WO2016003197A1 - Méthode et appareil de détection d'acide nucléique spécifique d'une séquence à l'aide d'une exonucléase, et kit utilisé à cet effet - Google Patents

Méthode et appareil de détection d'acide nucléique spécifique d'une séquence à l'aide d'une exonucléase, et kit utilisé à cet effet Download PDF

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WO2016003197A1
WO2016003197A1 PCT/KR2015/006775 KR2015006775W WO2016003197A1 WO 2016003197 A1 WO2016003197 A1 WO 2016003197A1 KR 2015006775 W KR2015006775 W KR 2015006775W WO 2016003197 A1 WO2016003197 A1 WO 2016003197A1
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nucleic acid
sequence
detection
bound
specific nucleic
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Korean (ko)
Inventor
프리이 다베비벡
이기환
김수성
이준서
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U-Gene&cell Co
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U-Gene&cell Co
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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
    • C12Q1/6844—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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • 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

Definitions

  • NAME OF THE INVENTION A method and apparatus for detecting sequence-specific nucleic acids using nucleic acid terminal enzymes and kits used therein
  • the present invention relates to a method and apparatus for detecting sequence-specific nucleic acids using nucleic acid protease with enhanced sensitivity and specificity, and a kit used therein.
  • information about specific DNA sequences can be used to identify genetic or infectious diseases.
  • FIG. 17 is a diagram for explaining a method of detecting a conventional target nucleic acid.
  • the method of FIG. 17B is at the end of two strands of the nucleic acid sequence.
  • the single-stranded nucleic acid sequence is hybridized to a capture probe having a complementary sequence bound to a stationary phase in the presence of a substance that dissociates the nucleic acid such as sodium hydroxide.
  • the background (backgound) value is so large that the nautical ball that remains fixed
  • a method for detecting sequence-specific nucleic acids using nucleic acid terminal enzymes having improved sensitivity and specificity may be provided.
  • a sequence-specific nucleic acid detection device using nucleic acid protease can be provided.
  • Kits for sequence-specific nucleic acid detection using nucleic acid protease can be provided.
  • Terminal is in a state that cannot be degraded by nucleic acid terminalase, and the 5 'terminal (second terminal) of the other strand is in a state that can be degraded by nucleic acid terminalase-combining with nucleic acid terminalase;
  • a method for detecting sequence-specific nucleic acids using nucleic acid terminal enzymes comprising detecting a target nucleic acid sequence localized in the capture probe.
  • the nucleic acid protease is characterized by degrading phosphate-bonded strands in a double-stranded amplified product amplified by the pair of primers. Kits for sequence-specific nucleic acid detection using nucleic acid protease may be provided.
  • the pair of primers has a phosphate group attached to the 5 'end of one primer.
  • the 5 'end of the pair of primers has a hydroxyl group present
  • the nucleic acid protease is characterized by degrading phosphate-bonded strands in a double helix amplified product amplified by the pair of primers.
  • Kits for sequence-specific nucleic acid detection using nucleic acid protease can be provided.
  • the double-stranded target nucleic acid sequence has a 5 'end (first end) of one strand of the double strand that cannot be decomposed by nucleic acid protease, and 5 ⁇ of the other strand.
  • the terminal (second end) is in a state that can be degraded by nucleic acid terminal enzyme-and
  • a mixed portion in which nucleic acid protease is combined is combined
  • a hybridization unit which hybridizes the mixed mixture mixed by the mixing unit with the probe; Y -1
  • a sequence-specific nucleic acid sequence detection device using a nucleic acid terminal enzyme can be provided, including a detection unit for detecting a target nucleic acid sequence from the hybridized hybridization by the hybridization unit.
  • the sensitivity and specificity are very high in detecting the target nucleic acid in the sample.
  • FIG. 1 is a flowchart illustrating a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention.
  • FIG. 2 is a diagram illustrating a sequence-specific nucleic acid detection apparatus using nucleic acid terminal enzyme according to an embodiment of the present invention.
  • 3 is a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention, showing an embodiment using a primer in which nothing is bound (in the presence of a hydroxyl group). -.
  • FIG. 4 shows an example of using a primer to which a detection material is bound as a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention.
  • FIG. 5 shows an example of using a ligand-bound primer as a sequence-specific nucleic acid detection method using nucleic acid protease according to an embodiment of the present invention.
  • FIG. 6 is a view showing the results of an experimental example to which the embodiment of FIG. 3 is applied.
  • FIG. 7 is a drawing showing the results of an experimental example to which the embodiment of FIG. 4 is applied.
  • FIG. 8 is a diagram showing the results of an experimental example to which the embodiment of FIG. 5 is applied.
  • FIG. 8 is a diagram showing the results of an experimental example to which the embodiment of FIG. 5 is applied.
  • FIG. 9 is a diagram showing the results of an experimental example in which the embodiment of FIG. 3 is applied but a detection probe is used.
  • FIG. 9 is a diagram showing the results of an experimental example in which the embodiment of FIG. 3 is applied but a detection probe is used.
  • FIG. 10 is a diagram showing the results of another experimental example in which the embodiment of FIG. 4 is applied.
  • FIG. 11 is a diagram showing the results of an experimental example in which the embodiment of FIG. 3 is applied but a detection probe is used.
  • FIG. 11 is a diagram showing the results of an experimental example in which the embodiment of FIG. 3 is applied but a detection probe is used.
  • FIG. 12 is a diagram showing the results of another experimental example in which the embodiment of FIG. 5 is applied.
  • FIG. 13 is a diagram showing the results of an experimental example using the detection probe coupled with a ligand, applying the embodiment of FIG. 3.
  • FIG. 14 is a diagram showing the results of another experimental example in which the embodiment of FIG. 3 is applied but using a detection probe coupled with a ligand.
  • FIG. 14 is a diagram showing the results of another experimental example in which the embodiment of FIG. 3 is applied but using a detection probe coupled with a ligand.
  • FIG. 15 is a diagram showing the results of another experimental example in which the embodiment of FIG. 4 is applied.
  • FIG. 15 is a diagram showing the results of another experimental example in which the embodiment of FIG. 4 is applied.
  • FIG. 16 is a diagram showing an experimental example and a result using a microparticle probe while applying the embodiment of FIG. 3.
  • FIG. 17 is a diagram for explaining a method for detecting a conventional target nucleic acid.
  • Nucleic acid proteases cannot be degraded, for example, they can be substances such as detectors or ligands.
  • '5' terminal decomposable state may mean any of the following.
  • FIG. 1 is a flowchart illustrating a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention.
  • the sequence-specific nucleic acid detection method includes the step of amplifying a sample (S101), the output authentication product of step S101, that is, the 5 'end (first end) of one strand of a double-stranded target nucleic acid sequence-a double-stranded helix.
  • the 5 'terminus (second terminus) of the other strand is incapable of digestion by nucleic acid terminase and can be resolved by nucleic acid terminase.
  • step S101 Hybridizing the resultant (hereinafter referred to as a mixture) with the capture probe (S105), and detecting the target nucleic acid sequence hybridized with the capture probe from the hybrid of step S105 (S107). ' ; ⁇ ⁇
  • step S101 amplification of a sample, which is a target to be checked whether a target (target nucleic acid sequence) is included.
  • the amplification method is one of techniques for amplifying a known or future known nucleic acid. This can be used.
  • Examples of conventionally known amplification technology stones are polymerase chain reactions.
  • PCR Reverse Transcription-Polymerase Chain Reaction
  • RPA Recombinase Polymerase Amplification
  • the primers used for amplification of nucleic acids may for example be constructed as follows.
  • One of the pairs of primers used to amplify the sample may be in an unconnected state (i.e., in the presence of hydroxyl groups) at the 5 'end of either primer, and the pair of primers 5 'terminal is phosphate
  • FIG. 3 shows a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention using an primer (without hydroxyl group) to which nothing is bound.
  • One pair of primers used to amplify a sample may have a blocker attached to the 5 'end of one primer, and the other may have a phosphate group attached to the 5' end of the primer.
  • a blocker attached to the 5 'end of one primer
  • the other may have a phosphate group attached to the 5' end of the primer.
  • FIG. 4 shows an example using a primer bound to a detection material as a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention.
  • one of the pair of primers used when amplifying a sample is used. Fluorescence at the 5 'end of the primer or
  • the amplification is shown by way of example in which a detection material such as luminescence is bound and a phosphate group is used at the 5 'end of the other primer.
  • One pair of primers used to amplify a sample may have a blocker attached to the 5 'end of one primer, and a phosphate group bound to the 5' end of the other primer.
  • a phosphate group bound to the 5' end of the other primer For example, it can be a ligand.
  • FIG. 5 illustrates an example of using a ligand-bound primer as a sequence-specific nucleic acid detection method using nucleic acid terminal enzyme according to an embodiment of the present invention.
  • An example of the amplification is shown when a ligand is bound to the 5 ends of the primer and a phosphate group is bound to the 5 'end of the other primer.
  • the sample amplified in step S101 may be inferred to contain, for example, DNA and / or RNA of a disease mediated organism.
  • the disease agent may be applicable not only to living things but also to DNA and / or RNA such as viruses.
  • step S103 the product authentication bomb of step S101, that is, the target nucleic acid sequence of the double helix, is mixed with the exonuclease.
  • step S103 exonucleases bind phosphate groups in the amplification of step S101. Only the strands are selectively decomposed, so that only a single strand to which a blocker is bound or no substance is bound (i.e., a hydroxyl group is present).
  • the exonuclease is a lambda.
  • It may be a lambda exonuclease, but
  • Nucleic acid protease is an example, and it is not limited to the present invention. Those skilled in the art belong to the present invention.
  • Nucleic acid protease (exonuclease) coupled with the amplification product performs the operation.
  • step S105 the mixture resulting from the performance of step S103 is hybridized with a capture probe.
  • the capture probe is specific to the target to be found in the sample.
  • the capture probe may be fixedly coupled via UV cross-linking or drying, but this is illustrative, and the present invention is not limited to such fixedly coupled capture probes. Will be understood.
  • a target coupled with a capture probe is illustratively shown.
  • step S107 as a result of performing step S105, a target coupled to the capture probe is detected.
  • a technique that is widely known or known in the future is known as a technique for detecting a target coupled to the capture probe. Can be used.
  • the technique for detecting a target bound to the capture probe may be a technique for detecting a substance such as fluorescence or luminescence bound to the target, for example.
  • step S107 is captured by detecting the detection material bound to the target.
  • the amount of target bound to the probe will be known (see Figure 4).
  • step S107 identifies a detection probe that is specifically bound to the target.
  • the amount of target bound to the capture probe may be known (see Figure 3).
  • the detection probe may have a fluorescence or
  • Detection substances such as luminescence may be bound, or ligands may be bound.
  • the detection probe is bound to the detection probe.
  • the amount of fluorescence or luminescence is the amount of fluorescence or luminescence
  • the amount of target bound to the capture probe can be known.
  • step S105 By detecting the amount of detection material such as fluorescence or luminescence bound to the receptor, the amount of target bound to the capture probe can be determined. In this case, the performance of step S105 and Additional steps can be performed to mix ligand receptors in which substances such as fluorescence or luminescence are bound together.
  • step S107 is performed when a target is bound to a ligand
  • Targets can be detected using ligand receptors combined with materials such as fluorescence or luminescence.
  • materials such as fluorescence or luminescence.
  • FIG. 2 is a view for explaining a sequence-specific nucleic acid detection apparatus using nucleic acid terminal enzyme according to an embodiment of the present invention.
  • the sequence-specific nucleic acid detection apparatus 100 may include an amplifier 101, a mixing section 103, a hybridization section 105, and a detection section 107.
  • the amplifier 101 amplifies the sample.
  • the amplification unit 101 may perform an amplification operation using any one of conventionally known or future known nucleic acid amplification techniques.
  • known amplification techniques include polymerase chain reaction ( polymerase chain reaction (PCR) technology, reverse transcription-polymerase chain reaction (RT-PCR) technology, or recombinase polymerase amplification (RPA) technology.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription-polymerase chain reaction
  • RPA recombinase polymerase amplification
  • Primers for example, a primer according to the first embodiment, a primer according to the second embodiment, or a blue primer according to the third embodiment
  • Primers can be used.
  • the amplification part 101 includes software for maintaining an area where a sample is located and amplified (called a “sample location area”); and a condition under which a sample existing in the sample location area can be amplified.
  • sample location area software for maintaining an area where a sample is located and amplified
  • hardware unit as a generic term, will be used.
  • the mixing unit 103 mixes the amplified product (hybridized nucleic acid sequence) mass-nucleic acid protease that has been amplified by the amplifying unit 101.
  • the mixing unit 103 the amplified by the amplification unit 101 and
  • the region in which the mixture containing the nucleic acid protease is located (referred to as the 'mixture position region'), and in the mixture present in the mixture position region,
  • the hybridization unit 105, the nucleic acid terminal enzyme is a phosphate group is bonded in the mixing unit 103
  • the capture probes can become active. Keep it.
  • the capture probe includes a sequence that is specifically bound to the target to be found in the sample.
  • the capture probe may be coupled to a stationary phase, but this is merely illustrative. It will be readily apparent to those skilled in the art that this is not sufficient.
  • the hybridization unit 105 is mixed in the mixing unit 103.
  • the mixture (the mixture in which the phosphate-linked strand is decomposed by the nucleic acid terminalase) and the region in which the capture probe is located ('hybrid position region'); the capture probe at the mass-, hybrid position region
  • the term "software and hardware” that maintains the conditions for the hybridization reaction to be performed will be used as a generic term.
  • the detection unit 107 detects a target coupled to the capture probe. For example,
  • the detection unit 107 can directly or indirectly detect a target coupled to the capture probe in the hybridized by the shake unit 105.
  • the detection unit 107 is a fluorescence (fluorescence) bound to the target or
  • the detection unit 107 when the ligand is coupled to the target,
  • Targets can be detected using ligand receptors incorporating materials such as fluorescence or luminescence.
  • the detection unit 107 is used as a generic term for both software and hardware units that directly or indirectly detect a target coupled to the capture probe.
  • the person skilled in the art further includes a means for the apparatus 100 to move a sample located in the sample location area to the mixture location area, and a means for moving a mixture located in the mixture location area to the hybrid location area. It can also be implemented.
  • the device 100 includes an amplifier 101.
  • the mixing part 103 As described above, except for the amplification part 101, the mixing part 103, the shaking part 105, and
  • the amplification operation is performed by an amplification device configured separately from the device 100,
  • the mixing section 103, the shaking section 105, and the detecting section 107 will perform the operation.
  • the apparatus 100 has been described as including a detector 107, but without the detector 107, the amplifier 101, the mixer 103, and the hybridizer 105. It is also possible to implement to include. In this case the detection operation may be performed by a detection device configured separately from the device (100).
  • 3 to 5 show time series flows when the method or apparatus for detecting sequence-specific nucleic acid using a nucleic acid terminal enzyme according to an embodiment of the present invention is applied.
  • the present invention will be described in terms of time series with reference to these drawings.
  • a sequence-specific nucleic acid detection method or apparatus is applied to an amplified sample using the primer according to the first embodiment described with reference to FIG. 1.
  • time series flow is represented.
  • nucleic acid proteases e.g. lambda nucleic acid proteases
  • the capture probe maintains a condition that can be hybridized and mixed with the detection probe to which the detection material is bound
  • the target coupled to the capture probe is hybridized to the detection probe to which the detection material is bound as shown in the drawing on the right side of FIG. Thereafter, the detection material bound to the detection pro- bodied in the target can be detected.
  • FIG. 4 a sample amplified using the primer according to the second embodiment described with reference to FIG. 1 according to an embodiment of the present invention.
  • the time-series flow is shown when a sequence-specific nucleic acid detection method or apparatus using nuclear trimethylase is applied.
  • nucleic acid proteases e.g. lambda nucleic acid proteases
  • the target of the capture probe is hybridized as shown in the first right side of FIG. 4. After that, the detection coupled to the hybridized target is detected. The substance can be detected.
  • FIG. 5 a sample amplified using a primer according to the third embodiment described with reference to FIG. 1 according to an embodiment of the present invention.
  • Time-series flow in the case of applying a sequence-specific nucleic acid detection method or apparatus using nucleic acid terminal enzyme is shown.
  • nucleic acid proteases eg lambda nucleic acid proteases
  • the ligand receptor is retained in the target ligand bound to the capture probe, as shown in the first right side of FIG. 5, when the ligand is bound to-and the capture probe is hybridized and mixed with the ligand receptor. Thereafter, the detection substance bound to the ligand receptor can be detected.
  • a control base sequence prepared by cloning the pGMT vector was synthesized by TA cloning method.
  • the forward primer used was 5'-CCTTGTTTGGCCCTTCCT-3 ', with a phosphate group bound to the 5' end. Is
  • the length of the control base sequence synthesized by polymerase chain reaction (PCR) is 90 base pairs.
  • the base sequence is as follows.
  • HCV Hepatitis C virus
  • the containing HCV fragment base sequence was synthesized (Cosmogenetech, South Korea) and linked to the T7 promoter to create a target plasmid for HCV.
  • RT-PCR Reverse transcription-polymerase chain reaction
  • FIG. 6 shows a forward primer having a phosphate group bonded to the 5 ′ terminal.
  • the electrophoresis results for the amplification of RT-PCR were obtained using a reverse primer of 5'-GGGAGAGCCATAGTGGTCT-3 'and no substance at the end of 5 (ie, in the presence of hydroxyl groups).
  • the time series flow of the present experiment is additionally shown in the upper part of FIG. Those skilled in the art will be able to easily understand the operation principle of the present experiment by referring to the time series flow shown in FIG.
  • the capture probe was dissolved in 14xSSC solution at a concentration of 100 pmol / ul, line-lined on nitrocellulose membrane using DCI-300 (ZETA Corporation, South Korea), and at 0.12 J with UV cross linker (Hoefer, USA).
  • a membrane for lateral flow assay was prepared by cross linking for 10 minutes.
  • hybridization buffer In addition to single-stranded DNA-90 ul of hybridization buffer was used. When using biotin-coupled primers, 10 ul of single-stranded DNA was mixed with 90 ul of hybridization buffer and 2 ul of streptavidin conjugated dark beads, and when Alexa detection probe was used. 90 ul of hybridization buffer and Alexa detection probe were injected into membrane cartridge in 10 ul of single stranded DNA. Hybridization reaction was performed at 40 ° C for 20 minutes to confirm the fluorescence value with a probe.
  • FIG. 8 shows a forward primer having a phosphate group bonded to five ends thereof.
  • Sensitive and specific targets can be detected.
  • RPA reaction was conducted for 15 minutes during the 38 ° C term.
  • the 80 ° C term added 10 U of lambda exonuclease, and the 38 ° C term reacted for 5 minutes. Single stranded DNA was synthesized.
  • the HBV target sequence made with RPA was 218 base pairs.
  • the base sequence of the capture probe attached to the stationary phase to detect it was 218 base pairs.
  • FIG. 10 shows an example of HBV detection in the case of using a reverse primer having Cy5 bonded to the 5 'end.
  • the upper part of FIG. The flow is additionally shown,
  • the following shows an example of the HBV detection experiment when the reverse prime conditioner is used.
  • the time series flow of the experiment is further illustrated in the upper part of FIG. U. Referring to this time series flow, the hybridization of the probe probe with the Cy5 coupled probe is performed. The principle of detecting a target can be easily understood.
  • FIG. 12 shows an example of HBV detection in the case of using a reverse primer coupled with a ligand at the 5 'end.
  • the upper part of FIG. 12 also shows the time series flow of the experiment. Further illustrated and referring to these time series flows, the principle of detecting targets hybridized to the capture probe can be easily understood by detecting the detection material bound to the ligand receptor.
  • a target primer is prepared by using a T7 RNA polymerase to form a target RNA, and a forward primer having a phosphate group bound to the 5 'end thereof.
  • Reverse transcription-recombinase polymerase amplification was performed using 5'-TAGCCATTACCTGCTAAAGTCATTCTTCCCAAA-3 '.
  • a 15-boom RT-RPA reaction was performed at 38 ° C using a TwistAmp Basic kit from Mycoplasma pneumoniae target RNA, reverse transcriptase, RNase inhibitor and TwistAmp (UK). After 10 minutes and half of the reaction at 80 ° C, 10 U of lambda exonuclease was added and the reaction was performed at 38 ° C for 5 minutes to synthesize single-stranded DNA.
  • the target sequence of Mycoplasma pneumoniae made with RT-RPA was 182 base pairs.
  • the base sequence of the capture probe attached to the stationary phase to detect this is
  • 5'-TTTTTTTnTTTTTTTTACAATGAGCGAAAGCTTGA-3 'and 5'-GCGTGAACGATGAAGGTCTT-C6-NH 2 -3' was used as a detection probe.
  • This red microparticle probe was manufactured as follows.
  • Figure 15 is a "Mycoplasma pnetwio in the case of using the sum passed Cy5 at the terminal, the detection result 5 of the reverse primer, Figure 16 (state in which that is, a hydroxyl group is present), the state not bonded any substance to the 5 'end
  • the invention may be applied to other assays.
  • a sequence-specific nucleic acid detection kit using a nucleic acid terminal enzyme can be provided, and such a sequence-specific nucleic acid detection kit may be provided as described above. Can be used in detection methods or devices.
  • sequence-specific nucleic acid detection kit For example, the sequence-specific nucleic acid detection kit, the sequence-specific nucleic acid detection kit, the sequence-specific nucleic acid detection kit, and the sequence-specific nucleic acid detection kit.
  • the 5 ′ end of the pair of primers is bound to a phosphate group and the 5 ′ end of the other primer of the pair of primers is a block.
  • the blocker is bound, and the nucleic acid protease may be to decompose the phosphate bound strand in a double helix amplification amplified by the pair of primers.
  • the blocker may be a detection material or a ligand.
  • sequence-specific nucleic acid detection kit is
  • the 5 'end of one primer is bound to a phosphate group
  • the 5' end of the other primer of the pair of primers is free of other substances (i.e., in the presence of a hydroxyl group), and It may be the breakdown of phosphate-bonded strands in a double helix amplified by a pair of primers.
  • kits may also be configured to further include a capture probe coupled to a stationary phase, a control probe coupled to a stationary phase, and / or a detection probe.
  • the detection probe may be a combination of a detection material or a ligand.
  • kits may further include samples.

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Abstract

L'invention concerne une méthode de détection d'acide nucléique spécifique d'une séquence à l'aide d'une exonucléase, la méthode de détection d'acide nucléique spécifique d'une séquence à l'aide d'une exonucléase comprenant les étapes consistant à : mélanger l'exonucléase avec une séquence d'acide nucléique double brin cible, pour laquelle, parmi les deux brins, l'extrémité 5' (une première extrémité) de l'un des brins n'est pas susceptible à la dégradation par l'exonucléase, et l'extrémité 5' (une seconde extrémité) de l'autre brin est susceptible à la dégradation par l'exonucléase ; hybrider le produit résultant de l'étape de mélange (mélange ci-après) avec une sonde de capture ; et détecter la séquence d'acide nucléique cible hybridée avec la sonde de capture.
PCT/KR2015/006775 2014-07-01 2015-07-01 Méthode et appareil de détection d'acide nucléique spécifique d'une séquence à l'aide d'une exonucléase, et kit utilisé à cet effet Ceased WO2016003197A1 (fr)

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