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  • 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
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/6823—Release of bound markers
• • C—CHEMISTRY; METALLURGY
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  • 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
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
• • 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
  • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

• the detection of nucleic acid hybridization has the advantage that, for. B. the infectious agent can be detected directly after infection and very sensitive.
• nucleic acid detection methods When detecting the presence or absence of the body's own nucleic acid within certain genomic loci and or their changes, such as. B. inherited, spontaneous or a mixture of inherited and spontaneous mutations, deletions, inversions, translocations, rearrangements or triplet expansions in the form of specific and / or polymorphic changes, the availability of specific and sensitive nucleic acid detection methods is also advantageous.
• Quantitative sensitive and specific determinations of the nucleic acids to be detected have so far been possible in the context of heterogeneous or homogeneous target-specific exponential nucleic acid amplification reaction formats in which the Nucleic acid amplification product is intercepted either by built-in labels or by hybridization with a probe which is specific for the nucleic acid to be detected or its complement in part of the sequence section formed by elongation.
• Exponential nucleic acid amplification reaction formats in which nucleic acid-binding dyes are intercalated are also sensitive, but are not sequence-specific.
• Shorter amplicon lengths have so far only been used to detect special sequences such as e.g. B. in triplet expansions, for in-situ studies or the detection of highly fragmented nucleic acids in the context of antiquity research.
• these short amplicon lengths were detected in more time-consuming gel formats or in-situ formats, which are characterized by a lack of sensitivity and / or a lack of quantification.
• Other special short sequences such as short tandem repeats, short interspersed repetitive elements microsatellite sequences or HLA-specific sequences have so far only been used as primer or probe binding sequences.
• a further object of the invention was to make the selection of the primer and probe sequences so flexible while maintaining the overall specificity that a determination of several different nucleic acids to be detected is possible in a unified reaction format using partially identical primer or probe sequences.
• the amplificates can have one or more further regions Y which lie outside the region which contains the sequence information derived from the nucleic acid to be detected.
• Such solid supports are e.g. B. solid supports with glass-containing surfaces (e.g. magnetic particles, glass fleece with glass-containing surfaces, particles, microtiter plates, reaction vessels, dip sticks or miniaturized reaction chambers, which in turn can also be part of integrated reaction chips).
• This solid support is preferably used for non-sequence-specific purification, i.e. no substantial isolation of the nucleic acids to be detected from other nucleic acids, but only a sample material (nucleic acid) concentration and possibly inactivation and or elimination of inhibitors for the subsequent nucleic acid amplification and detection reactions.
• These solid supports also make it possible to provide several nucleic acids to be detected, e.g. B. in the context of multiplexing, in a form accessible for nucleic acid amplification and detection reactions possible.
• Such propagation methods are used which permit an amplification of the nucleic acid sequence to be detected or its complement, which result in the formation of tripartite mini-nucleic acid amplification products.
• All nucleic acid amplification methods known in the prior art are available for this.
• Target-specific nucleic acid amplification reactions are preferably used.
• exponential target-specific nucleic acid amplification reactions are particularly preferably used, in which an antiparallel replication of the nucleic acid to be detected or its complement takes place, such as, for. B. elongation-based reactions such.
• B. the polymerase chain reaction PCR for deoxyribonucleic acids, RT-PCR for
• Ribonucleic acids or transcription-based reactions
• B. Nucleic Acid Sequence Based Amplification (NASBA) or Transcription Mediated Amplification (TMA).
• Thermocyclic exponential elongation-based nucleic acid amplification reactions such as, for. B. uses the polymerase chain reaction. The evidence to be used for propagation
• a primer in the sense of the present invention is understood to mean a molecule which can bind to a nucleic acid via base pairings and which can be extended, preferably enzymatically.
• Ohgonucleotides are preferred which can be extended at their 3 'end using the nucleic acid to be detected or a complement thereof as template nucleic acid.
• Monovalent or multivalent or monofunctional or multifunctional agents which permit nucleic acid-dependent elongation can be used as primers.
• Oligomers or polymers with a binding length of between 9 and 30 nt can preferably be used as primers, which bind antiparallel to the nucleic acid to be detected and which act as one of several reaction partners for an enzymatic replication of the nucleic acid to be detected or its complement.
• oligomers which, after addition of a multiplication reagent by addition of at least part of the primer to the nucleic acid to be detected or its complement, are directed replication of one or both strands of the nucleic acid to be detected or their complement initiate.
• An example of a particularly preferred primer is an oligonucleotide with a free 3 'hydroxyl end.
• nucleic acid sequence additions and / or other modifications such as.
• Preferred nucleotide equivalents are PNA monomers or PNA oligomers (WO92 / 20702) with or without positive and / or negative charges in the backbone and / or in the spacer.
• PNA oligomer probes with or without positive or negative charges in the backbone and / or spacers have the additional advantages that they are stable against the degradation of nucleases or proteases because of the different structure of the backbone and the H or NH 2 ends , have a higher melting point in binding complexes between nucleic acids and PNA than between two nucleic acid molecules and the hybrid complex is therefore more stable, can be used at low salt concentrations, has a higher difference in melting points in the case of mismatches, and thus better mismatch discrimination is possible, Sequences with secondary structures at low salt concentrations are more accessible, the competition between amplicon counter strand and probe is lower at low salt concentrations and thereby a higher signal yield is achieved and the potential for eliminating the amplicon denaturation step at low salt concentrations consists.
• a binding sequence is preferably understood to mean the sequence of bases that are between the outermost ones with a certain nucleic acid.
• a primer or a probe is based on base-base interaction binding bases of a particular nucleic acid, a primer or a probe, including these outermost bases.
• Regions A and C are, according to the invention, preferably so long that conditions can be found under which primers of a corresponding length can hybridize with the bases in these regions.
• the regions are therefore preferably longer than 8, particularly preferably longer than 12 nucleotides.
• preferred ranges also result with regard to the upper limit of the length of the regions A and C.
• the regions A and C are each preferably less than 30, particularly preferably less than 20 nucleotides.
• the length of the regions is limited by the fact that the primers should be able to hybridize to them in a manner that is unspecific for the nucleic acid to be detected. Therefore, the particularly preferred length of the binding sequences A and C is 12 to 20 nucleotides.
• the areas A and C on the nucleic acid to be detected do not overlap.
• Preferred nucleotides are dATP, dGTP, dCTP, dTTP and or dUTP, dITP, iso-dGTP, iso-dCTP, deaza-dGTP and ATP, GTP, CTP, UTP and / or ITP, deazaGTP, iso-GTP, iso-CTP.
• Equivalents are PNA monomers or PNA oligomers with or without positive and / or negative charge in the backbone and / or in the spacer.
• the elongation substrates can carry modifications as stated above.
• thermocyclic multiplication reactions e.g. PCR, RT-PCR
• 2- or 3-phase cycles are carried out, preferably 2-phase cycles.
• the strand separation of the nucleic acid amplification products is carried out at high temperature, preferably 85 ° C.-95 ° C., the common primer annealing and primer elongation at temperatures close to the melting point between primer and elongation counter strand, preferably between 55 ° C and 75 ° C.
• the strand separation is carried out by supplying energy and / or enzymatically, preferably by means of elevated temperature, microwaves or applying a voltage via a microelectrode, particularly preferably by means of elevated temperature.
• Up to 60 thermal cycles are carried out, preferably 32-42.
• Enzyme activity uracil deglycosylase preferably with a thermolabile embodiment of the enzyme activity in which the renaturation takes place more slowly after thermal denaturation of the enzyme activity, the fragmentation of the amplification product and thus its property as a nucleic acid amplification unit.
• the UMP-containing amplification product can be incubated following the nucleic acid amplification and detection reaction (sterilization) and / or before a renewed nucleic acid amplification reaction (carry over prevention).
• Direct spectroscopic or physical methods are e.g. B. melting temperature determinations, attachment of intercalating or nucleic acid-binding dyes or metal atoms or particles, mass spectroscopy, surface plasmon resonance or fluorescence-coupled surface plasmon resonance, or E-wave measurements.
• the probe When using the probe as a capture probe, the probe can either be covalently attached to the solid support or via a binding pair and the formation of the
• the increased tripartite mini amplicons are bound by nucleic acid capture probes or PNA capture probes, which are covalently immobilized on micro titer plates or magnetic particles.
• detection takes place after formation of the binding complex and washing via a biotin modification of one or both primers in the amplificate by addition of avidin-horseradish peroxidase and a mixture of TMB / TMF color substrates.
• one or more amplificates are detected after binding by one or more different covalently (for example anthraquinone: UV light coupling or gold surface: SH coupling) or coordinatively (for example Biotin: streptavidin) -bound capture probes, by washing and by detection of a fluorescence or chemiluminescence signal, which was excited either directly by primary light or via surface plasmon resonance or E-wave, with the aid of CCD cameras or confocal fluorescence scanners.
• covalently for example anthraquinone: UV light coupling or gold surface: SH coupling
• coordinatively for example Biotin: streptavidin
• ruthenium chelate-containing detection probes are bound to the amplificates which contain biotin modifications via one or both primers.
• the detection probes are either ruthenium-labeled oligonucleotides or ruthenium-labeled PNA oligomers.
• the complex is bound by a capture probe which is covalently immobilized on a microtiter plate or on magnetic particles.
• detection takes place after formation of the binding complex and washing via a biotin modification of one or both primers in the tripartite mini amplicon by addition of avidin-horseradish peroxidase and a mixture of TMB / TMF color substrates.
• detection probes When using homogeneous reaction formats, detection probes are used that contain either quenched fluorescent labels, internal base substitutions with double-strand complex-activatable fluorescent dyes or terminal energy donors or acceptors (in combination with corresponding energy donors or acceptors on adjacent primer ends: energy transfer Complexes). In these cases, the detection probe is added during the nucleic acid amplification. In the case of the quenched Fluorescence labels are activated by dequenching after binding the detection probe to the resulting tripartite mini-amplicon and exonucleolytic degradation and release of the fluorescent dye-modified nucleotide. In the case of internal base substitutions, the fluorescence signal is generated by forming the binding complex between the detection probe and the tripartite mini-amplicon that forms. In the case of the energy transfer complexes, a fluorescence signal is formed by adjacent attachment of the labeled primer and the labeled probe. The measurement of the resulting fluorescence signals is preferably carried out by real-time measurements.
• fluorescein and rhodamine or derivatives thereof are used as fluorescence and quencher components in the quenched detector probes.
• ruthenium or rhenium chelates and quinones or derivatives thereof are used as electrochemiluminescent and quencher components in the quenched detector probes.
• anthraquinone or derivatives thereof are used as internal base substituents of the detector probe.
• Cy-5 and fluorescein or derivatives thereof are used as energy transfer components.
• cyanine dyes such. B. SYBR Green or acridine dyes used.
• the invention also relates to a method for the specific detection of a nucleic acid comprising the steps of producing a multiplicity of amplificates of a section of this nucleic acid with the aid of at least two primers, bringing the amplificates into contact with a probe which can bind to the amplificate and detection of the formation of a hybrid from the strand of the amplificate and the probe, characterized in that at least one of the primers is not specific for the nucleic acid to be detected.
• region B can contain nucleotides which do not belong to the binding sequence E.
• the binding sequences of the primers and the probe can overlap.
• the primers at their 5 'end contain further sequences which connect to the primer sequences in the human genome. These sequences are between 1 and 100, particularly preferably between 5 and 80 nucleotides long. It is possible to modify one or both of the primers accordingly. The additional sequences are not so long that they hybridize the primers with the binding sequences on the nucleic acid to be detected, e.g. B. prevent the HCV genome.
• the primers bind to the binding sequences A or C, as described above, and the probe to a region B between the ends of the binding sequences A and C or the complement thereof.
• the overall specificity of the detection method is retained. If one of the primer sequences is not specific for the nucleic acid to be detected, it also binds to others Nucleic acids, no specific nucleic acid amplification product can be formed on the other nucleic acid because the second primer binding sequence on this other nucleic acid is missing. Unspecific nucleic acid amplification products on the other nucleic acid are not detected if the specific binding sequence for the probe is missing.
• Nucleic acid amplification products of the other nucleic acid that may be formed contain other sequences in the probe binding region and are therefore not detected. All three binding sequences for the two primers and the probe are not specific for the nucleic acid to be detected. no nucleic acid amplification product is formed if at least one of the two primer sequences is not in a nucleic acid amplification unit of the other nucleic acid. If the probe sequence is not in the nucleic acid multiplication unit of the two primer sequences for the other nucleic acid, a specific nucleic acid multiplication product of the other nucleic acid can be formed, but it cannot be detected.
• partial components (primers or probes) of the different primer-probe combinations can be identical for the different nucleic acids to be detected.
• the determination of several nucleic acid targets eg. B. possible for different viruses such as HBV, HTV and HCV with a single amplification reaction (multiplex).
• a technical advantage of the method according to the invention is that a high degree of agreement of the measured values is achieved with multiple determinations of a sample.
• HCV RNA from the 5 'untranslated region of the HC V genome in a copy number of 10 copies per test with a dynamic range of 105 due to an improved signal-to-noise ratio.
• primers and probes can be used in the test which have a primer / probe design which is not preferred for the person skilled in the art, namely e.g. B. sequence sections that tend to form primers-dimers, or base mismatches near the 3 ' end.
• the short probe has a melting point close to the test temperature, so that the person skilled in the art would not have expected stable binding of the probe to the nucleic acid amplification product.

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