WO2012070863A2 - Procédé de détection d'acides nucléiques à l'aide d'une enzyme à acide nucléique/marqueur moléculaire - Google Patents

Procédé de détection d'acides nucléiques à l'aide d'une enzyme à acide nucléique/marqueur moléculaire Download PDF

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WO2012070863A2
WO2012070863A2 PCT/KR2011/008968 KR2011008968W WO2012070863A2 WO 2012070863 A2 WO2012070863 A2 WO 2012070863A2 KR 2011008968 W KR2011008968 W KR 2011008968W WO 2012070863 A2 WO2012070863 A2 WO 2012070863A2
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nucleic acid
seq
target
molecule
enzyme
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WO2012070863A3 (fr
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박현규
롱장후
전경은
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한국과학기술원
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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • 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
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present invention relates to a nucleic acid detection method using a complex of a nucleic acid enzyme and a molecular beacon and a nucleic acid signal amplification method, and more specifically, a complex, a blocker nucleic acid and a nucleic acid polymerization of a molecular beacon which forms a hairpin structure with a nucleic acid enzyme having peroxidase activity.
  • the present invention relates to a nucleic acid detection method using an enzyme polymerization reaction.
  • a nucleic acid detection biosensor using a conventional molecular beacon type probe generates a fluorescent signal according to a polymerization product generated through a nucleic acid chain polymerization reaction specific to a target nucleic acid sequence, thereby inducing a structural change of the molecular beacon (Tyagi, S et al ., Nature biotechnology, 14: 303-308,1996).
  • phosphors and quenchers are modified at both ends of the molecular beacon probe, so when the hairpin structure is not fluorescence, the loop portion of the molecular beacon is complementary to the target nucleic acid. When combined with the hairpin structure, the fluorescent signal is displayed.
  • the target nucleic acid and the molecular beacon are quantitatively combined 1: 1 to generate a fluorescent signal. Therefore, accurate measurement is possible because one molecule of target nucleic acid generates one molecule of fluorescent material, but there is a limit in detecting a low concentration of target nucleic acid, and in this case, the molecular beacon must be modified with fluorescence. This costs
  • the molecular beacon does not bind to the substrate when maintaining the hairpin structure, but when the target nucleic acid is present, the hairpin structure of the molecular beacon is opened to bind to the substrate, and the substrate is cleaved by a nucleic acid enzyme having nuclease activity. It is a way. At this time, since both ends of the substrate are modified with phosphor and quencher, respectively, a fluorescent signal is emitted through decomposition reaction (Stojanovic, MN et al ., Chem BioChem., 2: 411-415,2001).
  • a biosensor using a peroxidase DNAzyme having a single strand of peroxidase activity may be used.
  • the nucleic acid enzyme In the absence of a target nucleic acid, the nucleic acid enzyme has a hairpin structure and exists in an inactive state.
  • the hairpin structure of the nucleic acid enzyme is opened and activated to give a color signal through peroxidase activity (Xiao, Y., et al ., J. Am. Chem. Soc., 126: 7430-7431 , 2004).
  • their nucleic acid detection sensitivity was not as high as satisfactory.
  • the present inventors have made intensive efforts to improve sensitivity in detecting nucleic acid.
  • the present inventors have used a complex probe consisting of a two-stranded nucleic acid enzyme-molecule beacon complex having a peroxidase activity and a blocker nucleic acid and a nucleic acid polymerase having a strand substitution activity.
  • a complex probe consisting of a two-stranded nucleic acid enzyme-molecule beacon complex having a peroxidase activity and a blocker nucleic acid and a nucleic acid polymerase having a strand substitution activity.
  • An object of the present invention is to provide a method for detecting a target nucleic acid with high sensitivity through a polymerization reaction of a nucleic acid polymerase having strand substitution activity of a probe consisting of a nucleic acid enzyme-molecule beacon complex and a blocker nucleic acid.
  • nucleic acid enzyme-molecule beacon complex i) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, complementary to a portion of the target nucleic acid at the other end, and contains a sequence complementary to the target nucleic acid substitution primer at either end Blocker nucleic acid; iii) target nucleic acid substitution primers; iv) hemin; v) nucleic acid polymerase; And vi) mixing the sample containing the target nucleic acid with the nucleic acid detection solution containing dNTP to separate the target nucleic acid from the blocker nucleic acid; And (b) adding a peroxidase substrate to a solution containing the nucleic acid enzyme-molecule beacon complex activated by the separated target nucleic acid to detect the target nucleic acid.
  • the present invention also has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, a sequence complementary to a portion of the target nucleic acid at the other end, and a sequence complementary to the target nucleic acid substitution primer at either end. It provides a blocker nucleic acid containing.
  • the present invention is also directed to a process for preparing i) a nucleic acid enzyme-molecular beacon complex; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, a sequence complementary to a portion of the target nucleic acid at the other end, and contains a sequence complementary to the target nucleic acid substitution primer at either end Blocker nucleic acid; iii) target nucleic acid substitution primers; iv) hemin; v) nucleic acid polymerase; And vi) a nucleic acid detection solution containing dNTP.
  • the invention also provides a composition
  • a composition comprising (a) i) a nucleic acid enzyme-molecular beacon complex; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence at the other end having a binding capacity with a target small molecule or protein, and at either end with a target small molecule or protein substitution primer Blocker nucleic acids containing complementary sequences; iii) target small molecule or protein substitution primers; iv) hemin; v) nucleic acid polymerase; And vi) mixing the sample containing the target small molecule or protein with the small molecule or protein detection solution containing dNTP to separate the target small molecule or protein from the blocker nucleic acid; And (b) detecting the target small molecule or protein by adding a peroxide enzyme to a solution containing the isolated nucleic acid enzyme-molecule beacon complex activated by the target small molecule or protein. to provide.
  • the present invention also has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, an aptamer sequence having a binding capacity with a target small molecule or protein at the other end, and a target small molecule or protein substitution at either end.
  • an aptamer sequence having a binding capacity with a target small molecule or protein at the other end and a target small molecule or protein substitution at either end.
  • blocker nucleic acids containing sequences complementary to primers.
  • the present invention is also directed to a process for preparing i) a nucleic acid enzyme-molecular beacon complex; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence at the other end having a binding capacity with a target small molecule or protein, and at either end with a target small molecule or protein substitution primer Blocker nucleic acids containing complementary sequences; iii) target small molecule or protein substitution primers; iv) hemin; v) nucleic acid polymerase; And vi) provides a small molecule or protein detection solution containing dNTP.
  • 1 is a diagram showing a principle of detecting target nucleic acid using a nucleic acid enzyme (DNAzyme) having a signal amplification principle and peroxidase activity by a target material.
  • DNAzyme nucleic acid enzyme
  • Figure 2 is a graph showing the absorbance intensity results according to the length of the primer when analyzing the target nucleic acid according to an embodiment of the present invention.
  • FIG 3 is a graph showing the change in absorbance intensity according to the presence or absence of addition of nucleic acid polymerase and deoxynucleotide (dNTP) and target nucleic acid concentration when analyzing target nucleic acid according to an embodiment of the present invention.
  • dNTP deoxynucleotide
  • Figure 4 is a graph showing the change in absorbance intensity according to the concentration of the target nucleic acid when analyzing the target nucleic acid according to an embodiment of the present invention.
  • 5 is a graph showing the change in absorbance intensity according to the type of target nucleic acid when analyzing target nucleic acid according to an embodiment of the present invention.
  • nucleic acid enzyme-molecule beacon complex i) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, complementary to a portion of the target nucleic acid at the other end, and contains a sequence complementary to the target nucleic acid substitution primer at either end Blocker nucleic acid; iii) target nucleic acid substitution primers; iv) hemin; v) nucleic acid polymerase; And vi) mixing the sample containing the target nucleic acid with the nucleic acid detection solution containing dNTP to separate the target nucleic acid from the blocker nucleic acid; And (b) adding a peroxidase substrate to a solution containing the isolated nucleic acid enzyme-molecule beacon complex activated by the target nucleic acid to detect the target nucleic acid.
  • the present invention has a sequence complementary to the loop portion of the nucleic acid enzyme-molecular beacon complex at one end, a sequence complementary to a portion of the target nucleic acid at the other end, and complementary to the target nucleic acid substitution primer at either end It relates to a blocker nucleic acid containing a sequence.
  • the present invention provides a kit comprising: i) a nucleic acid enzyme-molecular beacon complex; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, a sequence complementary to a portion of the target nucleic acid at the other end, and contains a sequence complementary to the target nucleic acid substitution primer at either end Blocker nucleic acid; iii) target nucleic acid substitution primers; iv) hemin; v) nucleic acid polymerase; And vi) a nucleic acid detection solution containing dNTP.
  • the present invention provides a composition
  • a composition comprising: (a) i) a nucleic acid enzyme-molecular beacon complex; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence at the other end having a binding capacity with a target small molecule or protein, and at either end with a target small molecule or protein substitution primer Blocker nucleic acids containing complementary sequences; iii) target small molecule or protein substitution primers; iv) hemin; v) nucleic acid polymerase; And vi) mixing the sample containing the target small molecule or protein with the small molecule or protein detection solution containing dNTP to separate the target small molecule or protein from the blocker nucleic acid; And (b) detecting the target small molecule or protein by adding a peroxidase substrate to a solution containing the isolated nucleic acid enzyme-molecule beacon complex activated by the target small molecule or protein. It is about.
  • the present invention has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, an aptamer sequence having a binding capacity with a target small molecule or protein at the other end, and at one end the target small molecule or A blocker nucleic acid containing a sequence complementary to a protein substitution primer.
  • the present invention provides a kit comprising: i) a nucleic acid enzyme-molecular beacon complex; ii) has a sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon complex at one end, and has an aptamer sequence at the other end having a binding capacity with a target small molecule or protein, and at either end with a target small molecule or protein substitution primer Blocker nucleic acids containing complementary sequences; iii) target small molecule or protein substitution primers; iv) hemin; v) nucleic acid polymerase; And vi) a small molecule or protein detection solution containing dNTP.
  • nucleotide sequence of the nucleic acid enzyme-molecule beacon complex may be selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 13.
  • the color development substrate is ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], TMB (3,3 ', 5,5'-tetramethyl benzidine) and OPD ( o-phenylenediamine) may be selected from the group consisting of.
  • Molecular Beacon is an oligonucleotide forming a hairpin secondary structure in which the 3 'end is tagged with a quencher material, and the molecular beacon probe is complementary to the template DNA in the annealing process. Specific hybridization in the area, and the distance between the fluorescent material and the quencher material is far from the suppression of the emission by the quencher material is fluorescence is emitted. On the other hand, unhybridized molecular beacons maintain secondary structure and are suppressed by the quencher and thus do not fluoresce.
  • the nucleic acid enzyme-molecular beacon complex in the present invention has a part of the target nucleic acid sequence in the loop portion, and both ends of the stem structure have a feature of forming a stem structure by itself only when hemin is present, and having hydrogen peroxide activity.
  • nuclease generally means a nucleic acid molecule having enzymatic activity.
  • aptamer refers to a polymer composed of nucleic acids (DNA, RNA, modified nucleic acid) that specifically binds to a target molecule, and artificially used as a diagnostic and medicine for the same concept as an existing antigen-antibody reaction. Mass production is possible.
  • the nucleic acid enzyme in the present invention has peroxidase activity, and the nuclease is inactivated by forming a complex with the molecular beacon and binding with the blocker nucleic acid. Thereafter, the blocker nucleic acid and the target nucleic acid bind to each other to activate the nucleic acid enzyme-molecule beacon complex, thereby displaying a color reaction through oxidation of ABTS.
  • hemin forms a system structure in both terminal portions of the complex of the nucleic acid enzyme-molecule beacon so that the nucleic acid enzyme-molecular beacon has peroxidase activity.
  • a of FIG. 1 shows a complex probe inactive by binding a nucleic acid enzyme-molecule beacon complex and a blocker nucleic acid.
  • the nuclease-molecule beacon complex has a portion of the target nucleic acid sequence in the loop portion and has a sequence capable of forming a two-stranded peroxidase-activated nucleic acid enzyme through the stem structure at both ends. Both terminal portions form a stem structure by themselves only in the presence of hemin and have peroxidase activity.
  • the blocker nucleic acid has a sequence complementary to the loop portion of the nuclease-molecule beacon, a sequence complementary to a portion of the target nucleic acid, and a sequence that is a binding site to the target nucleic acid substitution primer.
  • the nucleic acid enzyme-molecule beacon complex is designed to be inactivated by binding to a blocker nucleic acid, and the sequence that is the binding portion of the target nucleic acid is designed to be relatively longer than the sequence complementary to the loop portion of the nucleic acid enzyme-molecule beacon. By binding more strongly with the blocker nucleic acid, the nuclease-molecule beacon complex can be separated from the blocker nucleic acid and retain its original enzymatic activity.
  • step [B] of FIG. 1B ABTS (2,2'-azino-) by binding of a blocker nucleic acid and a target nucleic acid, activation of a nucleic acid enzyme-molecular beacon complex, and peroxidase activity of an activated nucleic acid enzyme-molecular beacon complex
  • the color reaction occurs through the oxidation of is- (3-ethylbenzothiazoline) -6-sulfonic acid), and in step [2], the target nucleic acid binds to the blocker nucleic acid, so that the primer is released as the binding site of the target nucleic acid substitution primer is revealed. It becomes possible to bind, and the DNA production
  • step [3] the complementary sequence of the blocker nucleic acid is synthesized according to the step [2] to form a nucleic acid pair complementary to the blocker, and the target nucleic acid is separated by the nucleic acid polymerase having strand substitution activity.
  • step 4 the isolated target nucleic acid is used for new signal generation to activate another probe of the nucleic acid enzyme-molecule beacon complex and the blocker nucleic acid.
  • the blocker nucleic acid of the present invention preferably comprises a sequence which binds to a target nucleic acid substitution primer to induce signal amplification through a polymerization reaction by a nucleic acid polymerase having strand substitution activity, and the target nucleic acid substitution primer sequence is a blocker nucleic acid.
  • the sequence of the nuclease-molecule beacon complex is also changed, and as a result, it affects not only the amplification efficiency but also the enzyme activity of the nuclease-molecule beacon complex.
  • the nucleic acid enzyme-molecular beacon complexes of SEQ ID NOs: 1, 4, 7, 10, and 13, the blocker nucleic acids of SEQ ID NOs: 2, 5, 8, 11, and 14, SEQ ID NOs: 3, 6, 9, 12, and A nucleic acid detection solution containing 15 target nucleic acid substitution primers, hemin, nucleic acid polymerase and dNTP, and a sample containing target nucleic acid were mixed to conduct a polymerization reaction.
  • the target nucleic acid attached to the blocker nucleic acid is separated as the sequence complementary to the blocker nucleic acid is separated, and the target nucleic acid separated from the blocker nucleic acid is separated from other inactive nucleic acid enzyme-molecular beacon complexes.
  • the absorbance of the absorbance was decreased as the base sequence length of the target nucleic acid substitution primer was reduced. It could be seen that the intensity increased.
  • the nucleic acid polymerase-molecule beacon and the blocker nucleic acid complex probe having the highest absorbance are used to include the nucleic acid polymerase and the dNTP, and the nucleic acid polymerase and the dNTP, respectively.
  • the change in intensity of absorbance was measured according to the concentration.
  • the absorbance intensity increased due to the increase of the nucleic acid enzyme-molecular beacon isolated from the inactive complex probe as the concentration of the target nucleic acid increased, but the signal by the reuse of the target nucleic acid when the nucleic acid polymerase and dNTP were included. Due to the amplification effect, the intensity of the absorbance was relatively high, indicating that the intensity of the signal varies depending on the presence or absence of the polymerization reaction.
  • the polymerization solution in order to determine the analysis limit according to the target nucleic acid concentration, is mixed with the assay buffer solution, and then the absorbance intensity is measured at the concentration of the target nucleic acid of 1pM ⁇ 10nM to perform a color analysis reaction. It was. As a result, it was confirmed that the absorbance increased linearly with the concentration of the target nucleic acid.
  • an experiment for detecting a nucleic acid in a sample containing a nucleic acid other than the target nucleic acid, a sample containing the target nucleic acid, a negative sample without the target nucleic acid was performed.
  • the final absorbance intensity is very low, similar to that caused by the hemin peroxidase activity of the background signal.
  • this detection method was confirmed to have a high specificity for the target nucleic acid.
  • the present invention uses a signal amplification method using a complex probe of a nucleic acid enzyme-molecule beacon complex / blocker nucleic acid and a polymerization reaction by a nucleic acid polymerase.
  • the target nucleic acid can also be detected, and since the strand substitution polymerization reaction used in the signal amplification is possible even in an isothermal state, there is an advantage that thermal cycling is not required.
  • the present invention can sensitively detect small molecules or proteins using a blocker nucleic acid comprising an aptamer sequence.
  • Aptamers are nucleic acid molecules that act like antibodies and can attach to small molecules or proteins in sequence. Therefore, since the blocker nucleic acid having an aptamer sequence attaches to a small molecule with a higher affinity than the nuclease-molecule beacon complex, it is separated from the complex probe of the nuclease-molecule beacon / blocker nucleic acid in the presence of the small molecule. Thus, nucleic acid enzymes become active and give off color signals.
  • a target nucleic acid substitution primer may be attached to a sequence complementary to a primer included in a blocker nucleic acid.
  • small molecules attached to the aptamer sequence of the blocker nucleic acid are released to activate other nucleic acid enzyme-molecule beacon complexes / blocker nucleic acid complexes.
  • the present invention can detect not only nucleic acids but also small molecules or proteins.
  • Example 1 Confirmation of signal amplification efficiency and nucleic acid enzyme-molecule beacon enzyme activity according to primer length
  • Nucleic acid enzymes SEQ ID NOs: 1, 4, 7, 10, and 13
  • blocker nucleic acids SEQ ID NOs: 2, 5, 8, 11, and 14
  • target nucleic acid substitution primers SEQ ID NOs: 3, 6, 9, 12, and 15
  • the nucleic acid detection solution was prepared using 1 ⁇ l of 25 ⁇ M hemin, 1.6 ⁇ l of Klenow fragment exo-, 2 ⁇ l of 0.2 mM dNTP, 8.4 ⁇ l water and 2 ⁇ l NEB (New England Biolabs) Buffer 2.
  • Ureaplasma urealyticum gene sequence (Gene bank No: AF085729.2), a genitourinary infection, was used as a target nucleic acid (SEQ ID NO: 16), mixed with the nucleic acid detection solution, and polymerized by polymerization at 37 ° C. for 2 hours. The resulting solution was obtained.
  • the resulting polymerization solution was mixed with analytical buffer solution (25 mM HEPES, 20 nM KCL, 200 mM NaCl, 0.05% Triton X-100, 1% DMSO), the mixed solution was stored at room temperature for 1 hour, and then ABTS / H
  • the 2 O 2 substrate solution was mixed with the mixed solution of the polymerization production solution and the analysis buffer solution, and the absorbance intensity at 415 nm was measured using a spectrophotometer after 10 minutes of reaction.
  • the absorbance increased as the length of the primer base sequence decreased from 12 (SEQ ID NO: 3) to 9 (SEQ ID NO: 12). In this case, it was confirmed that the intensity of absorbance decreased rather than the primer consisting of 9 bases (FIG. 2).
  • Example 2 Confirmation of the change according to the concentration of target nucleic acid and the presence or absence of nucleic acid polymerase and dNTP
  • the target nucleic acid concentrations of each of the target nucleic acid concentrations ( ⁇ ) and the non-nuclease (d) and dNTPs using the nucleic acid enzyme-molecular beacon / blocker nucleic acid complex showing the highest absorbance in Example 1 were The change in absorbance intensity with respect to 100 nM) was measured.
  • nucleic acid detection solution of Example 1 It was prepared by the composition (divided into the case containing and not containing nucleic acid polymerase and dNTP).
  • the nucleic acid detection solution was mixed with the target nucleic acid to meet the respective target nucleic acid concentration, and then polymerized at 37 ° C. for 2 hours to prepare a polymerization solution.
  • the obtained polymerization product solution and the assay buffer solution 25 mM HEPES, 20 nM KCL, 200 mM NaCl, 0.05% Triton X-100, 1% DMSO) were mixed and stored at room temperature for 1 hour, followed by color reaction analysis.
  • the absorbance intensity increased due to the increase in the nuclease-molecule beacon isolated from the inactive complex probe by the target nucleic acid as the concentration of the target nucleic acid increased (FIG. 3).
  • the absorbance of the target nucleic acid is relatively high. Therefore, from the above results, it was confirmed that the target nucleic acid was isolated from the blocker nucleic acid through the nucleic acid polymerization reaction by the nucleic acid polymerase having the strand substitution activity, and the signal was amplified by reuse for the new nucleic acid enzyme activity.
  • the nucleic acid detection solution of the same composition used in Example 1 and the target nucleic acid solution corresponding to the concentration of 1pM ⁇ 10nM were mixed, and then polymerized at 37 ° C for 2 hours.
  • the polymerization solution thus obtained was mixed with analytical buffer solution (25mM HEPES, 20nM KCL, 200mM NaCl, 0.05% Triton X-100, 1% DMSO) and stored at room temperature for 1 hour, and then at a target nucleic acid concentration of 1pM ⁇ 10nM.
  • Absorbance intensity was measured to carry out colorimetric analysis.
  • the absorbance was confirmed to increase linearly with the concentration of the target nucleic acid, in the present invention, the value of the analysis limit at 1pM, which is significantly lower than the previous nucleic acid detection method using other molecular beacons It could be confirmed that having.
  • a sample comprising a negative sample without target nucleic acid, a target nucleic acid (a portion of Ureaplasma urealyticum gene sequence (Gene bank No: AF085729.2)) (SEQ ID NO: 16) (100 nM), a target nucleic acid
  • a target nucleic acid a portion of Ureaplasma urealyticum gene sequence (Gene bank No: AF085729.2)
  • SEQ ID NO: 16 100 nM
  • the absorbance obtained as a result of analyzing a sample containing nucleic acid other than 100 nM was measured.
  • a portion of the Chlamydia trachomatis gene sequence (Gene bank No: X06707.3) (SEQ ID NO: 17) was used as the nucleic acid instead of the target nucleic acid.
  • the sample containing the target nucleic acid (Urea nucleic acid) and the sample containing the nucleic acid other than the target nucleic acid, the sample without the target nucleic acid in the nucleic acid detection solution was polymerized at 37 °C for 2 hours, and then analyzed It was mixed with the buffer solution and the absorbance at 415 nm was measured using a spectrophotometer.
  • the detection method of the present invention was confirmed to have a high specificity for the target nucleic acid (Fig. 5).
  • the nucleic acid detection method according to the present invention can detect the target nucleic acid with high sensitivity by allowing the target nucleic acid to be repeatedly reused in the signal generation process through the color reaction using the strand replacement activity of the nucleic acid polymerase. It may also be applied to the detection of proteins and small molecules.

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Abstract

La présente invention concerne un procédé de détection d'acides nucléiques à l'aide d'un composite marqueur moléculaire/enzyme à acide nucléique et d'un procédé d'amplification de signal d'acide nucléique, et plus particulièrement, un procédé de détection d'acides nucléiques cibles à l'aide d'un composite d'une enzyme à acide nucléique ayant une activité peroxydase et d'un marqueur moléculaire ayant une structure en épingle à cheveux, et la réaction d'acides nucléiques bloqueurs et d'une polymérase d'acide nucléique. Le procédé de détection d'acides nucléiques selon la présente invention permet à des acides nucléiques cibles d'être utilisés de façon répétée dans le processus de génération de signal par l'intermédiaire d'une réaction colorimétrique en utilisant l'activité de déplacement de brin de la polymérase d'acide nucléique, permettant ainsi la détection d'acides nucléiques cibles à sensibilité élevée, et par conséquent, le procédé de la présente invention peut être appliqué à la détection de protéines et de petites molécules, ainsi qu'à la détection d'acides nucléiques.
PCT/KR2011/008968 2010-11-23 2011-11-23 Procédé de détection d'acides nucléiques à l'aide d'une enzyme à acide nucléique/marqueur moléculaire WO2012070863A2 (fr)

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