EP1415003A2 - Dispositif d'analyse d'acide nucleique - Google Patents

Dispositif d'analyse d'acide nucleique

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Publication number
EP1415003A2
EP1415003A2 EP02794485A EP02794485A EP1415003A2 EP 1415003 A2 EP1415003 A2 EP 1415003A2 EP 02794485 A EP02794485 A EP 02794485A EP 02794485 A EP02794485 A EP 02794485A EP 1415003 A2 EP1415003 A2 EP 1415003A2
Authority
EP
European Patent Office
Prior art keywords
control
analysis
nucleic acid
analyte
amplification
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Withdrawn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
EP02794485A
Other languages
German (de)
English (en)
Inventor
Christian Rudolf Mittermayr
Bernhard Ronacher
Florian Winner
Karl Zehethofer
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.)
Lambda Labor fur Molekularbiologische Dna-Analysen GmbH
Original Assignee
Lambda Labor fur Molekularbiologische Dna-Analysen GmbH
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 Lambda Labor fur Molekularbiologische Dna-Analysen GmbH filed Critical Lambda Labor fur Molekularbiologische Dna-Analysen GmbH
Publication of EP1415003A2 publication Critical patent/EP1415003A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers
    • G01N35/00594—Quality control, including calibration or testing of components of the analyser
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/521—Single-layer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers
    • G01N35/00594—Quality control, including calibration or testing of components of the analyser
    • G01N35/00693—Calibration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/028—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having reaction cells in the form of microtitration plates

Definitions

  • the invention relates to a device for analyzing at least one nucleic acid sequence, a method for amplifying nucleic acid sequences, a method for identifying nucleic acid sequences, an oligonucleotide primer and an analysis kit according to the features in the preambles of claims 1, 24, 28, 30 and 31 and the use of the device or the analysis kit according to claims 32 to 35.
  • Periodontitis an inflammatory disease of all parts of the periodontium caused by bacterial deposits with progressive loss of supporting tissue.
  • Periodontitis is a common problem that affects a large percentage of the population.
  • the disease can usually be treated well using antibiotics.
  • antibiotics Recently, however, the frequent and sometimes untargeted use of antibiotics to treat various diseases has led to an increase in the development of resistance in many bacteria.
  • a delayed bacteriological finding first requires broad-spectrum antibiotic therapy, which means more side effects for the patient and is more expensive.
  • further therapeutic measures may be necessary, which consequently results in reduced effectiveness, a protracted course of the disease and an inefficient one
  • Microbiological examinations enable the detection of physiological features with indicator media that e.g. B. indicate a change in pH, an exact determination of the type or type of bacteria.
  • indicator media e.g. B. indicate a change in pH, an exact determination of the type or type of bacteria.
  • the disadvantage that can be seen in microbiological analysis systems is that only live germs are to be detected, which requires careful sampling and a corresponding sample transport, and furthermore that the analysis takes several days to weeks.
  • Serological tests can detect protein components from bacteria that react with antibodies.
  • the use of monoclonal antibodies enables the detection of individual types and types of bacteria. Antibody-coupled enzyme reactions enable detection.
  • Such an identification system consists in that, as shown for example in US Pat. No. 4,741,999 A, the use of monoclonal antibodies serves for the detection and determination of the concentration of certain microorganisms relating to the etiology of human periodontal diseases.
  • a monoclonal antibody is specifically described for an antigen from Actinobacillus actinomycetemco-mitans. Actinobacillus actinomycetemcomitans mostly occurs in juvenile periodontitis.
  • the serological systems have the disadvantage that only a few epitopes of an antigen can be detected with monoclonal antibodies.
  • the number of available antibodies is therefore limited to certain types of bacteria.
  • WO 00/52203 A describes a method for identifying bacteria which describes the amplification of part of the 23S rDNA using the primer 5 'GCGATTTCYGAAYGGGGRAACCC-3' and the primer 5'-TTCGCCTTTCCCTCACGGTACT-3 '.
  • the amplificate is hybridized with various oligonucleotides on a nylon membrane.
  • This method allows the identification of at least 8 types of bacteria in one test, the bacteria being selected from a group comprising bacteria such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus spp., Klebsiella spp., Enterobacter spp., Proteus spp., Pneumococci and coagulase include negative staphylococci.
  • this system has the disadvantage that the 23S rDNA is variable and therefore suitable primary sequences for the bacterial genera and species to be analyzed have to be found in a few consensus areas to identify the bacterial genera and species. This in turn limits the number of types of bacteria that can be analyzed.
  • the species present is identified by sequence comparison.
  • a restriction analysis of the amplification product in the sense of an RFLP restriction fragment length polymorphism
  • RFLP restriction fragment length polymorphism
  • the main disadvantage of this system is that direct sequencing takes a long time and is expensive, and that RFLP does identify sequence variations but not many types of bacteria can be distinguished.
  • Another disadvantage is that the oligonucleotide primers only find complementary sequences in the genera Helicobacter and Wolinella, and therefore can only detect and analyze these two genera.
  • the object of the invention is therefore to provide a possibility for reproducible, quick and simple analysis of nucleic acid sequences. It is also a subtask of the
  • the object of the invention is achieved by the device according to the features in the characterizing part of claim 1.
  • the advantage of this device is that for the analysis of various analytes there are control systems on the device which go through the same process steps as the specific binding partners for the analyte and thus the user has the possibility of the quality of the analysis result via these without additional expenditure of time Assess or improve control systems. This is particularly advantageous if the individual process steps for the analysis of several people are possibly carried out at different locations and these do not communicate with each other. Unclear results can be traced back to possible sources of error.
  • Another advantage is that the process costs using the device are hardly or not increased compared to processes without controls.
  • the species-specific, amplified and hybridized analytes can be archived together with the quality controls, so that the process sequence can be checked at any time and the evaluation with consistent quality can be repeated.
  • the development according to claim 4 is advantageous, which provides a control option for the alignment of the device both during the evaluation and after the evaluation.
  • the evaluation device can already recognize the wrong orientation of the wearer.
  • the evaluation can be terminated immediately and thus the costs for this and / or subsequent process steps can be saved.
  • An embodiment according to claim 6 proves to be advantageous, according to which a complex error search can be carried out by specifically detecting an error in the amplification of the analyte
  • the analysis result can be limited to the amplification step and the entire analysis does not have to be searched for the source of the error.
  • the embodiment according to claim 7 also proves to be advantageous because mix-ups of samples to be analyzed, which took place before the start of the analysis, can be detected quickly. Furthermore, it proves advantageous that the source from which the at least one analyte originates can be determined, and thus the sample can still be assigned even after the analysis.
  • the further development according to claim 8 proves to be advantageous, whereby by connecting an at least one further specific binding partner to the analyte to be analyzed as well as to a further binding partner, the analyte to be identified can be detected if an additional further specific binding partner is present for the further analyte.
  • the signal of the analyte to be analyzed results from the difference between the detectable signals.
  • a further development according to claim 9 is also advantageous, wherein the binding of the same specific binding partner to two different analytes, which have a very similar structure, can be demonstrated.
  • a further binding partner that is specific only for the one analyte the presence of another but undesired analyte that also binds to the same binding partner can be identified.
  • detection of contamination of the sample with, for example, infectious material is made possible.
  • the analyzing or medical staff can be informed and warned at an early stage, thus minimizing the risk of infection and the possible consequential costs.
  • An embodiment according to claim 10 is also advantageous, according to which at least a semi-quantitative determination of the analyte in the original sample is made possible because the quantity and quality of the isolation can be determined by adding a precisely defined amount of a substance with behavior similar to that of the analyte.
  • the application of the specific binding partner to the carrier can be carried out immediately after this step but before the start of the analysis, after which the quality of the analysis can be ensured at a high level by the use of only perfect carriers and thus unnecessary costs by the use be avoided by faulty devices.
  • An embodiment according to claim 13 proves to be advantageous, according to which a uniform intensity of the signals can be guaranteed when evaluating the analysis and thus results do not falsify the result due to different intensities of the signals.
  • the surface of the device can check for unspecific deposits of analytes and thus a signal which arises from an unspecific binding of an analyte and would lead to a wrong result regarding the identification of the analyte.
  • the device can thus be used universally regardless of an expected concentration of the analyte. It is also advantageous that the different concentrations of the binding partners quantify the analytes, such as. B. gene expressions.
  • a configuration according to claim 18 is also advantageous, according to which the analysis can be carried out with greater certainty by arranging identical binding partners on the carrier in several analysis and / or control areas. Doubtful results in a certain analysis and / or control area can be confirmed or refuted by the result in at least one other analysis and / or control area.
  • a further advantage is that the multiple arrangement of the same positive controls, both in different and in the same concentrations, enables normalization to be carried out within or between different measurements. So z. For example, by calculating a correction factor, the measurements in the analysis areas of a device can be compared with one another on one or more other devices. Of course, the measurements in the analysis areas can also be made on the same
  • Device can be normalized by calculating a correction factor.
  • an embodiment according to claim 19 proves advantageous, according to which, for. B. defective spots on the carrier not recognized during the manufacture of the device and / or uneven evaluation conditions over at least one further analysis and / or control area on the device due to the non-adjacent arrangement of the same binding partner in at least one further analysis and / or control area of the wearer can be recognized or compensated.
  • nucleic acid sequences of many different types and types of bacteria can be analyzed simultaneously in any combination and thus a corresponding time saving or increased throughput can be realized in the laboratory.
  • a further development of the device according to claim 22 is advantageous, with which only an oligonucleotide primer or primer a quality control for various process steps, in particular the amplification, is available. Furthermore, by using at least one of these oligonucleotide sequences, the necessary steps for optimizing the successful amplification can be minimized.
  • different target nucleic acid sequences can be analyzed in an advantageous manner using only one oligonucleotide primer or primer pair, because by appropriately selecting the oligonucleotide sequences on the support, amplified target nucleic acid sequences simultaneously have complementary regions and can therefore hybridize.
  • the object of the invention is also independently achieved by a method according to the features in the characterizing part of claims 24 and 28.
  • the advantage of this is that several target nucleic acid sequences with only one primer sequence, in particular bacterial genera or species associated with periodontitis, can be amplified or identified simultaneously.
  • the process costs for the laboratory can be reduced and the feasibility of the method can be simplified, since the risk of confusion regarding different oligonucleotide primers for different target nucleic acid sequences is minimized.
  • the process can also be carried out by less experienced people.
  • the storage can be simplified and the costs for a laboratory caused thereby can be reduced by reducing the number of different reagents. Due to the greater consumption of a primer, the acquisition costs for chemicals can also be reduced and the larger sales of this primer can reduce the storage time and thus the risk of the same being unusable.
  • the further development according to claim 26 is also advantageous, because by using the gene sequences for the 16S rRNA with only one Prime ⁇ aar sequences different Bacterial species can be amplified.
  • the gene sequence for the 16S rRNA has on the one hand highly conserved consensus regions to which the oligonucleotide primers bind and on the other hand gene or species-specific regions in between.
  • the amplification of partial sequences of the genes which encode the 16S rRNA makes the use of many different oligonucleotide primer sequences and the optimization of the amplification conditions for the respective oligonucleotide primer sequence superfluous.
  • the hybridization products ie. H. the amplificate bound to the complementary oligonucleotide can generate a signal. This increases the degree of automation and thus a
  • the object of the invention is also achieved independently by an oligonucleotide primer according to the features in the characterizing part of claim 30.
  • the advantage of this is that amplification products, created by the amplification with this primer or a prime pair formed from them, hybridize to a carrier with a complementary oligonucleotide sequence for at least one positive control and / or target nucleic acid sequence and the resulting signal enables the method and / or quality to be checked identification of the types of bacteria is made possible.
  • the object of the invention is also achieved independently by an analysis kit according to the features in the characterizing part of claim 31.
  • the advantage of this is that the analysis kit contains both the device and the oligonucleotide primers for the simultaneous amplification of different types of bacteria.
  • the analysis result also contains results of the checks carried out during the analysis, so that any errors can be recognized before or with the analysis of the analytes and thus a lengthy research into the causes and, if necessary, a necessary optimization effort can be reduced.
  • the object of the invention is also independent through the use of the device solved according to the features in the characterizing part of claims 32 and 33.
  • the advantage of this is that analytes of bacterial species associated with periodontitis can be identified and thus an exact analysis of the associated germs can be achieved.
  • the object of the invention is also achieved independently by using the analysis kit according to the features in the characterizing part of claims 34 and 35. It has proven to be advantageous that a complete set for identifying various types of bacteria or different gene expression patterns is already available through the use of the analysis kit and that the components do not have to be purchased individually and coordinated with one another. A further advantage is that it can quickly detect germs associated with periodontitis, in particular periodontitis, and therefore the consequential costs of a primarily untargeted antibiotic therapy can be prevented.
  • FIG. 1 shows a device with binding partners, arranged in analysis or control areas in a schematically simplified representation.
  • This device comprises a carrier 1, preferably in the form of a plate, with a surface 2 on which a plurality of analysis areas 3 and a number of control areas 4 are arranged.
  • a carrier 1 preferably in the form of a plate
  • surface 2 on which a plurality of analysis areas 3 and a number of control areas 4 are arranged.
  • control areas 4 it is of course possible that in a minimal version only one analysis or control area 3, 4 or that an analysis area 3 and several control areas 4 or vice versa are arranged.
  • Alternative embodiments of the device are carrier 1 in a platelet-shaped configuration, with two recesses being formed on its surface, such as, for. B. Chambersli- des.
  • Another embodiment of the carrier 1 are microtitre plates according to dimensions in accordance with the recommendations of the SBS (Society of Biomolecular Screening).
  • the analysis area (s) 3 and / or the control area (s) 4 can be arranged on the surface 2 of the carrier 1 at any predetermined position.
  • a silanized glass support can preferably be used as the support 1. But it is it is also possible to form the carrier 1 from plastic, stone, metal, etc. or carriers which have been surface-treated with aldehyde, aminosilane, streptavidin, biotin, thiol, magnetic materials can also be used.
  • FIG. 1 shows only one example of the device according to the invention.
  • Other shapes such as e.g. B. a cube, a ball, or other cross-sections of the platelet-shaped formation, such as square, round, etc., can be used for the device.
  • these devices For example, possible at least approximately spherical devices with several analysis or To provide control areas 3,4 superficially, these devices subsequently being placed in a liquid sample solution to be analyzed. If necessary, these devices designed in this way can be provided with a layer of a magnetic material, so that the devices can be easily removed from the sample solution after the target nucleic acids have been bound.
  • the device is preferably in the form of a plate and the sample (s) to be examined is or are applied to this device.
  • the device with at least one cover layer on at least one of the surfaces 2 of the carrier 1 in order to protect the underlying surface 2 from unintended external influences, e.g. from scratches and thus destruction of surface areas.
  • This cover layer can also be arranged to be at least partially removable.
  • nucleic acid nucleic acid sequence and target nucleic acid sequence can be selected by a term from a group comprising nucleic acids, such as e.g. DNA, RNA, PNA (peptide nucleic acids), proteins, e.g. Antibodies, epitopes, antigens, scaffold proteins, fusion proteins, in particular recombinant
  • Fusion proteins signal proteins, transmitters, enzymes, substrates, hormones, peptides, lipids, carbohydrates, such as mono-, di-, oligo- and polysaccharides, and their modified forms, etc., can optionally be replaced.
  • the term oligonucleotide can of course also be extended to include first or further binding partners, the binding partner being a molecule selected from a group comprising nucleic acids, such as eg DNA, RNA, PNA (peptide nucleic acids), proteins, such as, for example, antibodies, epitopes, antigens, scaffold proteins, fusion proteins, in particular recombinant fusion proteins, signal proteins, transmitters, enzymes, substrates, hormones, peptides, lipids, carbohydrates, such as, for example, mono -, Di-, oligo- and polysaccharides, and their modified forms, etc., can represent.
  • the device according to the invention is preferably used in the analysis of analytes, in particular nucleic acids of bacterial species, in particular of those associated with periodontitis.
  • analytes in particular nucleic acids of bacterial species, in particular of those associated with periodontitis.
  • the invention also includes the amplification of the target nucleic acid sequence using a primer or Prime ⁇ aares and their hybridization to oligonucleotides in the analysis or. Control areas 3.4.
  • the device can be taken by various precautions, such as. B. Recesses in the surface can also be used directly for the amplification of nucleic acid sequences.
  • the device can, at least in some areas, include a device for increasing the temperature, e.g. comprise a heating layer, e.g. To enable temperature change programs.
  • oligonucleotides complementary to the target nucleic acid sequence and / or to the positive controls can preferably be attached to the support 1 in the analysis or control areas 3, 4 using a nanoplotter by piezoelectric contactless sample transfer.
  • other methods for oligonucleotide distribution can also be used, such as needle printers, ring and pin printers, electro addressing printers, top spoters, etc., or these can also be applied manually. Other methods are of course possible and are known to the person skilled in the art in this field.
  • the oligonucleotides can be used for the identification of different nucleic acid sequences, in particular of nucleic acid sequences of different types of bacteria. Ideally, the respective oligonucleotide sequence should contain only one bacterial species and their strains hybridize. As can be seen from WO 00/52203 A, several oligonucleotide sequences are sometimes necessary to identify individual bacterial species in order to identify the species specified in this document.
  • the invention can preferably be used for the identification of bacteria associated with periodontitis.
  • the device according to the invention preferably has several, for example 20 different, oligonucleotides therefor.
  • 3 oligonucleotides can be used to identify Actinobacillus actinomycetemcomitans, Actinomyces odontolyticus, Actinomyces viscosus, Bacteroides forsythus, Campylobacter concisus, Campylobacter gracilis (Bacteroides gracilis), Campylobacter rec nucleatum, Peptostreptococcus micros, Po ⁇ hyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Streptococcus constellatus, Streptococcus gordonii, Streptococcus mitis, Treponema denticola and Veillonella parvula can be used.
  • oligonucleotides can also be used for the identification of different nucleic acid sequences, in particular of gene expression patterns of different organisms.
  • the oligonucleotides preferably consist of 10 to 120 nucleotides, but can also consist of 15 to 100 nucleotides, but in particular 20 to 50 nucleotides, for example 33 nucleotides.
  • the detection of short nucleic acid sequences of amplified DNA is a simple method and can therefore also be carried out on a much larger scale, e.g. to identify viral nucleic acid sequences or gene variations, etc., as described here.
  • the oligonucleotide sequences are synthesized de novo.
  • the oligonucleotides are modified at at least one end in such a way that they adhere to the support 1 in the analysis or control areas 3, 4, in particular bind chemically via this modified end.
  • the 5 'end can preferably be modified with the amino modifier C6 MMT (6- (4-monomethoxytritylamino) hexyl- (2-cyanoethyl) - (N, N-diisopropyl) phosphoramidite with the following structural formula.
  • C6 MMT 6- (4-monomethoxytritylamino) hexyl- (2-cyanoethyl) - (N, N-diisopropyl) phosphoramidite with the following structural formula.
  • NtvITN HC H2CH 2CH 2CH 2CH 2CH 2CH 2CH 2CH
  • MMT monomethoxytrityl and Pr isopropyl.
  • modifiers can also be used, e.g. Succinyl, DNP (2,4-dinitrophenyl), etc.
  • oligonucleotides In order to be able to apply the oligonucleotides to the analysis or control areas 3, 4, they are used in solution.
  • a solvent e.g. a Tris-EDTA buffer, or water can be used.
  • Other solvents that can be used are e.g. physiological sodium chloride solution (0.9% NaCl), PBS, alcohol dilutions, Tris buffer, DMSO in a concentration selected from a range with an upper limit of 30%, preferably 25%, in particular 20% and a lower limit of 1% , preferably 2%, especially 5% etc.
  • the oligonucleotides are added to the solvent in an amount such that they are present in a concentration in the range between 1 nM to 1 mM, for example 10 nM to 500 ⁇ M, preferably 100 nM to 500 ⁇ M. Concentrations in the
  • Range between 0.5 uM to 100 uM, in particular 1 uM to 50 uM proven.
  • binding partners can be presented in the same or different concentrations in the control areas 4. Binding partners can be applied, which control the orientation of the support 1, e.g. in an evaluation device, e.g. one
  • binding partners for the control areas 4 can be used, which allow an assessment of the quality of an amplification, e.g. PCR and / or hybridization of the analytes can be used.
  • a specific binding partner such as a nucleotide sequence or an antibody of a specific organism, is used to demonstrate the sample identity or type or matrix.
  • a specific variety or breed such as cattle, sheep, pigs, or birch, palm, fir, etc.
  • Identification can also be done by adding material to the sample such as. for example, nanoparticles, dyes, etc., which are differentiated at the control areas 4.
  • the identity can also result from a combination of different markers.
  • the same label is used for each analyte from the same sample. This is done either by directly marking the
  • Analytes with a substrate such as e.g. Enzyme, biotin, digoxigenin, radioactive labeling, fluorescent or chemiluminescent labels, chemical substances with a specific spectrum, e.g. Infrared, etc. or by adding a precisely defined molecule, the molecule having different characteristics for each sample of different origins and being clearly identifiable in the further analysis.
  • a substrate such as e.g. Enzyme, biotin, digoxigenin, radioactive labeling, fluorescent or chemiluminescent labels, chemical substances with a specific spectrum, e.g. Infrared, etc. or by adding a precisely defined molecule, the molecule having different characteristics for each sample of different origins and being clearly identifiable in the further analysis.
  • the infected host organism can be detected by proving the origin or the source from which viruses originate, for example. This is particularly important when identifying virus strains that affect different host organisms. By immobilizing a binding partner that makes a phylogenetic distinction, e.g. Complementary sequences to the Cytrom B gene, or by immobilizing cytochrome B corresponding antibodies, the different host organisms are detected. Both for the analysis and for the further procedure, it can be decisive from which source this virus was isolated in order to be able to take immediate measures, for example to prevent the pathogen from spreading further.
  • prions come from the nervous system of cattle or from sheep in order to be able to quarantine the affected farm from which the sick animal comes.
  • an individual-specific binding partner such as short tandem repeats (STR), variable number of tandem repeats (VNTR), single nucleotide polymorphism (SNP), etc.
  • STR short tandem repeats
  • VNTR variable number of tandem repeats
  • SNP single nucleotide polymorphism
  • the cross reaction can thus be detected and the two types can be distinguished from one another.
  • the same principle can also be used for antibodies which bind to two different antigens, a further antibody being present for the second antigen, from which it has been demonstrated that it is a specific binding partner only for the second type.
  • Specific binding partners for contamination can also be immobilized on the carrier.
  • a specific binding partner for, for example, highly infectious viruses By applying a specific binding partner for, for example, highly infectious viruses, the presence of these viruses in the sample, which also contains the analyte, can be detected, and medical personnel are therefore warned in good time of potential infection options after receiving the result of the analysis.
  • the immobilization of a specific binding partner for the isolation control makes it possible to determine the amount of analyte that was lost during the isolation.
  • the amount lost through the isolation work can be determined. It is important that the added substance is very similar to the analyte in its behavior in order to obtain a representative statement.
  • DNAse is free.
  • a specific binding partner is arranged on the carrier 1, which binds a normalization of the intensity of the signal, which is indicated by markings, such as by enzymatic marking, silver coloring or marking with gold particles, by fluorescence or chemiluminescent color substances, biotin, digoxigenin, radioactive labels, etc., are allowed.
  • markings such as by enzymatic marking, silver coloring or marking with gold particles, by fluorescence or chemiluminescent color substances, biotin, digoxigenin, radioactive labels, etc.
  • gold particles are applied to carrier 1 in a defined amount, which then serve as a crystallization point, analogous to the detection system, where, for example, the primer is via a biotin Streptavidin system is labeled with the gold particles. Accordingly, a defined number of binding sites for dendrimer signal amplification or for enzymatic or similarly effective catalytic signal amplification systems are applied to the carrier 1.
  • a specific binding partner for the hybridization control which is added, for example, only after the isolation or amplification of the analyte from the sample, is immobilized either parallel to each specific binding partner.
  • a hybridization control can also be available in which the specific binding partner for the analyte and the specific binding partner for the hybridization probe are coupled and are therefore present in the same amounts, the problem of the different ratio of the specific binding partner to the analyte and of the specific one Binding partner to the hybridization probe, if these are available separately, is eliminated.
  • a label e.g. a fluorescent or chemiluminescent label is covalently bound.
  • the first determination of the print quality takes place after the immobilization of the specific binding partners on the support 1 and after the hybridization an evaluation is carried out again using the hybridization probe. This procedure enables a separation into control for the printing process and into the control for hybridization.
  • Another embodiment variant marks the specific binding partner with three different markings, e.g. Fluorescence or chemiluminescence markings that result in different signals during detection.
  • one label is the control for the immobilization of the specific binding partner
  • the other label is a control for the hybridization
  • the third label is a control for the detection of the binding of the specific binding partner to the analyte.
  • Control areas 4 make it possible to compare results that were made on different devices or at different times even when the device has aged in the meantime (for example, the life of a PMT or CCD chip is limited). It can also Results are compared that are made with different parameter settings, such as voltage for the PMT, measurement time for the CCD chip, etc.
  • control areas 4 can also be applied so that the measuring function can be determined. This determines the sensitivity, sensitivity, detection limit, linearity or the dynamic range of the measurement function; this enables comparability between different device types.
  • These control areas 4 should of course be used for the markings used in each case. When using substances with a broader spectrum (wavelengths), the quality of the filters and other optical properties can also be compared.
  • the oligonucleotides for the orientation control can be present in concentrations in the range from 1 ⁇ M to 100 ⁇ M, in particular in the range from 1 ⁇ M to 50 ⁇ M, preferably in a concentration of 10 ⁇ M.
  • the oligonucleotides for the amplification control can be in a concentration in the range between 0.1 nM to 0.5 mM, for example 1 nM to
  • the oligonucleotides for the hybridization control can be present in concentrations in the range between 0.1 nM to 500 ⁇ M, for example in the concentration range from 1 nM to 100 ⁇ M, in particular from 10 nM to 50 ⁇ M. Concentrations in the range between 25 nM to 25 ⁇ M, in particular 40 nM to 10 ⁇ M, have also proven to be advantageous.
  • oligonucleotides other than those specified above can be used for the control areas 4, e.g. Oligonucleotides as a positive control with a sequence complementary to the oligonucleotide primer sequence which is used for the amplification of the target nucleic acid sequence or oligonucleotides as a negative control with a sequence of other organisms which do not have the gene for coding the 16S rRNA.
  • the oligonucleotides can be present in the control areas 4 in dilution series, one concentration per control area 4 being used in each case.
  • the highest concentration of the oligonucleotides can be 500 ⁇ M, for example 250 ⁇ M, in particular 100 ⁇ M and preferably 10 ⁇ M.
  • a dilution series can comprise 2 to 24 concentrations, for example 3 to 20 concentrations, in particular 4 to 18 concentrations. 5 to 15, in particular 6 to, have also proven advantageous
  • the statements relating to the oligonucleotides for the control areas 4 can of course be appropriately transferred to the oligonucleotides for the analysis areas 3, so that only the corresponding concentrations or concentration ranges are given here.
  • the concentration of the oligonucleotides in the analysis areas 3 are in the range from 100 nM to 1 mM, for example from 1 ⁇ M to 500 ⁇ M, in particular from 10 ⁇ M to 100 ⁇ M and preferably from 25 ⁇ M to 45 ⁇ M.
  • the oligonucleotides are preferably selected in volumes from a range with a lower limit of 1 pl, in particular 10 pl, preferably 200 pl and with an upper limit of 5 ⁇ l, in particular 1 ⁇ l, preferably 0.3 ⁇ l, per analysis region 3 and / or Control area 4 applied.
  • the arrangement of the oligonucleotides on the carrier 1 can be predefined.
  • Each oligonucleotide can have an area of 50 to 500 ⁇ m in diameter, in particular from 200 to 250 ⁇ m.
  • the surface 2 of the device used for the analysis which e.g. B. can be designed as a slide for microscopy, an area of 0.1 cm 2 to 50
  • oligonucleotides with a low density enables simple hybridization conditions, such as. B. small temperature fluctuations, homogeneous buffer distribution, etc. and uncomplicated evaluation methods, because an overlap of signals is not normally expected.
  • the oligonucleotides consist of sequences which are complementary to certain partial sequences of the target sequence and / or are complementary to at least one positive control.
  • the oligonucleotide for the hybridization control serves as proof of quality for the hybridization.
  • a partial sequence of the bacteriophage genome is inserted as a linker sequence at the 3'-end and the 5'-end is modified with the amino modifier C6 MMT mentioned.
  • Any chemical compound which creates the necessary spatial distance and thus avoids any steric hindrances which may occur can also be used as a linker sequence.
  • the oligonucleotide for the orientation control can be used to determine or determine the orientation of the carrier 1 during the manufacture of the device or in later analysis steps. Furthermore, the orientation control for determining or setting the limits of the field to be evaluated, in particular defined by the analysis and control areas 3, 4, can be used on the carrier 1 and for checking the localization of the reading field. It can preferably be sequence-identical to the oligonucleotide of the hybridization control and additionally bears, for example, a label at the 3 'end, for example digoxigenin, biotin, radioactive labels, such as, for. B. 33 P, or fluorescent dyes to deliver a well evaluable signal even without hybridization.
  • the orientation control is preferably arranged in two control areas 4, which can be arranged at certain predefined locations on the support 1, for example during the application of these oligonucleotides.
  • the oligonucleotides for the control areas 4 and for the analysis areas 3 can be arranged in several areas of the device, for example in at least 2 areas, but also in 3, 4, 5, 6, 7, 8, 9 or 10 areas.
  • Oligonucleotides such as e.g. B. the orientation control and / or the PCR control and / or orientation control in the same analysis or control area 3.4, as the target nucleic acid sequence are present and by their different labeling such. B. different dye derivatives can be distinguished during the detection.
  • the oligonucleotides are hybridized with nucleic acid sequences of the target sequence and subsequently generate a signal. Since an oligonucleotide sequence is preferably arranged in several regions on the device, only those hybridized oligonucleotides for which more than one signal can be observed are preferably and positively evaluated, as a result of which the safety and the reproducibility of the result can be improved.
  • the negative control is a control area 4 on the surface 2 of the support 1, to which no oligonucleotide is bound. This makes it possible to detect non-specific adhesions of various nucleic acid sequences.
  • the oligonucleotides of the analysis areas 3 and / or control areas 4 are mixed with the hybridization control in a certain ratio before application to the support 1.
  • the ratio of the oligonucleotides to the hybridization control can be in a ratio of 1: 1 to 1: 100.
  • the mixing ratio of 1:10 has proven to be advantageous.
  • the hybridization control serves as a control of the hybridization that has taken place for each individual analysis and / or control area 4. False negative results can be excluded by means of the hybridization control.
  • the target nucleic acid sequence can be labeled with a different dye derivative than the hybridization probe sequence, and therefore the signal of the target nucleic acid sequence can be evaluated from the signal of the hybridization probe by z. B.
  • the hybridization probe is a nucleic acid sequence complementary to the hybridization control and can be labeled, for example, with various dye derivatives such as, for example, Cyl, Cy 2, Cy 3, Cy 4 and / or Cy 5. It is also advantageous that if there is no hybridization with a complementary target nucleic acid sequence, a signal, namely that of the hybridization probe, can be detected, whereby it can be ascertained that the missing signal has not occurred due to an analysis method error.
  • the unbound oligonucleotides are removed from the support 1.
  • the unbound Oligonucleotides can be removed by washing the carrier 1 in an SDS solution with a concentration in the range from 0.01% to 2%, in particular in the range from 0.1% to 1%, preferably in a concentration of 0.2% ,
  • the detergent remaining on the surface 2 of the carrier 1 is washed with a washing liquid, such as. B. water, PBS, TE, etc. removed.
  • the carrier 1 is dried with air and in a liquid such as.
  • Hydrogen-releasing reagents such as LiAlH 4 , Na BtLj, etc. are added to the solution in an amount of 0.001 g to 0.5 g, in particular 0.01 g to 0.25 g, preferably 0.01 g to 0.1 g Volume of 500 ml, preferably 100 ml, in particular 50 ml, added.
  • a volume of 40 ml, in particular 30 ml, has proven to be advantageous.
  • the different washing steps take place at a temperature in a range from 0 ° C. to 100 ° C., in particular from 10 ° C. to 98 ° C., preferably from 20 ° C. to 95 ° C.
  • DNA sequences of possible oligonucleotides of the target nucleic acid sequences or the positive controls from 5 'to 3' are listed:
  • Target organism Actinobacillus actinomycetemcomitans
  • Target organism Actinomyces odontolyticus
  • Target organism Actinomyces viscosus
  • Target organism Bacteroides forsythus
  • Target organism Campylobacter concisus
  • Target organism Capnocytophaga gingivalis
  • Target organism Eubacterium nodatum
  • Target organism Fusobacterium nucleatum
  • Target organism Peptostreptococcus micros
  • Target organism Porphyromonas gingivalis
  • Target organism Prevotella intermedia
  • Target organism Streptococcus constellatus
  • Target organism Streptococcus gordonii
  • Target organism Streptococcus mitis
  • Target organism Treponema denticola
  • Target organism Veillonella parvula
  • single-stranded nucleic acids can be used which are either obtained from double-stranded nucleic acid sequences by denaturation or which have already been produced from single-stranded nucleic acid sequences, advantageously by amplification with a primer gradient.
  • the primer gradient can represent a ratio of the forward primer to the reverse primer of 1: 100, preferably 1:50, in particular 1:10, and leads to an asymmetrical PCR. If only one primer is present during the amplification of the target nucleic acid sequence, single-stranded DNA molecules are produced, which are then available for hybridization on the DNA chip.
  • the target nucleic acid sequence is DNA or RNA isolated from a biological sample, such as. B. bacterial cells, viruses or eukaryotic cells. Tissue samples (blood cells, biopsies, etc.) and liquid samples (blood, saliva, urine, pleural or peritoneal fluid, etc.) can be used. All methods available can be used to isolate the nucleic acid. It is often desirable to amplify the target nucleic acid sequence before hybridization.
  • special new oligonucleotide primers of at least 15 base pairs in length, which are characterized in that they have newly defined consensus sequences for the 16S rRNA, were used for the analysis or identification of the above-mentioned types of bacteria
  • the DNA sequences of these primers comprise at least 15 nucleotides of the sequences listed below from 5 'to 3'.
  • the sequences of the primers and oligonucleotides which are mentioned here are given in the standard IUB / IUPC nucleic acid code.
  • the reverse primer listed above contains the symbol Y.
  • Y stands for C and T.
  • Y indicates nucleotide permutations in "wobble" regions of the sequences.
  • the reverse primer is therefore used as a degenerate primer set with C and T as suitable nucleotides for the Inko ⁇ oration provided in the Woobleregions.
  • the primers are marked for signal detection.
  • markings can e.g. B. digoxigenin, biotin, radioactive labels such as 33 P, fluorescent dyes such as Cy 1, Cy 2, Cy 3, Cy 4, and / or Cy 5, etc. or a combination thereof. Any other marking system can also be used. Of course, this also applies to all the markings mentioned in this description.
  • the nucleotides used for the amplification can already be marked.
  • the oligonucleotides on the support 1 can also be labeled with digoxigenin, biotin, radioactive labels, such as P, fluorescent dyes, etc.
  • the marked reverse primer is therefore a component of each double-stranded amplicon, as well as the single-stranded amplicon produced by the asymmetric PCR.
  • primers can be used to amplify both gram-negative and gram-positive bacteria nucleotide sequences.
  • the amplified nucleic acid sequences can be used directly for the hybridization, which means that purification steps can be saved.
  • the oligonucleotides on the support 1 form stable hybrid duplex with complementary base pairing with the, in particular amplified, target sequence.
  • the carrier 1 can be selected over a period of time from a range with a lower limit of 1 minute, in particular 2 minutes and an upper limit of 30 minutes, in particular 20 minutes, preferably 5 Minutes, at a temperature selected from a range with a lower limit of 20 ° C, in particular 30 ° C, and an upper limit of 95 ° C, in particular 75 ° C, preferably at 60 ° C.
  • the labeled amplification products can be in different
  • Volumes selected from a range with a lower limit of 0.5 ⁇ l, in particular 1 ⁇ l, and an upper limit of 20 ⁇ l, in particular 15 ⁇ l, preferably in a volume of 5 ⁇ l for hybridization in a solution consisting of sodium citrate and / or NaCl can be used.
  • the applied to the carrier 1 solution with the amplificates can, for. B. with wax, oil, glass plates, etc., are layered over to ensure uniform conditions, such as maintaining a constant temperature and / or a constant air humidity.
  • the incubation can take place at temperatures in a range from 20 ° C. to 75 ° C., in particular from 30 ° C. to 70 ° C., and preferably at 50 ° C. to 60 ° C.
  • nucleic acid sequences which do not form a hybrid duplex are washed away under stringent conditions and the hybridized nucleic acid sequences which are analyzed remain on the support 1.
  • Nucleic acids are denatured by increasing the temperature or lowering the salt concentration of the buffer. Under low stringent conditions, such as low temperature and high salt concentration, duplexes even form which do not have an exactly complementary sequence. This reduces the specificity of hybridization under low stringent conditions. Under highly stringent conditions such as high temperature and low salt concentration in the buffer, the specificity of the hybridization is increased.
  • the unbound target sequences can be removed with a solution consisting of sodium citrate and / or NaCl in a first step at temperatures from 40 ° C. to 75 ° C., in particular from 45 ° C. to 70 ° C. and preferably at 50 ° C. to 60 ° ° C. In a further step, the same solution is used at temperatures to select from a range with a lower limit of 4 ° C., in particular 8 ° C., and an upper limit of 30 ° C., in particular 20
  • the hybridized nucleic acid sequences can be detected by means of the label which is introduced into the target nucleic acid sequences and or into the oligonucleotides.
  • the markers are preferably already incorporated into the target sequence during the amplification. It can e.g. B. during the PCR labeled primers or labeled nucleotides can be used. Alternatively, the marker can also be attached directly to the target sequence or after amplification, e.g. B. by nick translation or end marking, such as. B. Kinination of the nucleic acid sequence and subsequent ligation of a linker that connects the nucleic acid sequence with the marker.
  • Markings can be detected using spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or the like methods.
  • the evaluation of the carrier 1 can take place, for example, by measuring the intensity of a certain light wavelength after excitation of a fluorescent molecule with at least approximately, preferably by means of a laser, generated monochromatic light.
  • the location data (coordinates), such as the location and the limits of the reading field, can also be coordinated with the measured intensities depending on the wavelength by means of the oligonucleotides for the orientation control on the read-in carrier 1.
  • the reader used is equipped with two different lasers. The lasers and photomultipliers are matched for the dye derivatives Cy3 and Cy5 used. Operation and software acquisition is part of the device operation.
  • the result of the hybridization is obtained by querying the light intensities measured with the reader in the
  • optical, electrical, electrochemical, magnetic, photochemical, photoelectric or enzymatic detection and measurement methods can also be used. These methods can of course also be combined with one another.
  • unlabeled binding partners or analytes can also be detected using surface plasmon resonance technology.
  • the production of a device according to the invention in the form of a DNA chip is described below.
  • the preparation of the DNA chip blank of the carrier 1 takes place at room temperature. All oligonucleotides to be applied are provided in a master plate in 150 ⁇ l portions for the nanoplotter. At the same time, this mast plate serves for storage. All DNA oligonucleotides are dissolved in 10% DMSO. 350 pl per oligonucleotide are applied per spot (location of the probe placement on the DNA chip). The resulting spot forms a circular area with a diameter of 300 ⁇ m and contains about 10 fmol oligonucleotide for the analysis areas 3 and 1 fmol oligonucleotide for the control areas 4.
  • the orientation control is carried out in a concentration of 100 ⁇ M, the hybridization control in a concentration of 0. 8 ⁇ M to 80 ⁇ M and the PCR control applied in a concentration of 0.1 nM to 100 ⁇ M.
  • the oligonucleotides are applied complementarily to the target sequences in the concentration of 100 ⁇ M of the corresponding oligonucleotide and 0.5 ⁇ M of the hybridization control. Excess oligonucleotides are washed off the DNA chip by washing for 5 minutes followed by a short swivel) in 0.1% SDS at 60 ° C. Immediately afterwards, the DNA chip is immersed in 94 ° C. warm H 2 O for 5 minutes.
  • the DNA chip After removing any water drops with an air spray, the DNA chip is completely dried at room temperature. A five-minute immersion bath in 0.01 g NaBH 4 per 40 ml PBS and 20 ml ethanol completes the process of covalently binding the oligonucleotides to the chip and deactivates unused reactive groups. Before storage, the DNA chip is immersed again in warm H 2 0 for 10 seconds and dried with compressed air. Storage is protected from light at 4 ° C.
  • the oligonucleotides for the positive control for determining the orientation of the support 1 can, for example, at the points of intersection of the first column with the first and last
  • the oligonucleotides for positive controls for hybridization can, for example, be arranged in decreasing concentration in the first column at the intersection with the second row starting from the penultimate row.
  • the oligonucleotides for the positive control for determining the quality of the PCR can, for example, in increasing concentration on
  • the negative control can be arranged, for example, beginning at the intersection of the last row with the second column up to the last column.
  • the oligonucleotides complementary to the target sequence are arranged alternately in every second analysis area 3, for example.
  • the analysis areas 3 extend, for example, from
  • oligonucleotides of the same nucleotide sequence which are complementary to the target sequence, occur with up to three repetitions in different analysis areas 3.
  • Total DNA is isolated from clinical samples. This is done using commercially available kits (e.g. QIAGEN GmbH, Hilden, Germany) in accordance with the manufacturer's instructions.
  • the amplification of the target nucleic acid sequences can be carried out as described below as an implementation example. 1/10 to 1/1000 of the totally prepared DNA are amplified. For this, 2 ul 10-fold PCR buffer (100 mM Tris-HCl; pH 8.3; 500mM KC1;
  • the amplification takes place in a thermal cycler from Perkin Elmer (Gene Amp PCR System 9700). 0.2 ml MicroAmp ® reaction tubes are used.
  • the temperature steps required for the amplification proceed as follows. The first step is a 5 minute denaturation phase at 94 ° C, followed by 30 cycles with 40 seconds at 94 ° C, 60 seconds at 62 ° C and 40 seconds at 72 ° C. The cycles are followed by an extension phase of 5 minutes at 72 ° C.
  • An amplification product of approximately 300 base pairs is formed, depending on the sequence of the particular type of bacteria amplified.
  • the description of the amplification is not to be seen as restrictive, and alternative amplification conditions can of course also be selected.
  • the amplificate is used directly for hybridization or previously cleaned using commercially available kits (e.g. QIAGEN GmbH, Hilden, Germany) in accordance with the manufacturer's instructions.
  • the DNA chip for the hybridization is pretreated as follows. A pre-incubation takes place for 10 minutes at 80 ° C and saturated air humidity. During this time, 10 ⁇ l of the hybridization solution (1 ⁇ SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7) and 10 fmol / ⁇ l hybridization probe) are mixed with 10 ⁇ l PCR amplificate in a DNAse-free reaction vessel at room temperature. After the pre-incubation period, the hybridization mixture is pipetted into the hybridization area on the chip and immediately covered with a cover slip. Hybridization takes place during a 20 minute incubation at 50 ° C and saturated air humidity. detection
  • the cover slip is removed and the DNA chip is immediately immersed in a washing solution (1 ⁇ SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7). After briefly swirling, the incubation is carried out at 40 ° C. for 2 minutes Any liquid residues are removed from the chip surface 2 with compressed air.
  • the dry DNA chip can be stored protected from light at room temperature for a few days until evaluation with the reader.
  • the DNA chip is inserted into the scanner (Genepix 4000A from Axon Instrument) and measured by excitation of the Cy3 and Cy5 dye molecules with monochromatic light.

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Abstract

La présente invention concerne un dispositif destiné à l'analyse d'au moins un analyte issu d'un échantillon, ledit dispositif comprenant un support (1) doté d'une surface (2) sur laquelle se trouvent au moins une zone d'analyse (3) pouvant être prédéfinie et au moins une zone de régulation (4) pouvant être prédéfinie, la zone d'analyse (3) pouvant être prédéfinie contenant au moins un premier partenaire de liaison qui est immobilisé dans ladite zone et qui se lie spécifiquement à au moins un analyte. La/les zone(s) de régulation (4) contiennent au moins un autre partenaire de liaison destiné à au moins une régulation permettant la détermination de la qualité de l'analyse.
EP02794485A 2001-08-09 2002-08-08 Dispositif d'analyse d'acide nucleique Withdrawn EP1415003A2 (fr)

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AT0124701A AT411174B (de) 2001-08-09 2001-08-09 Verfahren und chip zur analyse von nukleinsäuren
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WO2003014382A8 (fr) 2004-04-08
AT411174B (de) 2003-10-27
WO2003014382A2 (fr) 2003-02-20
ATA12472001A (de) 2003-03-15
WO2003014382A3 (fr) 2003-10-23
AU2002332940A1 (en) 2003-02-24
WO2003014382B1 (fr) 2004-02-12

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