example, for 10 min at 10,000 s / t 2 * 9.81 to separate the phases of protein precipitation.
  • the aqueous solution is transferred to a new reaction vessel.
  • a DNA-precipitating solution for example. Isopropanol with a volume of 50% of the volume of the transferred aqueous phase.
  • the reaction vessel is centrifuged, for example, for 20 min at 10 000 s / t 2 * 9.81 and 4 ° C to sediment the DNA.
  • the supernatant is removed.
  • the sedimented DNA is dried, for example by blowing with a moderate air flow.
  • the DNA is dissolved in buffer solution or water, for example bi-distilled water.
  • a PCR reaction is set up in a PCR-suitable reaction vessel, for example consisting of 2.5 ⁇ l of 10 ⁇ PCR buffer, 2.5 ⁇ l of a 2 mM solution of the four deoxynucleotide triphosphates (dNTPs), 2.5 ⁇ l of magnesium chloride solution (20 mM).
  • a thermostable DNA polymerase for example Taq DNA polymerase
  • 1 ⁇ l of, for example, a 25 mM solution of the PCR primer t3B (5'-AGGTCGCGGTTCGAATCC-3 ', SEQ ID No. 36).
  • the primer is designed so that it can bind to the microsatellite t3B.
  • the mixture is made up to 25 ⁇ l with water.
  • thermocycler takes place according to the device-specific modalities.
  • an incubation for example.
  • incubation is carried out at, for example, 94 0 C, for 1 min.
  • the batch is cooled to a temperature, for example.
  • Figure 3B in which the primers can hybridize to the microsatellite t3B; see. Fig. 3 C.
  • the batch is incubated at the working temperature of the polymerase, for example. At 72 ° C for 2 min; see.
  • FIG. 3D The binding of the polymerase is shown in partial FIG. 3 E. It comes to the synthesis of new for Peronospora swinglei sl specific, ie organism-specific DNA fragments flanked by the repetitive DNA sequences of the t3B microsatellite; see. Fig. 3F.
  • Steps A to F of Fig. 3 are then repeated 20 to 30 times; see. Fig. 3G.
  • the amplicons specific for Peronospora swinglei sl obtained in the PCR are separated, for example by means of gel electrophoresis in an agarose gel; see. Fig. 3H.
  • This can be prepared, for example, by placing 1 g of agarose in a vessel, making up to 100 ⁇ l with an ion-containing liquid, for example with Tris-borate-EDTA buffer (TBE). Thereafter, the PCR reaction solution is placed in a preformed well of the agarose gel, for example. After addition of 1/6 vol. Glycerol and possibly dyes, which in the electrophoresis in the same direction as the DNA fragments in the agarose gel and the control of the progress of the electrophoresis these.
  • the electrophoresis itself takes place in an electrophoresis chamber provided for this purpose, which is filled with an ion-containing solution containing isotonic acid, preferably with the same used for the preparation of the agarose gel.
  • an ion-containing solution containing isotonic acid preferably with the same used for the preparation of the agarose gel.
  • a voltage for example 140 V
  • the migration of the DNA fragments is forced through the agarose gel, which is separated by the selective retention capacity, which is based in particular on the size of the migrating fragments.
  • staining with a dye associating with or binding to nucl ⁇ ic acids, for example ethidium bromide, for example by incubating the agarose gel for half an hour in a dyebath containing ethidium bromide. 4. Isolation of an amplificate
  • a particularly well amplified band here for example in the range of 400 to 900 bp, is cut out of the agarose gel; see. Fig. 31.
  • PCR fragment cloning vector introduction and amplification of the resulting nucleic acid molecule into a PCR fragment cloning vector by means of a commercially available PCR fragment cloning kit, for example the Invitrogen TOPO TA cloning kit according to the manufacturer's instructions.
  • a commercially available PCR fragment cloning kit for example the Invitrogen TOPO TA cloning kit according to the manufacturer's instructions.
  • the vector together with included for Peronospora swinglei s.l. specific nucleic acid fragment are isolated, for example by means of a commercial Plasmidpreparationskits, such as the GeneElute HP Plasmid Midiprep Kits Sigma, according to the manufacturer's instructions.
  • Sequencing of the Peronospora swinglei sl-specific nucleic acid fragment contained in the vector is carried out by means of vector-specific sequencing primers and conventional methods, for example by means of the Big-Dye v3.1 Terminator Cycle Sequencing Kit from Applied Biosystems, according to the instructions of the manufacturer and subsequently Elucidation of the sequence of the nucleic acid molecule, for example by means of a capillary sequencer, for example the AbiPrism 3730x1 DNA Analyzer from Applied Biosystems, according to the instructions of the manufacturer; see. Fig. 3K. A section of the chromatogram of the sequencing is shown in the partial FIG. 3L. In the case of this embodiment, the specific for Peronospora swinglei sl nucleotide sequence SEQ ID NO. 2 determined.
  • Table 1 gives some further examples of nucleotide sequences from the genome of various plant pathogens flanked both 5 'and 3' by microsatellites such as the t3B, or single sequence repleats (SSRs).
  • SSRs single sequence repleats
  • Table 1 Examples of nucleic acid molecules or nucleotide sequences which are suitable for producing an organism-specific hybridizable nucleic acid molecule. 6. Selection of a section suitable for producing a specific hybridizable nucleic acid molecule
  • nucleotide sequence of the Peronospora swinglei s.l. specific nucleic acid molecule two sections were selected which are suitable for the preparation of PCR primers; see. Fig. 3M. Accordingly, the nucleotide sequence elucidated in step 5 serves as a template for the preparation of two hybridisable nucleic acid molecules which are used as PCR primers for detecting an infection of a plant with Peronospora swinglei s.l. can be used.
  • nucleotide sequences are chosen to hybridize specifically to the state of the art and also to be sorted by database alignment (GenBank) organism - i. Peronospora swinglei s.l. are specific because they are found individually in parts only in a few and combined in no sequence of a GenBank cataloged nucleic acid molecule.
  • the preparation of the two PCR primers used to detect infection of a plant with Peronospora swinglei s.l. can be used, for example, by means of a 96 Column DNA Synthesizer Workstation of PolyGen company according to the manufacturer; see. Fig. 3N. Numerous commercial suppliers, for example Sigma-Aldrich, offer the preparation of nucleotide oligomers and polymers.
  • the hybridizable nucleic acid molecule designated S_Ps_F, that can be used as a forward PCR primer has the sequence 5'-TGCTCTCGCAGGTCTGACTACC-3 '(SEQ ID NO: 11).
  • hybridizable nucleic acid molecules Two additional hybridizable nucleic acid molecules were designed as primers for nested or nested PCR, using a second amplification based on the first amplification to further increase sensitivity.
  • the hybridizable nucleic acid molecule, designated S_Ps_Fn, that can be used as a forward PCR primer has the sequence 5'-GTCTTAACGAGACCGCGTGGTC-S '(SEQ ID NO: 13).
  • the hybridizable nucleic acid molecule, designated S_Ps_Rn that can be used as a reverse PCR primer has the sequence 5'-ATGACGGCAGGTGCTGTCGTTC-S '(SEQ ID NO: 14).
  • Table 2 below lists some examples of PCR primers generated by the method of the invention.
  • Table 2 Examples of species-specific hybridizable nucleic acid molecules for the detection of selected plant pathogens.
  • Example . 1 . 2 Use of the nucleic acid molecule according to the invention (PCR primer) for the specific detection of Peronospora swinglei sl in basil seed
  • the detection method essentially comprises the following 5 steps: (a) digestion of the seed; (b) DNA extraction; (c) quality inspection of the recovered DNA solution; (d) application of PCR-based detection and (e) electrophoretic separation of the PCR reaction solutions.
  • the basil seed of seven different seed lots was mechanically digested for 5 min under liquid nitrogen prior to extraction.
  • other digestion techniques such as e.g. comminution in mills, mixers or the enzymatic degradation of the structuring cell components.
  • the extraction solution was incubated for 30 min with gentle shaking on ice. After sedimentation of the insoluble components by centrifugation for at least 9,000 s / t 2 * 9.81 for 5 minutes, the supernatant liquid was transferred completely to a new centrifugation tube. After adding 0.6 volume of isopropanol (100%), the extraction solution was incubated for 10 minutes with gentle shaking at room temperature. By centrifuging for at least 9,000 s / t 2 * 9.81 for 10 minutes, the now insoluble DNA was sedimented. After complete supernatant lift, the sediment was washed twice with ethanol (70%) and then dried.
  • the dried sediment was resuspended in 5 ml of TE buffer and then admixed with 500 ⁇ l of a 1% spermidine solution. After a 15 minute incubation on ice, the insoluble constituents were sedimented by centrifugation for 10 minutes, the supernatant was discarded. After resuspension of the sediment by addition of 1 ml of Spermidine Removal Buffer (sodium acetate: 300 mM, magnesium acetate: 10 mM) and 3 ml of cold ethanol (100%), the mixture is incubated for 1 hour on ice with simultaneous gentle shaking. By centrifuging for at least 9,000 s / t 2 * 9.81 for 10 minutes, the insoluble DNA was sedimented.
  • Spermidine Removal Buffer sodium acetate: 300 mM, magnesium acetate: 10 mM
  • the concentration and purity of the isolated DNA molecules were determined by absorption measurement. The concentration determination results from the measured value of the optical absorption in the UV range at 260 nm. The purity of the isolated DNA was determined from the ratio of the absorption measurement at 260 and 280 nm (BioPhotometer, Eppendorf, Germany). In parallel, the quality of the isolated DNA was determined by electrophoretic separation in agarose gel (1%).
  • the DNA was purified using a commercially available DNA purification kit, e.g. from GE Healthcare in the form of the "il-lustra GFX TM PCR DNA and Gel Band Purification Kit", after which it has been cleaned up and retested.
  • a commercially available DNA purification kit e.g. from GE Healthcare in the form of the "il-lustra GFX TM PCR DNA and Gel Band Purification Kit", after which it has been cleaned up and retested.
  • a PCR reaction is carried out in a PCR-suitable reaction vessel, for example consisting of 2.5 ⁇ l of 10 ⁇ PCR buffer, 2.5 ⁇ l of a 2 mM solution of the four deoxynucleotide triphosphates (dNTPs), 2 ⁇ l of magnesium chloride solution (20 mM).
  • a thermostable DNA polymerase for example Taq DNA polymerase
  • 1 ⁇ l of, for example, a 25 mM solution of the PCR primer S_Ps_F 5'-TGCTCTCGCAGGTCTGACTACC -3 '; SEQ ID NO: 11) and S Ps R ( 5'-CACGCAAATCCCTACCTCTGCC-S '; SEQ ID NO: 12).
  • the primers are designed to match the identified ones for Peronospora swinglei s.l. specific intergenic DNA segment (SEQ ID NO: 2).
  • the mixture is made up to 25 ⁇ l with water.
  • the PCR is carried out, for example with the following conditions:
  • the incubation in a thermocycler takes place in accordance with the device-specific modalities. First, for example, an incubation at 94 ° C for 5 min. This is followed by incubation at eg. 94 0 C for 30 sec. After incubation at 65 ° C for 30 sec. This is followed by incubation at 72 0 C for 40 sec. The last three steps are then for example 45 times. Repeated. A final incubation at 72 ° C for 4 minutes may then be performed.
  • a second detection reaction (nested PCR).
  • 1 ⁇ l of the first detection reaction and 1 ⁇ l of, for example, 25 mM solution of the nested primer S_Ps_Fn (5'-GTCTTAACGAGACCGCGTGGTC-3 'SEQ ID No. 13) and S_Ps_Rn (5'-ATGACGGCAGGTGCTGTCGTTC-3' SEQ ID no. 14) to the other solutions or reagents described in Example 1 (10x PCR buffer, dNTPs, magnesium chloride solution and Taq DNA polymerase) in a new PCR-suitable reaction vessel.
  • the incubation in a thermocycler takes place according to the device-specific see modalities.
  • the amplificates obtained in the detection reaction 2 are separated, for example by means of gel electrophoresis, for example in an agarose gel.
  • This can be prepared, for example, by placing 1 g of agarose in a vessel, making up to 100 ⁇ l with an ion-containing liquid, for example with Tris-borate-EDTA buffer (TBE). Thereafter, the PCR reaction solution is placed in a preformed well of the agarose gels, for example. After addition of 1/6 vol. Glycerol and optionally dyes, which in the electrophoresis in the same direction as the DNA fragments in the agarose gel and the control of the progress of the electrophoresis serve.
  • TBE Tris-borate-EDTA buffer
  • the electrophoresis itself is carried out in a dedicated electrophoresis chamber filled with an isotonic ion-containing solution to the gel, preferably with the same, which has been used to prepare the agarose gel.
  • a voltage for example 140 V
  • the migration of the DNA fragments is enforced by the agarose gel, which is separated by the selective retention, which is based in particular on the size of the migrating fragments.
  • staining with a dye which associates or binds to nucleic acids for example ethidium bromide, for example by incubating the agarose gel for half an hour in a dyebath containing ethidium bromide.
  • a dye which associates or binds to nucleic acids
  • the traces of the 1.5% agarose gel were as follows: Lane 1: Seed Sample 1 (contaminated); Lane 2: seed sample 2; Lane 3: seed sample 3 (contaminated); Lane 4: seed sample 4; Lane 5: seed sample 5; Lane 6: seed sample 6; Lane 7: seed sample 7; Lane 8: reference DNA from Peronospora swinglei sl (positive control); Lane 9: Reference DNA of Ocimum basilicum (basil) (negative control 1); Lane 10: PCR approach without DNA (negative control 2); Lane 11: DNA size marker (standard) 1.
  • the reference sample of Peronospora swinglei s.l. shows a clearly visible amplicon in the size of 500 nt (A).
  • the white band is the organism-specific hybridizable nucleic acid molecule of Peronospora swinglei sl.
  • the same amplificate is found in the seed mixtures 1 (lane 1) and 3 (lane 3) PCR assays, but the amplificate is not present in seed samples 2, 4-7 (Lanes 2, 4 -7) and in the negative controls 1 and 2 (lanes 9 and 10).
  • the pathogen Peronospora swinglei s.l. be clearly detected in the seed samples 1 and 3.
  • the smear appearing in lanes 4, 6 and 7 is a nonspecific side reaction typical of nested PCR approaches, which is not to be confused with the specific amplificate in Lanes 1, 3 and 8. False negative reactions can be excluded by the previous quality check. False positive reactions are excluded by the inclusion of the negative controls 1 and 2.
  • the application of the method according to the invention in the form of a PCR-based detection leads to a clearly interpretable result.
  • the seed lots contaminated with the pathogen Peronospora swinglei sl can be safely identified and removed from the market.
  • the proliferation of the target organism, in this case a dangerous pest is prevented.
  • the detection method according to the invention has a number of advantages over pattern analysis. It is particularly characterized by its flexible use (line 11) and its high adaptability to new organisms (line 12). By the manufacturing method according to the invention the user is able to develop new organism-specific detection methods within a short period of time and to use them for the reliable identification of organisms.

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Abstract

La présente invention concerne un procédé de production d'une molécule d'acide nucléique hybridable spécifique d'un organisme, une molécule d'acide nucléique produite selon ce procédé, un nécessaire présentant au moins une des molécules d'acide nucléique, l'utilisation d'une séquence nucléotidique située entre deux éléments sur l'information génomique d'un organisme, pour produire une molécule d'acide nucléique hybridable spécifique d'un organisme, ainsi qu'un procédé d'identification d'un organisme dans un échantillon biologique.
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EP3109328A1 (fr) 2016-12-28
WO2008101701A2 (fr) 2008-08-28
US20100055703A1 (en) 2010-03-04
EP2471955B1 (fr) 2016-07-27
WO2008101701A3 (fr) 2009-04-02
EP2471955A3 (fr) 2012-09-05
EP2471955A2 (fr) 2012-07-04

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