JP2007330134A - Method for detecting nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method by which the sequence of the nucleic acid can clearly be detected in good repeatability, and to provide a reagent therefor. <P>SOLUTION: This method for detecting the nucleic acid comprises denaturing a double strand nucleic acid into a single strand nucleic acid, hybridizing a portion of the single strand nucleic acid with at least one complementary oligonucleotide probe (D) for detection, elongating the single strand nucleic acid as a template to form the complex (P) of the single strand nucleic acid with the elongation product of the oligonucleotide probe (D), and detecting the presence of the complex (P). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for detecting a nucleic acid using an oligonucleotide probe in a form in which a nucleic acid elongation reaction can occur. The present invention is particularly useful for diagnosis of genetic diseases, identification of causative substances in infectious diseases, and the like.

  Technologies for detecting specific nucleic acid sequences present in biological samples include analysis at the nucleic acid molecule level of proteins expressed and functioning in specific organs, research on protein expression control in information transmission in the nerve, brain, or immune system, etc. It is extremely important in genetic diagnosis such as detection of mutant genes for genetic diseases, diagnosis of cancer, detection of virus-related genes.

  However, the conventional technique is complicated and includes many steps, and requires skill, so that a considerable amount of time is often required depending on the operation.

  In addition, since genetic diagnosis is used as a definitive diagnosis, errors are not allowed and rapidity is also required, so it must have high accuracy while maintaining rapidity. It's not enough.

  As a typical technique for detecting a specific nucleic acid molecule present in a sample, there is a hybridization method (see, for example, Non-Patent Document 1). Nucleic acid hybridization analysis is a technique for detecting a very small number of target nucleic acids (DNA and RNA) from various types of nucleic acids using probes. In hybridization methods, highly sensitive reporters (enzymes, fluorescent dyes, Even if a radioisotope or the like is used, it is difficult to detect a target nucleic acid molecule having a low copy number (1-1000). Furthermore, since the hybridization method has a big problem of non-specific reaction, operations such as carrier blocking and removal of non-specific signal are indispensable for accurately detecting a specific nucleic acid molecule. Effort is required.

In addition, as a method for solving the problem of non-specific reaction in the past, a competitive hybridization method (for example, for suppressing non-specific reaction by adding similarly prepared non-labeled DNA to labeled DNA prepared from target nucleic acid) Patent Document 1 and Non-Patent Document 2) have been developed. However, the competitive hybridization method is cumbersome to operate and is not sufficient in terms of detection sensitivity, so it is particularly applicable to genetic diagnosis such as detection of mutant genes in genetic diseases, diagnosis of cancer, detection of virus-related genes, etc. Can not.
Japanese Patent No. 2982304 Sambrook et al., Molecular Cloning, A Laboratory Manual, Third Edition, Chapter 10, 10.1-10.52 (2001) Nicolas et al., Anal. Biochem. 205, 193, (1992)

  An object of the present invention is to provide a method and a reagent therefor that can solve the above-described problems and can detect a nucleic acid sequence in a sample clearly and reproducibly.

  In view of the above circumstances, the present inventors have completed the present invention as a result of intensive studies.

That is, the present invention has the following configuration.
1. A method for detecting a target nucleic acid sequence in a double-stranded nucleic acid, comprising at least the following steps (1) to (3):
(1) a first step of denaturing the double-stranded nucleic acid to form a single-stranded nucleic acid;
(2) The single-stranded nucleic acid obtained in the first step is hybridized with at least one kind of detection oligonucleotide probe (D) complementary to a part of the single-stranded nucleic acid, and the single-stranded nucleic acid A second step of forming a complex (P) of the extension product of the oligonucleotide probe (D) and the single-stranded nucleic acid by performing an extension reaction using as a template,
(3) A third step of detecting the presence of the complex (P).
2. The method for detecting a target nucleic acid sequence according to claim 1, wherein the oligonucleotide probe (D) is labeled with a ligand.
3. The method for detecting a target nucleic acid sequence according to 1 or 2, wherein at least one nucleotide from the 3 'end of the oligonucleotide probe (D) is labeled with a ligand.
4). 5. The method for detecting a target nucleic acid sequence according to any one of 1 to 3, wherein the 5 ′ end of the oligonucleotide probe (D) is labeled with a ligand.
5). 4. The method for detecting a target nucleic acid sequence according to any one of 2 to 4, wherein the ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.
6). The target nucleic acid sequence detection method according to any one of 1 to 5, wherein the third step includes at least the following steps (i) and (ii):
(I) capturing the complex (P) on the solid phase using the ligand of the oligonucleotide probe (D);
(Ii) A step of detecting the complex (P) captured on the solid phase.
7). 6. The method for detecting a target nucleic acid sequence according to 6, wherein the step (i) includes a step of removing a single-stranded nucleic acid not hybridized with the oligonucleotide probe (D).
8). The method for detecting a target nucleic acid sequence according to any one of 1 to 7, wherein an oligonucleotide probe (D) is present in the first step.
9. The method for detecting a target nucleic acid sequence according to any one of 1 to 8, wherein the double-stranded nucleic acid is a double-stranded nucleic acid obtained by an amplification reaction.
10. 9. The method for detecting a target nucleic acid sequence according to 9, wherein the amplification reaction is PCR.
11. 9. The method for detecting a target nucleic acid sequence according to 9 or 10, wherein an oligonucleotide probe (D) is present in the amplification reaction.
12 The method for detecting a target nucleic acid sequence according to any one of 9 to 11, wherein the Tm value of the oligonucleotide probe (D) is lower than the Tm value of the primer used in the amplification reaction.
13. The method for detecting a target nucleic acid sequence according to any one of 1 to 12, wherein the denaturation in the first step is heat denaturation.

  The present invention provides a method capable of clearly and easily detecting a nucleic acid sequence in a sample nucleic acid. Since the method of the present invention does not require a complicated operation as in the conventional methods, results with good reproducibility can be obtained quickly and easily.

Hereinafter, the present invention will be described in detail.
In the present invention, the “double-stranded nucleic acid” refers to a nucleic acid having a sequence complementary to a target nucleic acid sequence, that is, a chromosome containing a nucleic acid sequence to be analyzed or a fragment thereof, a sequence complementary to an amplified nucleic acid or a fragment thereof. The nucleic acid etc. which have.

A nucleic acid having a sequence complementary to the chromosome or a fragment thereof can be prepared from any material such as bacteria, animal or plant tissue, and lysates derived from individual cells. A method for preparing a nucleic acid having a sequence complementary to the chromosome or a fragment thereof is not particularly limited. For example, it may be prepared from a patient's blood or tissue by a known method. Typical examples include phenol / chloroform extraction method (Biochimica et Biophysica acta 72, 619-629, 1963), alkaline SDS method (Nucleic Acid Research 7, 1513-1523, 1979). There is a method of performing in the liquid phase. Further, as a system using a nucleic acid binding carrier for nucleic acid isolation, a method using glass particles and a sodium iodide solution (Proc. Natl. Acad. USA, Vol. 76-2, 615-619, 1979). ) And a method using hydroxyapatite (Japanese Patent Laid-Open No. 63-263093). Examples of other methods include a method using silica particles and chaotropic ions (J. Clinical Microbiology Vol. 28-3, 495-503, 1990, JP-A-2-289596).

The nucleic acid having a sequence complementary to the amplified nucleic acid or a fragment thereof may be prepared, for example, by a known amplification method. As typical nucleic acid amplification methods, PCR (Molecular Cloning, A Laboratory Manual, Third Edition (Sambrook et al., Chapter 8, 8.1-8.126, 2001)), NASBA (Nucleic acid sequence-based amplification) method; Nature 350, 91 (1991)), LCR (International Publication No. 89/12696, JP-A-2-2934), SDA (Strand Displacement Amplification: Nucleic acid research 20, 2091) (1992)), RCA (International Publication No. 90/1069), TMA (Transcription mediated amplification method; J. Clin. Microbiol. Vol. 31, p. 3270 (1993)), LAMP (loop-mediated isothermal) amplification method: J Clin Microbiol. Volume 42: 1,956 (2004)), ICAN (isothermal and chimeric primer-initiated amplification of nucleic acids: Kekkaku 78, 533 (2003)).

  In particular, the PCR method repeats a cycle consisting of three steps of denaturation, annealing, and extension in the presence of a sample nucleic acid, four types of deoxynucleoside triphosphates, a pair of oligonucleotide primers, and a heat-resistant DNA polymerase. This is a method of exponentially amplifying a sample nucleic acid region sandwiched between a pair of oligonucleotide primers. That is, the nucleic acid of the sample is denatured in the denaturation step, each oligonucleotide primer is hybridized with a region on the single-stranded sample nucleic acid complementary to each in the subsequent annealing step, and each oligonucleotide primer is then synthesized in the subsequent extension step. The DNA strand complementary to each single-stranded sample nucleic acid serving as a template is extended by the action of DNA polymerase from the starting point to form double-stranded DNA. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of oligonucleotide primers is theoretically amplified to 2 n times. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis. Therefore, if a gene amplification method is used, it is possible to detect even a very small amount of sample nucleic acid that could not be detected in the past (one molecule is acceptable), which is a very widely used technique recently. .

  In the present invention, the “target nucleic acid sequence” refers to a nucleic acid sequence including a target nucleic acid, that is, a nucleic acid sequence to be analyzed. Examples of the target nucleic acid include an Alu sequence, a ribosomal gene, an exon, intron, and promoter of a gene encoding a protein. More specifically, genes related to various diseases including genetic diseases, drug metabolism, lifestyle-related diseases (hypertension, diabetes, etc.) can be mentioned. For example, the CYP2C19 (Cytochrome P450 2C19) gene can be mentioned as a gene related to drug metabolism.

  In the present invention, the “primer” used in the amplification reaction is an oligonucleotide having a sequence complementary to a chromosome containing a target nucleic acid sequence or a fragment thereof, and is a known amplification method such as PCR, NASBA, There is no particular limitation as long as it can be used for the LCR, SDA, RCA, TMA, LAMP, ICAN and UCAN methods. The primer chain length may be 9 to 35 bases, and preferably 11 to 30 bases. A plurality of the primers may be mixed depending on the nucleic acid sequence to be detected. For example, in order to simultaneously amplify a plurality of types of nucleic acids from a chromosome containing a target nucleic acid sequence or a fragment thereof, primers corresponding to the respective nucleic acid sequences may be mixed and used.

  “At least one type of detection oligonucleotide probe (D) complementary to a part of a single-stranded nucleic acid” obtained by denaturing the double-stranded nucleic acid used in the present invention is a target in a double-stranded nucleic acid. Oligonucleotides complementary to a portion of the nucleic acid sequence can be used. More preferably, a ligand bound to an oligonucleotide complementary to a part of the target nucleic acid sequence in the double-stranded nucleic acid can be used.

  In the present invention, “denaturation” of a double-stranded nucleic acid was formed in the double-stranded nucleic acid in the double-stranded nucleic acid at least in a region capable of hybridizing with the oligonucleotide probe for detection (D). This means that the base pair has been eliminated and it has become a single strand. As a method for denaturing the double-stranded nucleic acid, a conventionally known method such as a method by heating (thermal denaturation) or a method of adding formamide can be used, but thermal denaturation is preferable from the viewpoint of simplicity.

  One of the important disclosures of the present invention is that a single-stranded nucleic acid obtained by denaturing a double-stranded nucleic acid has at least one type of oligonucleotide probe for detection complementary to a part of the single-stranded nucleic acid ( When D) is hybridized, an extension reaction is performed using the single-stranded nucleic acid as a template to form a complex (P) of the extension product of the oligonucleotide probe (D) and the single-stranded nucleic acid. The production amount of the complex (P) was greatly improved as compared with a conventionally known hybridization method without the extension reaction, and a remarkable effect was found that the target nucleic acid could be clearly and easily detected. There is.

  Also, as a part of the target nucleic acid sequence in the double-stranded nucleic acid as at least one kind of detection oligonucleotide probe (D) complementary to a part of the single-stranded nucleic acid that can be obtained by denaturing the double-stranded nucleic acid When a ligand is bound to an oligonucleotide complementary to the ligand, it is preferable to bind the ligand at a position that does not inhibit the extension reaction. As the position for binding the ligand, it is preferable to bind the ligand to at least one nucleotide after the 3 ′ end of the oligonucleotide probe (D), and to the 5 ′ end of the oligonucleotide probe (D). More preferably, the ligand is bound.

  In the amplification reaction, an oligonucleotide probe (D) may be present. In that case, the oligonucleotide probe (D) is preferably designed so as not to inhibit the amplification reaction using the primer used in the amplification reaction. Furthermore, in order to suppress nonspecific reaction, it is preferable to design the oligonucleotide probe (D) so that the nucleic acid extended from the oligonucleotide probe (D) is not used for the amplification reaction. For example, the oligonucleotide probe (D) may be designed to have a Tm value lower than that of the primer used in the amplification reaction.

  The Tm value of the oligonucleotide primer is a temperature at which 50% of the double-stranded DNA molecules forming the hybrid are separated from each other, and the calculation method is not particularly limited as long as it is a known method, but the Neighbor Neighbor method, It is obtained by either the Wallace method or the GC% method and needs to satisfy the characteristics of the present invention. Note that a particularly preferable Tm value is calculated by the Nearest neighbor method. The difference in Tm value between the oligonucleotide probe (D) and the primer used in the amplification reaction is not particularly limited as long as it is 1 ° C. or higher, but is preferably 5 ° C. or higher. In addition, the difference of Tm value should just be 50 degrees C or less, Preferably 40 degrees C or less is good. The probe chain length may be 5 to 30 bases, and preferably 8 to 25 bases.

  In the present invention, at least one kind of detection oligonucleotide probe (D) complementary to a part of a single-stranded nucleic acid obtained by denaturing a double-stranded nucleic acid is separate from the denaturation of the double-stranded nucleic acid. Or simultaneously.

  When acting separately, for example, the oligonucleotide probe (D) is added after the denaturation of the double-stranded nucleic acid, and after heat denaturation at 98 ° C. for 3 minutes, the temperature is gradually increased to near room temperature. Can be lowered.

  When acting simultaneously, as an example, in the mixed state of the above double-stranded nucleic acid and the oligonucleotide probe (D), heat denaturation at 98 ° C. for 3 minutes and then gradually lower the temperature to near room temperature. Can do.

  Until the complex (P) is formed from the nucleic acid amplification method, a homogeneous system may be used. For example, a cycle consisting of three steps of denaturation, annealing, and extension in the presence of a sample nucleic acid, four types of deoxynucleoside triphosphates, a pair of oligonucleotide primers, a detection oligonucleotide probe (D) and a heat-resistant DNA polymerase. Repeat the nucleic acid amplification reaction. Then, after heat denaturation at 98 ° C. for 3 minutes, the temperature is gradually lowered to near room temperature, and the detection oligonucleotide probe (D) and the single-stranded nucleic acid obtained by denaturing the double-stranded nucleic acid are hybridized. Let me. Thereafter, an extension reaction of the oligonucleotide probe for detection (D) is carried out using a single-stranded nucleic acid as a template by a thermostable DNA polymerase present in the reaction system, and the extension product of the oligonucleotide probe (D) and the single-stranded nucleic acid A complex (P) is formed. Thereafter, the complex (P) may be detected.

  In the method using the nucleic acid amplification method as described above, after the reaction, a single-stranded nucleic acid obtained by denaturation of the double-stranded nucleic acid, and a detection oligonucleotide probe complementary to a part of the single-stranded nucleic acid ( A complex (P) comprising D) is formed.

  In the present invention, it is only necessary to confirm that the single-stranded nucleic acid and the oligonucleotide probe (D) coexist in the formed complex (P). For example, as the oligonucleotide probe (D), a ligand bound to an oligonucleotide complementary to a part of the target nucleic acid sequence in a double-stranded nucleic acid is used, whereby the ligand capture agent is bound. The complex (P) can be captured and detected on the phase.

  The ligand is not particularly limited as long as it does not interfere with the detection of the nucleic acid sequence, but is preferably selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme, avidin, biotin, and digokesigenin. Can do. Antigens, fluorescent substances, biotin, digoxigenin, and the like are preferable, and antigens, fluorescent substances, biotin, and digoxigenin are particularly preferable.

  As the solid phase, any material such as a metal plate, a piece of wood, a plastic plate, a glass plate, a rubber plate, a polystyrene foam, a film, a membrane, a liquid-permeable filter, and a gel can be used, but a membrane and a liquid-permeable filter are preferable. If necessary, the surface and / or the inside of the solid phase may be modified to a form capable of capturing the ligand capture agent.

  The ligand capture agent can be appropriately selected from antibodies, oligonucleotides, receptors, nucleic acid-specific binding substances, avidin, biotin, and digokesigenin. An antibody and avidin are preferable, and avidin is particularly preferable. For example, when the ligand is biotin, the ligand capture agent may be avidin.

  The method for detecting the complex (P) is not particularly limited. As an example, a substance capable of selectively binding to a conventionally known double-stranded nucleic acid represented by ethidium bromide or cyber green is used as the complex (P). Fluorescence can be detected by binding to P) and emitting ultraviolet light.

  As another example, when a ligand is bound to a single-stranded nucleic acid obtained by denaturation of a double-stranded nucleic acid, an oligonucleotide probe (D) is allowed to act to form the complex (P). Thereafter, at least one type of physiologically active substance that can bind to the ligand and is labeled may be bound, and the label of the physiologically active substance may be detected.

  The physiologically active substance to be used is not particularly limited as long as it has an affinity for the ligand bound to the single-stranded nucleic acid, but is preferably an antibody, oligonucleotide, receptor, nucleic acid-specific binding. It can be selected from the group consisting of substance, avidin, biotin and digokesigenin. An antibody and avidin are preferable, and an antibody is particularly preferable. For example, when the ligand is FITC, the physiologically active substance may be an anti-FITC antibody.

  The label bonded to the physiologically active substance can be selected from the group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance. Preferred are fluorescent chemical substances, fluorescent proteins, luminophores, enzymes and conductive substances, more preferred are fluorescent chemical substances, fluorescent proteins and enzymes, and particularly preferred are enzymes.

  The detection of the label is not particularly limited as long as it is a known method. However, when the label is a fluorescent chemical substance or a fluorescent protein, light of a specific wavelength is irradiated to excite the fluorescent chemical substance or the fluorescent protein to bring it to the ground state. It is possible to measure the amount of fluorescence of a specific wavelength generated when converted. Examples of the fluorescent chemical substance used in these include FITC, FAM, TAMRA, Texas Red, VIC, Cy3, Cy5, HEX, and the like, and examples of the fluorescent protein include GFP, YFP, and RFP. When the label is an enzyme, it can be measured by detecting the reaction product produced by adding the enzyme substrate. The combinations of enzyme and substrate used in these include alkaline phosphatase and paranitrophenyl phosphate, CDP-star, AMPPD, DDAOphosphate, BCIP-NBT, etc., peroxidase and TMB, Lumi-Light (Roche Diagnostics) ), Combinations of SAT-1 (Dojindo), combinations of diaphorase and NTB, various oxidases and substrates, combinations of various dehydrogenases and substrates, etc. It can be used without any problems. When the label is a magnetic substance, measurement can be performed by detecting magnetism. The label can be measured by detecting a conductive substance and a current value.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1
Detection of Cytochrome P450 2C19 (CYP2C19) Gene (1) Synthesis of Oligonucleotide to Prepare CYP2C19 Double-Stranded Nucleic Acid Oligonucleotides having the indicated base sequences (hereinafter referred to as oligos 1 to 4) were synthesized. The synthesis was performed according to the manual, and the deprotection of each oligonucleotide was carried out overnight at 55 ° C. with ammonia water. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, DNA commissioned companies (Nippon Bioservice, Sigma-Aldrich Japan, Operon Biotechnology, etc.) were requested.
Oligo 1 is the sense strand, oligo 2 is the antisense strand, oligo 1 and oligo 2 are used as oligonucleotides for the amplification reaction, and the 5 ′ end of oligo 2 is labeled with FITC. Oligo 3 is a detection oligonucleotide probe (D) labeled with biotin at the 5 ′ end, and oligo 4 is a detection oligonucleotide probe (D) labeled with biotin at the 3 ′ end.

(2) Preparation of CYP2C19 double-stranded nucleic acid by PCR method Using a DNA solution extracted from human leukocytes by phenol-chloroform method as a sample, the following reagents are added, and double-stranded containing human CYP2C19 gene under the following conditions: Nucleic acids were prepared.

Reagents A 25 μl solution containing the following reagents was prepared.
Taq DNA polymerase reaction solution Oligo 1 5 pmol,
Oligo 2 (5 ′ end labeled with FITC) 5 pmol,
X10 buffer 2.5 μl,
2.5 μl of 2 mM dNTP,
1.5 μl of 25 mM MgCl ₂ ,
Taq DNA polymerase 1.3U,
Extracted DNA solution 100ng

Amplification conditions 95 ° C, 5 minutes 95 ° C, 30 seconds,
60 ° C for 30 seconds,
72 ° C, 30 seconds (35 cycles)
25 ° C for 15 minutes.

(3) Detection of CYP2C19 gene The following reagent was added to the reaction solution containing the prepared CYP2C19 double-stranded nucleic acid, and the detection probe was bound to the double-stranded nucleic acid under the following conditions.

Reagents A 30 μl solution containing the following reagents was prepared.
Detection probe reaction solution Oligo 3 (5 ′ end labeled with biotin) or Oligo 4 (3 ′ end labeled with biotin) 5 pmol,
PCR product 25 μl

Reaction conditions 98 ° C, 3 minutes,
65 ° C for 1 minute,
55 ° C for 1 minute,
45 ° C for 1 minute,
35 ° C for 1 minute,
25 ° C for 15 minutes.

(4) Detection using liquid-permeable filter 15 μl of the detection probe reaction solution was added to 30 μl of a solution of POD-labeled anti-FITC antibody (manufactured by DAKO Cytomation) and reacted at room temperature for 5 minutes. As a result, the POD-labeled anti-FITC antibody binds to the amplified CYP2C19 gene fragment. When this reaction solution is added to a liquid-permeable filter to which avidin is bound, oligo 3 or 4 which is a biotin-labeled oligonucleotide bound to the amplified CYP2C19 gene fragment is captured on the filter. Next, a washing solution and a POD substrate solution were sequentially added from above, and the color development on the filter surface was measured with a color difference meter (CR-221 manufactured by Minolta). The Lab mode a value was used for measurement, and a white plate attached to the color difference meter was used for correction of the measurement value. In Table 1, Sample No. The obtained measured values are shown for the samples indicated by 1 to 7 and the case of no sample.

  As described above, the gene could be clearly and easily detected quickly and easily. As the oligonucleotide probe (D), it was possible to detect specifically clearly in the oligo 3 designed in such a manner that a ligand was bound to the 5 'end and a nucleic acid elongation reaction could occur.

According to the present invention, the nucleic acid sequence in the sample nucleic acid can be clearly and easily detected, and does not require a complicated operation as in the conventional method, so that a result with high reproducibility can be obtained quickly and easily. However, it is expected to greatly contribute to the industry.

Claims (13)

A method for detecting a target nucleic acid sequence in a double-stranded nucleic acid, comprising at least the following steps (1) to (3):
(1) a first step of denaturing the double-stranded nucleic acid to form a single-stranded nucleic acid;
(2) The single-stranded nucleic acid obtained in the first step is hybridized with at least one kind of detection oligonucleotide probe (D) complementary to a part of the single-stranded nucleic acid, and the single-stranded nucleic acid A second step of forming a complex (P) of the extension product of the oligonucleotide probe (D) and the single-stranded nucleic acid by performing an extension reaction using as a template,
(3) A third step of detecting the presence of the complex (P). The method for detecting a target nucleic acid sequence according to claim 1, wherein the oligonucleotide probe (D) is labeled with a ligand. The method for detecting a target nucleic acid sequence according to claim 1 or 2, wherein at least one nucleotide from the 3 'end of the oligonucleotide probe (D) is labeled with a ligand. The method for detecting a target nucleic acid sequence according to any one of claims 1 to 3, wherein the 5 'end of the oligonucleotide probe (D) is labeled with a ligand. The method for detecting a target nucleic acid sequence according to any one of claims 2 to 4, wherein the ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme. The method for detecting a target nucleic acid sequence according to any one of claims 1 to 5, wherein the third step includes at least the following steps (i) and (ii).
(I) capturing the complex (P) on the solid phase using the ligand of the oligonucleotide probe (D);
(Ii) A step of detecting the complex (P) captured on the solid phase. The method for detecting a target nucleic acid sequence according to claim 6, wherein the step (i) includes a step of removing a single-stranded nucleic acid not hybridized with the oligonucleotide probe (D). The method for detecting a target nucleic acid sequence according to any one of claims 1 to 7, wherein an oligonucleotide probe (D) is present in the first step. The method for detecting a target nucleic acid sequence according to any one of claims 1 to 8, wherein the double-stranded nucleic acid is a double-stranded nucleic acid obtained by an amplification reaction. The method for detecting a target nucleic acid sequence according to claim 9, wherein the amplification reaction is PCR. The method for detecting a target nucleic acid sequence according to claim 9 or 10, wherein an oligonucleotide probe (D) is present in the amplification reaction. The method for detecting a target nucleic acid sequence according to any one of claims 9 to 11, wherein the Tm value of the oligonucleotide probe (D) is lower than the Tm value of the primer used in the amplification reaction. The method for detecting a target nucleic acid sequence according to any one of claims 1 to 12, wherein the denaturation in the first step is heat denaturation.