JPH03130097A - Detection of nucleic acid and detection reagent - Google Patents

Abstract

PURPOSE:To quickly and safely determine a nucleic acid in high specificity by hybridizing a single stranded nucleic acid with a complimentary single stranded nucleic acid in a specimen, removing the unhybridized single stranded nucleic acid and detecting the hybridized double stranded nucleic acid with a binder substance labeled with a labeling substance. CONSTITUTION:A specific nucleic acid in a specimen is detected by using a known single stranded nucleic acid as a probe and hybridizing the nucleic acid with a complimentary single stranded nucleic acid in the specimen. The process is carried out as follows. The single stranded nucleic acid left in the specimen in unhybridized state is removed by decomposing with an enzyme. etc., the double stranded nucleic acid produced by the hybridization is treated with a binder substance such as protein labeled with a labeling substance such as enzyme to bond the nucleic acid to the labeled binder substance and the labeled substance of the binder substance is detected to detect the nucleic acid in the specimen. The single stranded nucleic acid is preferably detected in a state immobilized to a supporting member.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to methods and reagents for detecting nucleic acids.

[Prior Art] With recent advances in genetic engineering, the frequency of detecting nucleic acids having specific base sequences has increased.

Conventional nucleic acid detection methods involve cutting nucleic acids with restriction enzymes, fractionating them by electrophoresis using agar or polyacrylamide gel, and physically or electrically transferring this to an adsorption membrane such as nitrocellulose. A known method is to denature such a transcript into a single strand, hybridize it with a single stranded nucleic acid labeled with a radioactive isotope, and detect this by autoradiography. Alternatively, immunological detection methods using antibodies against nucleic acids are known.

[Problems to be solved by the invention] However, conventional detection methods require complicated and time-consuming operations such as adsorption of sample nucleic acid onto a membrane, gel preparation, electrophoresis, transfer to a membrane, and autoradiography, and are difficult to automate. This is inappropriate and poses safety problems. Furthermore, since nucleic acids have a structure common to all biological species, specificity is very poor.

[Means for Solving the Problems] In view of the above-mentioned problems, the present inventors have conducted intensive studies to develop methods and reagents that can detect hybridized nucleic acids extremely specifically, rapidly, and safely, and that can be automated. As a result, we have arrived at the present invention. That is, the present invention is a method for detecting a specific nucleic acid by hybridizing a single-stranded nucleic acid with a complementary single-stranded nucleic acid in a sample. A nucleic acid detection method characterized by detecting stranded nucleic acids with a binding substance labeled with a labeling substance, a substance for removing uncrossed single-stranded nucleic acids, and a double-stranded nucleic acid after hybridization. A nucleic acid detection reagent used in the detection method according to any one of claims 1 to 9, comprising a binding substance labeled with a labeling substance that reacts with the nucleic acid.

The term "nucleic acid" as used in this specification refers to both single-stranded and double-stranded acids, and one or the other will be specified if necessary. Furthermore, the term "nucleic acid" includes all nucleic acids having one or more bases, regardless of the number of bases.

Furthermore, "nucleic acid" as used herein includes naturally occurring nucleotides as well as those in which nucleotides, which are unit components of nucleic acids, are artificially modified.

Furthermore, the term "single-stranded nucleic acid" literally includes a single-stranded nucleic acid portion formed by hybridizing a single-stranded nucleic acid itself and single-stranded nucleic acids having different chain lengths.

The single-stranded nucleic acid in the present invention includes a nucleic acid having a sequence complementary to the base sequence of a specific nucleic acid to be measured.

In the case of deoxyribonucleic acid (DNA), natural double-stranded nucleic acids can be separated and purified into single strands, or chemical methods can be used to sequentially link four nucleotides: adenine, guanine, cytosine and thymine. It may be synthesized in any way.

Preferably, it is manufactured by chemical method: A because of its purity and ease of mass production.

In the case of ribonucleic acid (RIIA), single-stranded nucleic acid can be used as is.

The number of bases in a single-stranded nucleic acid is usually 1 to 10,000, and a longer number is preferable in terms of accuracy and sensitivity, but a certain number of bases,
For example, there is not much change at approximately 5 (1G bases or more).

Preferably, the length is 5 to 100 bases, which allows recognition of complementary strands and ensures accuracy and sensitivity.

Furthermore, although single-stranded nucleic acids can be used in solution, it is preferable to use them after being immobilized on a support in advance from the viewpoint of reproducibility and simplicity. Examples of the support include, but are not limited to, synthetic resins such as nitrocellulose, polyethylene, polypropylene, polystyrene, polyvinyl chloride, nylon, silicone, polyurethane, and polyvinylidene fluoride, magnetic materials, or inorganic materials such as glass, silica gel, and bentonite. It's not something you can do. . Preferred are nitrocellulose, polyvinylidene fluoride, polystyrene, nylon, or glass in terms of retention capacity.

Immobilization methods include physical adsorption methods and chemical bonding methods. As a method using physical adsorption,
Specifically, a solution containing the single-stranded nucleic acid to be adsorbed (
For example, contacting a predetermined support with a phosphate buffer solution containing 10 μg/+nl of nucleic acid, etc. can be mentioned. If necessary, a support may be added (for example, at 80° C. for 120 minutes) to make the adsorption stronger after the reaction. On the other hand, as a method using chemical bonding,
Specifically, a support into which a functional group such as an azino group or a carboxyl group has been introduced and a single-stranded nucleic acid are combined with a bifunctional crosslinking agent (e.g., glutaraldehyde, etc.) or a bifunctional activating agent (e.g., Examples include a method of covalent bonding using water-soluble carbodiimide, carbodiimidazole, N-hydroxysuccinimide, bis(sulfosuccinimidyl) substrate, disulfide succinimidyl tartrate, etc.). Among these methods, chemical bonding is preferred because it increases the binding strength and stability of single-stranded nucleic acids and improves the reactivity during hybridization.

Also, during covalent bonding, maintaining a certain distance between the support surface and the binding site (terminal base) of the single-stranded nucleic acid improves the reactivity during hybridization, which ultimately leads to increased sensitivity. Therefore, it is preferable. That is, when a support and a single-stranded nucleic acid are bonded using a crosslinking agent or an activating agent, a functional group moiety introduced in advance into at least one of the surface of the support and the binding site of the single-stranded nucleic acid, It is preferable that a compound (for example, a polymethylene group having 3 or more carbon atoms, a polyether group having 4 or more carbon atoms, etc.) is inserted so as to have a certain molecular length (for example, inn, etc.).

In addition, the type of single-stranded nucleic acid referred to here may be one type for detecting one target, or two or more types (for example, 2 to 30 types) for simultaneously detecting multiple targets. Good too. For example, diseases caused by foreign pathogenic substances such as bacteria, viruses, rickettsiae, protozoa, spirochetes, etc. and/or mutations in the base sequence of nucleic acids, that is, base substitutions,
It is possible to select a plurality of arbitrary targets among single-stranded nucleic acids that are specific to genetic diseases caused by insertions, deletions, rearrangements, and the like. Furthermore, even if the same species of disease has several different base sequences, mutant strains of bacteria or viruses or the above-mentioned genetic diseases can also be used as targets. Specifically, pathogenic substances include bacteria such as Shigella, Diphtheria, Clostridium botulinum, and Salmonella, viruses such as human adenovirus species of the Adenoviridae family, influenza virus species of the Ortomycunviridae family, and human papillomavirus species of the Babovaviridae family. However, hereditary diseases include, but are not limited to, familial hypercholesterolemia, empty hyperlipidemia, thalassemia, hemophilia, diabetes, and cancer.

Complementary single-stranded nucleic acids in a sample are related to naturally occurring double-stranded nucleic acids and are derived from microorganisms, viruses, fish, birds, plants, animals, especially blowhole animals (including humans) Examples include nucleic acids.

Viruses include viruses consisting of single-stranded nucleic acids and viruses consisting of double-stranded nucleic acids, and these may be mixed or each may exist independently.

Since there are various analytes in the sample used here,
In addition to nucleic acids that are complementary to single-stranded nucleic acids, non-complementary nucleic acids are also included. In addition, there are cases where non-complementary nucleic acids are not present.

These nucleic acids are typically extracted from cells and treated with restriction enzymes (e.g. EcoRi, old ndI [[, Pst I, Bgl
Nucleic acids are fragmented by treatment with chemical means such as I) or physical means such as ultrasound. The preparation method using restriction enzymes is usually 100mM containing 0-1.0M NaCl.
- Nucleic acid sample 0.1-1 in Trls buffer (pH 7,5)
This can be done by adding 00 μg and 0.1 to 100 units of restriction enzyme and shaking at 20 to 45°C for 10 to 120 minutes. These methods are described, for example, by Evatom Maniatis, Molecular Cloning, Cold Spring Harbor Laboratory (Tom
Manlatls, MOLECULARCLONI
NG, Cold Sprlng Mouth Arbor Labo
p104.1982.

Furthermore, when the amount of natural nucleic acid is small, the amount of nucleic acid can be amplified in advance in a test tube using various nucleic acid synthases until it can be detected.

Methods for preparing complementary single-stranded nucleic acids include denaturing fragmented nucleic acids with heat or alkali. Specifically, in the case of heating, about 60-150°C1 about 1-30 minutes, preferably about 80-100"C1 about IQ ~
In the case of alkali denaturation in 20 minutes, methods include, but are not limited to, adding an appropriate amount of an aqueous solution containing sodium hydroxide, leaving it for about 1 to 30 minutes at room temperature, and finally returning to neutrality. isn't it.

Examples of the hybridization method include a method in which the above-mentioned sample is mixed with a single-stranded nucleic acid, and the mixture is heated and shaken usually at about 25 to 80°C for about 109 to 10 hours. Moreover, these methods are easier if carried out using a commercially available reagent kit (Rapid Hybridization System "Multi Prime" manufactured by Amerjam Japan).

Methods for removing unhybridized nucleic acids include a method of digestion and decomposition with enzymes, since specific treatment is required. Examples of enzymes include enzymes that act specifically on single-stranded nucleic acids, such as Nis-1 nuclease (Sl-N
tlCLEASE), Magbean Nuclease (MLIN
G BEAN NUCLEASE), PI-NUCLEASE, PAL 31-NUCLEASE (BAL~3l-NUCLEASE), EXONUCLEASE, ■, ■ (EXONIICLEASE-I, III
, ■), deoxyribonuclease (DEOXYRIB)
ONUCLEASE-I), U Ho3Crace H
(RIBONUCLEASE-11) and other single-strand cleaving enzymes. Preferred are Nis 1 nuclease, Magbean nuclease, and Pal 31 nuclease in terms of specificity.

The method of decomposition using these enzymes usually uses approximately xpg
~! 0.001-10 for samples containing μg of hybridized nucleic acids.
Add 00unlts of single strand cutting enzyme, 20-50℃
This can be easily done by shaking for 5 to 120 minutes. Specifically, Tom Maniatis, Molecular Cloning, Cold Spring Harbor Laboratory (Ton+ ManjaHs, lll0L)
ECULAR CLONING, Co1d Spring
Harbor Laboratory) p237.
1982.

Binding substances that react with the hybridized nucleic acids include substances that are specifically incorporated into molecules of the hybrid, especially by hydrogen bonding, such as chemical reagents such as ethidium bromide and propidium diiodide, or proteins, e.g. Examples include antibodies, double-stranded nucleic acid binding proteins, and the like. Proteins are preferred from the viewpoint of substance safety and stability of produced compounds, and antibodies are particularly preferred. In actual detection, nucleic acids that have not hybridized with each other are removed from the system through a decomposition process, so it is not necessary that the antibodies specifically react only to hybrids. The antibody may be either a polyclonal antibody or a monoclonal antibody.

These antibodies are prepared using conventional methods.

For example, it is carried out by the method described in Nippon Biochemistry Kinsen, "Sekibiochemistry Experiment Course S-Immunobiochemistry Research Methods" 1-84, Tokyo Kagaku Doujin.

Examples of the labeling substance that can be used to label these antibodies include substances selected from the group consisting of radioactive isotopes, fluorescent substances, luminescent substances, enzymes, and compounds that can indirectly bind these substances. Specifically, as a radioactive isotope [32p]
, [36S, [3)1, and [+40], fluorescein inthiocyanate (FITC) is used as a fluorescent substance.
, tetramethylrhodamine inthiocyanate (RIT)
C), acridine orange, fluorescein, and ethidium bromide, luminescent substances such as luminol and luciferin, enzymes such as peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase, and compounds that can be indirectly bound to biotin (avidin is bound to Examples include, but are not limited to, antibodies (to which an antigen and a secondary antibody can be bound), etc. Preferably, from the viewpoint of safety, a labeling substance excluding radioactive isotopes is used.The labeling method is, for example, published by Nippon Seikagaku Kinsen, "Seki Biochemistry Experiment Course 5-Immuno Biochemistry Research Methods J 102-112, Tokyo Kagaku Doujin. It is easily carried out by the method described in .

Methods for detection using substances that bind to nucleic acids include methods similar to ordinary immunoassays (enzyme immunoassays or radioimmunoassays). Namely, examples include a method of detecting a hybridizing nucleic acid with a pre-labeled antibody, a method of detecting with a labeled secondary antibody against an antibody bound to a nucleic acid, and the like. For example, Tokugansho [13-211i
No. 2479, Japanese Patent Application No. 6328 [1071, Japanese Patent Application No. 1999]
This is a method detailed in each publication of No. 0211i379.

In addition to the essential components, the detection reagent of the present invention can contain various auxiliary agents to facilitate the use of the detection reagent. For example, a solubilizing agent to dissolve solids, a cleaning agent to remove unnecessary components during the reaction, a substrate to measure the enzyme activity if the standard substance is an enzyme, and a substance to stop the enzyme reaction. Reaction terminators and the like can be included in any combination.

[Example] The present invention will be further explained below with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Example 1 Detection of specific nucleic acids l) Preparation of complementary single-stranded nucleic acids in samples Commercially available pBR3
22 (manufactured by Takara Shuzo) and the restriction enzyme BglI (manufactured by TOYOBO, which cuts pBR322 into 3 fragments) under the reaction conditions recommended by the manufacturer, purified through normal operations such as phenol extraction and ethanol precipitation, and further purified. The sample was heat denatured at 100'C for 10 minutes to give a concentration of 100 μg.
/ml was used as the nucleic acid reagent (A) in the following study.

2) Preparation of immobilized single-stranded nucleic acid As the single-stranded nucleic acid, commercially available pBR322PrlmerH (manufactured by Takara Shuzo Co., Ltd., has 16 bases and a complementary base sequence to pBR322) was used. (1.1 p g/ml-Pr1merl111 dissolved in 0.1 M carbonate buffer (pH 9,8)
00 μI was placed in a polystyrene test tube and shaken at room temperature for 2 hours. Then 0. Contains O1% Tveen 0.02M
Phosphate buffer (1) H7,5) (0,01% Twee
Washed twice with 500μI of n-PH5), added ■% EISA dissolved in PBS, heated and shaken at 37°C for 10 minutes, and used as immobilized single-stranded nucleic acid reagent CB) for the following detection. .

3) Preparation of labeled antibodies A commercially available anti-nucleic acid antibody (manufactured by Chemi-Con International) was prepared according to the literature [Nisu Yoshitake, M. Imagawa, E. Ishikawa, Etol; J. Biochem (S, YO5).
ITAKE, M, 1MAGAWA, E, l5IKAIr
A et al: J, Biocbem, ), vol. 9
2 (1982) 1413-1424 to obtain a labeled antibody. This was diluted to 10 μg/ml with a buffer containing 1% bovine serum albumin (BSA) and used as a labeled antibody reagent (C) below.

4) Before hybridization, heat at 95°C for 5 minutes and immediately cool on ice (
500μl of PBS containing 0.5.10 and 20μl of A)
1 and (B) were mixed, heated and shaken at 60°C for 2 hours, and then hybridized with 0.01% tveen-PBS (500°C).
Washed with μIX (2 times).

5) Enzymatically decomposed commercially available Sl Nuclease (manufactured by Takara Shuzo) as recommended by the manufacturer.
A fresh solution prepared at a concentration of 10 units/ml was used as the degrading enzyme reagent (D) below. These 10
Add 500 μl of the above buffer containing μm to the reaction product of 4), react for 37°CXIG minutes, and then add 0.01%
Washed with tveen-containing PBS (500 μl XZ times).

8) Add (C) to the reaction product obtained in step 5) at 100 μm and PflS4
After adding 0Qμm and shaking while heating at 37°C x go minutes, the mixture was washed with physiological saline. To this was added 500 μm of a substrate solution (hydrogen peroxide-containing orthophenylenediamine solution), and after heating and shaking in a 37”CX for 30 minutes, 3 ml of 1.
The reaction was stopped with 5N sulfuric acid. The absorbance of this liquid at 492 nm was measured to determine the activity of the enzyme bound to the test tube.

Figure 1 shows the results of nucleic acid detection by this detection method.

Comparative Example 1 Detection was carried out in the same manner as in Example 1, except that the nucleic acids that did not hybridize in Example 1 were not decomposed with enzymes.

FIG. 1 shows the detection results according to Comparative Example 1.

The results show that only nucleic acids complementary to single-stranded nucleic acids can be specifically, safely, and rapidly detected by enzyme treatment.

Example 2 A detection reagent of the present invention was produced by filling the following reagents into separate containers and sealing the containers.

■) Nucleic acid reagent (A) 1.2m12
) Immobilized single-stranded nucleic acid reagent (B) 100 pieces 3) Labeled antibody reagent (C) +211114) Degrading enzyme reagent (D) ■, 211115)
Degrading enzyme buffer & (1m18) PBS
120+a17)0.0
1%TveerrPBS 240m18
) Physiological saline 1000ml19) Substrate solution [10ml110) Sulfuric acid (1
, 5N) 350ml Example 3
Detection of multiple specific nucleic acids ■) Preparation of complementary single-stranded nucleic acids in a sample Commercially available nucleic acid pB
R322 DNA and old 3mp19RF DNA (both manufactured by Takara Shuzo) were treated with the restriction enzyme old ndIn (manufactured by Takara Shuzo).
Complete digestion under the reaction conditions recommended by the manufacturer, purification through normal operations such as phenol extraction and ethanol precipitation, further heat denaturation at +00°C for 10 minutes, mixing each sample, and final The respective concentrations were adjusted to 1008 g and 7 ml and used as the nucleic acid reagent (E) in the following study.

2) Preparation of immobilized single-stranded nucleic acids The single-stranded nucleic acids were commercially available pBR322PrlmerB (manufactured by Takara Shuzo, with 18 bases and a complementary base sequence to pBR322) and old 3Pr1merM3 (manufactured by Takara Shuzo, with 17 bases and a complementary base sequence to pBR322). with complementary base sequences) was used. Gets 0.1 N of each primer dissolved in 0.1 M carbonate buffer (pH 9, 8).
Mix to make I, then 100μ! was placed in a polystyrene test tube and shaken at room temperature for 2 hours.

Then 0. 0.02M phosphate buffer containing O1% Tveen (
pH7,! li) (0,01%Tveen-P13
The sample was washed twice with 500 μl of S), 1% BSA dissolved in PBS was added, and the mixture was heated and shaken at 37°C for 10 minutes, which was used as an immobilized single-stranded nucleic acid reagent CF) for the following detection.

In addition, (B) used in Example 1 was also used in combination.

3) Preparation of labeled antibody Same as Example 1.

4) Before hybridization, heat at 95°C for 5 minutes and immediately cool on ice (
E) Q, 5. PBS5 (to
μl and (B) or (F) were mixed, heated and shaken at 60°C for 2 hours, and then mixed with 0.01% tveen-P.
Washed with BS (500 It l x 2).

5) Enzyme degradation Same as Example 1.

6) Detection Same as Example 1.

Figure 2 shows the results of nucleic acid detection using this detection method.

Comparative Example 2 Detection was carried out in the same manner as in Example 3, except that the nucleic acids that did not hybridize in Example 3 were not decomposed with enzymes.

FIG. 2 shows the detection results according to Comparative Example 2.

The results show that only nucleic acids complementary to single-stranded nucleic acids can be specifically, safely, and rapidly detected by enzyme treatment.

Example 4 A detection reagent of the present invention was produced by filling the following reagents into separate containers and then sealing the containers.

I) Nucleic acid reagent (E) 2.4m12
) Immobilized single-stranded nucleic acid reagent CF) +oo books 3) Immobilized single-stranded nucleic acid reagent (B) 100 pieces 4) Labeled antibody reagent (C) 24m15) Degrading enzyme reagent (D) 2.4116) Degrading enzyme Buffer solution 1201117) PBS
240m+18)0.01%Tve
en-PBS 480ml 13) Physiological saline 2000 old 10) Substrate solution 1) Sulfuric acid (1,5N) 20ml 00ml Example 5 Detection of specific nucleic acid 1) Preparation of complementary single-stranded nucleic acid in sample In Example 1 same.

2) Preparation of immobilized single-stranded nucleic acid First, single-stranded nucleic acid was prepared using commercially available pBR322PrlmerH.
An oligonucleotide having the same base sequence as the above was obtained by converting the 5'-terminal hydroxyl group to aminoalkyl using carbodiimidazole and hexamethylene diamine.

In addition, as a support, ground glass beads with an outer diameter of 8 m+++ whose surface had been aminated in advance with γ-aminoproblesilane were used, and this and disuccinimidyl substrate (DSS) and the above aminoalkylated The nucleotides were mixed and allowed to react at room temperature for 24 hours. Next, the nucleotide concentration of this reaction product was adjusted to 0.1 Ng/ml and treated as in Example 1 to obtain an immobilized single-stranded nucleic acid reagent (G), which was used for the following detection.

3) Preparation of labeled antibody Same as Example 1.

4) PBS500 containing 0.5.10 and 20 μl of (^) heated at 95°C for 5 minutes and immediately cooled on ice before hybridization.
μl and (G) were mixed, heated and shaken at 60°C for 2 hours, and then b) was added with 0.01% tween-PBS (
Washed with 500 μl x Z times).

5) Enzyme degradation Same as Example 1.

6) Detection Same as Example 1.

Figure 3 shows the results of nucleic acid detection by this detection method.

Comparative Example 3 Detection was carried out in the same manner as in Example 5, except that the nucleic acids that did not hybridize in Example 5 were not decomposed with enzymes.

Figure 3 shows the detection results according to Comparative Example 3.

The results show that the presence of a certain molecular length between the support and the single-stranded nucleic acid provides good sensitivity, and that only nucleic acids complementary to the single-stranded nucleic acid can be detected specifically, safely, and quickly by enzyme treatment.

Example 6 A detection reagent of the present invention was produced by filling the following reagents into separate containers and then sealing the containers.

l) Nucleic acid reagent (A) 1.2m12
) Immobilized single-stranded nucleic acid reagent (c) too much 3) Labeled antibody reagent (C) 12m14) Degrading enzyme reagent (D) 1.2m15) Degrading enzyme buffer Hml [1] PBS
120m17) 0.01%Twee
n-PBS 240ml 18) Physiological saline 1000ml 19> Substrate solution
60ml110) Sulfuric acid (1,5N)
350+111 [Effects of the Invention] The detection method and detection reagent of the present invention have higher specificity than conventional detection methods, and enable rapid and safe measurements.

In conventional detection methods, when the target nucleic acid is immobilized and a radioactive isotope such as 32p is used as a labeling substance for the double-stranded nucleic acid, the detection sensitivity is very high;
There are many problems in terms of operational effort and safety. Furthermore, in detection methods using antibodies, since the immunogenicity of nucleic acids is weak, it is difficult to obtain antibodies specific to hybridized nucleic acids, resulting in poor detection accuracy. The present invention provides a method for significantly overcoming these drawbacks.

Furthermore, when multiple single-stranded nucleic acids are used, viruses and bacteria with mutant strains can be measured with the same test reagent, and a variety of diseases can be detected simultaneously. In general, viruses and bacteria easily form mutant strains due to various external factors, which often makes diagnosis and treatment difficult. For example, papillomaviruses and influenza viruses have many mutant strains, and the latter in particular changes its strain type every year and becomes prevalent. Therefore, in the case of a reagent that can only detect one type of nucleic acid,
It may also lead to incorrect diagnosis. The testing method of the present invention can eliminate this error and is very convenient for mass screening.

In view of the above, the present invention can specifically detect target nucleic acids present in extremely small amounts in serum or other body fluids, which require highly sensitive and highly accurate measurements, or nucleic acids derived from biological contamination in foods. Moreover, the detection time can be significantly shortened. This is particularly preferred for application to clinical testing and food production, which are the main uses of the present invention.

[Brief explanation of the drawing]

Figure 1 shows the detection results of nucleic acids in Example 1 and Comparative Example 1, Figure 2 shows the detection results of nucleic acids in Example 3 and Comparative Example 2, and Figure 3 shows the detection results of nucleic acids in Example 5 and Comparative Example 3. It is a graph showing each detection result. Drawing strategy - Figure (A) μl Figure 2 Rollo; When (B) is used in Comparative Example 2

Claims (1)

[Claims] 1. A method for detecting a specific nucleic acid by hybridizing a single-stranded nucleic acid with a complementary single-stranded nucleic acid in a sample, in which the uncrossed single-stranded nucleic acid is removed and the hybridization is performed. A method for detecting nucleic acids, which comprises detecting double-stranded nucleic acids using a binding substance labeled with a standard substance. 2. The detection method according to claim 1, wherein the removal is carried out by decomposition. 3. The detection method according to claim 2, wherein the decomposition is carried out using an enzyme. 4. The detection method according to claim 3, wherein the enzyme is an enzyme that specifically acts on single-stranded nucleic acids. 5. Claims 1 to 4, wherein the single-stranded nucleic acid is immobilized on a support.
Detection method described in any of paragraphs. 6. The detection method according to any one of claims 1 to 5, wherein the single-stranded nucleic acid is a nucleic acid synthesized by a chemical method. 7. The detection method according to any one of claims 1 to 6, wherein the binding substance is a protein. 8. The detection method according to claim 7, wherein the protein is an antibody. 9. The detection method according to any one of claims 1 to 8, wherein the labeling substance is an enzyme. 10. Claims 1 to 9 containing a substance that removes single-stranded nucleic acids that have not hybridized and a binding substance that is labeled with a labeling substance that reacts with the hybridized double-stranded nucleic acids. A nucleic acid detection reagent used in any of the detection methods described above.