Method for detecting a target nucleic acid

Abstract

The present invention is a method for detecting a target nucleic acid, in which the target nucleic acid can be detected distinctively from a non-target nucleic acid for which the nucleotide sequence or modification state is partially different from that for the target nucleic acid, the method comprising: performing a nucleic acid amplification reaction using a region in the non-target nucleic acid which is different from a corresponding region in the target nucleic acid as a corresponding target region and a region in the target nucleic acid which is different from a corresponding region in the non-target nucleic acid as a target region, also using a nucleic acid sample of interest as a template, and also using a primer capable of hybridizing with both of the target nucleic acid and the non-target nucleic acid in the presence of a molecule capable of binding specifically to the target region in the non-target nucleic acid under temperature conditions under which the molecule can bind to the non-target nucleic acid; and detecting the target nucleic acid on the basis of the presence or absence of an amplification product.

Description

Technical Field

[0001] The present invention relates to a method for detecting a target nucleic acid by discriminating a plurality of nucleic acids having similar base sequences and structures such as gene mutations, gene polymorphisms, and nucleic acid modifications, a method for detecting a nucleic acid-binding molecule or evaluating its nucleic acid-binding ability, and a kit used for these methods. This application claims priority based on Japanese Patent Application No. 2019-191409 filed in Japan on October 18, 2019, and incorporates the content herein by reference.

Background Art

[0002] Many gene mutations in which the base sequence of a gene changes involve the insertion, deletion, or conversion to other bases of only 1 to several bases. Therefore, in detecting gene mutations, it is necessary to discriminate and detect wild-type nucleic acids and mutant nucleic acids having the same base sequence except for the mutation site of 1 to several bases.

[0003] As methods for detecting mutations of 1 to several bases in DNA or RNA, various techniques exist. As a mutation detection method using PCR (polymerase chain reaction), for example, there is a PCR method using a modified oligonucleotide probe with a fluorescent substance and a quencher. In this method, a reaction for PCR amplification of a DNA fragment containing a DNA region where a mutation is expected is carried out in the presence of an oligonucleotide probe modified with a fluorescent substance and a quencher, which hybridizes to the mutant DNA region where a mutation is expected. For mutant DNA, the probe anneals, and the probe is cleaved by the exonuclease activity of the DNA polymerase used in PCR, resulting in the separation of the fluorescent substance and the quencher, and fluorescence is generated in the reaction system. In the case of wild-type DNA, since the probe does not anneal, no fluorescence is generated. However, in a sample in which wild-type and mutant DNAs are mixed, complicated operations are required for quantitative evaluation, so it is not easy to use for detecting heterozygous mutations.

[0004] There is also a Surveyor assay that uses Surveyor nuclease (see Non-Patent Document 1). In this assay, PCR-amplified control DNA and test DNA are mixed in a test tube, heat-denatured and re-double-stranded, and the 3' side of the mismatched base is cleaved using Surveyor nuclease to detect whether the test DNA contains a base different from the control DNA. This method is relatively simple and is mainly used to extract DNA from a cell population that has undergone genome editing and to assay the efficiency of genome editing. Usually, since the genome editing efficiency does not reach 100% and the ways of performing genome editing are various, there is no need to add control DNA. However, in the detection of individual genome-edited cells, in order to detect homozygous mutations, it is necessary to mix DNA derived from wild-type cells. Also, when analyzing DNA derived from individual cells, since the method of determining the nucleotide sequence by the Sanger method or the like is more direct, the Surveyor assay is usually not used.

[0005] In addition, there is an ORNi-PCR method in which a short-chain RNA (oligoribonucleotide, ORN) of about 17 to 29 bases complementary to the DNA region amplified in the PCR reaction is added to the reaction system (see, for example, Non-Patent Document 2). In this method, the PCR amplification of the DNA region where ORN hybridizes is specifically inhibited. That is, only the DNA to which ORN does not hybridize is PCR-amplified.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0008] The main object of the present invention is to provide a method for detecting nucleic acid differences or modifications, detecting a molecule having DNA-binding ability, or evaluating DNA-binding ability by utilizing the inhibition of nucleic acid amplification reaction by a molecule that binds to a nucleic acid serving as a template.

Means for Solving the Problems

[0009] As a result of intensive research, the present inventors have found that in a method capable of amplifying a nucleic acid fragment having a target base sequence under isothermal conditions such as the RPA (Recombinase Polymerase Amplification) method, by allowing a molecule that specifically binds to a nucleic acid having a specific base sequence or modification state to be present in the reaction system, only a nucleic acid fragment that does not bind to the molecule can be specifically detected, and thus the present invention has been completed.

[0010] That is, the method for detecting a target nucleic acid according to the present invention, and the kit used therefor are as follows. [1] A method for identifying and detecting a target nucleic acid from a non-target nucleic acid having a base sequence different from a part of the target nucleic acid, is wherein a region of the non-target nucleic acid that is different from the base sequence of the target nucleic acid is used as a target region, a region of the target nucleic acid that is different from the base sequence of the non-target nucleic acid is used as a corresponding target region, is a test nucleic acid sample is used as a template, in the presence of a molecule that specifically binds to the target region in the non-target nucleic acid (excluding a molecule consisting only of nucleic acid), is using a primer that hybridizes to both the target nucleic acid and the non-target nucleic acid, performing a nucleic acid amplification reaction under temperature conditions under which the molecule can bind to the non-target nucleic acid, detecting the target nucleic acid based on the presence or absence of an amplification product, wherein the molecule is a complex of a DNA strand cleavage activity-deficient Cas9 protein and a gRNA, and the nucleic acid amplification reaction is performed under temperature conditions of 65°C or lower, a method for detecting a target nucleic acid. ​​​[2] The method for detecting a target nucleic acid according to [1] above, wherein the gRNA specifically recognizes and binds to DNA having a base sequence complementary to the target region in the non-target nucleic acid. [3] A target nucleic acid is subjected to a nucleic acid sequence analysis using a part of the target nucleic acid. is 1. A method for discriminating and detecting a non-target nucleic acid from different non-target nucleic acids, comprising: The target nucleic acid and the base sequence in the non-target nucleic acid is Different regions are targeted regions, The base sequence of the target nucleic acid and the non-target nucleic acid is The different regions are corresponding target regions; A test nucleic acid sample is used as a template, In the presence of a molecule that specifically binds to the target region in the non-target nucleic acid (excluding molecules consisting of nucleic acid alone), Using a primer that hybridizes to both the target nucleic acid and the non-target nucleic acid, conducting a nucleic acid amplification reaction under temperature conditions that allow the molecule to bind to the non-target nucleic acid, and detecting the target nucleic acid based on the presence or absence of an amplification product; The molecule is a complex of an RNA strand cleavage activity-deficient Cas13a protein and a gRNA, A method for detecting a target nucleic acid, wherein the nucleic acid amplification reaction is carried out at a temperature of 65°C or lower. [4] The method for detecting a target nucleic acid according to [1] above, wherein the gRNA specifically recognizes and binds to RNA having a base sequence complementary to the target region in the non-target nucleic acid. [5] The method for detecting a target nucleic acid according to any one of [1] to [4] above, wherein, when an amplification product is obtained by the nucleic acid amplification reaction, the test nucleic acid sample contains the target nucleic acid. [6] The method for detecting a target nucleic acid according to any one of [1] to [5] above, wherein the nucleic acid amplification reaction is an isothermal nucleic acid amplification reaction. [7] The method for detecting a target nucleic acid according to any one of [1] to [6] above, wherein the nucleic acid amplification reaction is a Recombinase Polymerase Amplification method. [8] The method for detecting a target nucleic acid according to any one of [1] to [7], wherein the corresponding target region in the target nucleic acid and the target region in the non-target nucleic acid have different nucleotide sequences. [9] The method for detecting a target nucleic acid according to [8], wherein the target region is a mutation site of a gene mutation or a polymorphism site of a gene polymorphism. 10 A kit used for the method for detecting a target nucleic acid according to [1] or [2], comprising: A primer that hybridizes with both the target nucleic acid and the non-target nucleic acid; A DNA strand cleavage activity-deficient Cas9 protein; A gRNA; and a kit for detecting a target nucleic acid. 11 Further comprising a recombinase, a single-stranded DNA binding protein, and a DNA polymerase, the kit for detecting a target nucleic acid according to 10 . 12 A kit used for the method for detecting a target nucleic acid according to [3] or [4], comprising: A primer that hybridizes with both the target nucleic acid and the non-target nucleic acid; An RNA strand cleavage activity-deficient Cas13a protein; A gRNA; and a kit for detecting a target nucleic acid.

Advantages of the Invention

[0011] By the method for detecting a target nucleic acid according to the present invention, the target nucleic acid can be detected as a positive signal of a nucleic acid amplification product without the need for a thermal cycler for temperature control as required in PCR. Further, by the kit for detecting a target nucleic acid according to the present invention, the method for detecting the target nucleic acid can be more simply implemented to detect the target nucleic acid.

[0012] By the method for detecting a nucleic acid binding molecule and the method for evaluating nucleic acid binding ability according to the present invention, the nucleic acid binding ability can be detected and evaluated using the decrease amount of the nucleic acid amplification product as an index. ​​​In addition, the kit for detecting a nucleic acid binding molecule and the kit for evaluating nucleic acid binding ability according to the present invention enable the detection method of the nucleic acid binding molecule and the evaluation method of nucleic acid binding ability to be carried out more simply.

Brief Description of Drawings

[0013]

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Modes for Carrying Out the Invention

[0014] <Method for Detecting Target Nucleic Acid> In the present invention and the present specification, the "target nucleic acid" is a nucleic acid to be detected. The target nucleic acid is not particularly limited as long as its base sequence is specified to such an extent that primer design for a nucleic acid amplification reaction using a primer and a polymerase is possible. Specifically, single-stranded DNA, double-stranded DNA, single-stranded RNA, RNA-DNA hybrid (a nucleic acid in which single-stranded RNA and single-stranded DNA form a double strand), etc. can be used as the target nucleic acid. The target nucleic acid also includes nucleic acids having a specific modification state. Examples of the modification state include methylation, fluorination, phosphorothioation, phosphorodithioation, sugar addition, PEG (polyethylene glycol) addition, peptide addition, and the like.

[0015] In the present invention and the specification of the present application, the "non-target nucleic acid" means a nucleic acid having a structure common to the target nucleic acid, but partially different in base sequence or modification state from the target nucleic acid. The non-target nucleic acid is not particularly limited as long as the difference in structure such as the base sequence and modification state from the target nucleic acid is clear and the nucleic acid can be distinguished from the target nucleic acid. For example, if the target nucleic acid is a nucleic acid modified by CpG methylation, a nucleic acid having the same base sequence as the target nucleic acid but not modified by CpG methylation can be used as the non-target nucleic acid.

[0016] In the present invention and the specification of the present application, the "corresponding target region" means a region in the target nucleic acid that has a different structure from the non-target nucleic acid, and the "target region" means a region in the non-target nucleic acid that has a different structure from the target nucleic acid. The position where the corresponding target region exists in the target nucleic acid corresponds to the position where the target region exists in the non-target nucleic acid. For example, when the difference in structure between the target nucleic acid and the non-target nucleic acid is a difference in base sequence, the site (difference site) where the base sequence is different from the non-target nucleic acid in the target nucleic acid is the corresponding target region, and the site (difference site) where the base sequence is different from the target nucleic acid in the non-target nucleic acid is the target region. Similarly, when the difference in structure between the target nucleic acid and the non-target nucleic acid is a difference in modification state, the site (difference site) where the modification state is different from the non-target nucleic acid in the target nucleic acid is the corresponding target region, and the site (difference site) where the modification state is different from the target nucleic acid in the non-target nucleic acid is the target region. The number of corresponding target regions contained in the target nucleic acid may be one or two or more.

[0017] In the present invention and the specification of the present application, the "target base sequence" means the base sequence of a region containing the corresponding target region in the target nucleic acid. The target base sequence may be a base sequence consisting only of the corresponding target region, a base sequence of a partial region of the target nucleic acid containing the corresponding target region, or a base sequence of the entire length of the target nucleic acid.

[0018] In the present invention and the specification of the present application, "the base sequences are homologous" means "the base sequences are identical", and "the base sequences are complementary" means "the base sequences are complementary to each other".

[0019] In the detection of a nucleic acid containing a specific base sequence, the region containing the base sequence is amplified by a nucleic acid amplification reaction, and the detection sensitivity can be enhanced by detecting the amplification product. In such a detection method using a nucleic acid amplification reaction, not only the nucleic acid fragment consisting of the base sequence to be detected but also a nucleic acid fragment having a base sequence similar to the base sequence may be amplified. In the nucleic acid amplification reaction, by suppressing the amplification of a nucleic acid fragment having a base sequence similar to the base sequence to be detected, the detection accuracy of the nucleic acid fragment consisting of the base sequence to be detected can be enhanced.

[0020] The method for detecting a target nucleic acid according to the present invention uses a test nucleic acid sample as a template, performs a nucleic acid amplification reaction using a primer and a polymerase, and detects the target nucleic acid in the test nucleic acid sample as a nucleic acid amplification product. In the method for detecting a target nucleic acid according to the present invention, this nucleic acid amplification reaction is carried out in the presence of a molecule (excluding a molecule consisting only of nucleic acids) that binds to the target region in the non-target nucleic acid and does not bind to the corresponding target region in the target nucleic acid. Hereinafter, the "molecule (excluding a molecule consisting only of nucleic acids) that binds to the target region in the non-target nucleic acid and does not bind to the corresponding target region in the target nucleic acid" may be referred to as a "target region-specific binding molecule". In the nucleic acid amplification reaction using the non-target nucleic acid bound with the target region-specific binding molecule as a template, the annealing of the primer to the template nucleic acid and the nucleic acid elongation reaction by the polymerase are inhibited by the target region-specific binding molecule bound to the target region. Thereby, the nucleic acid amplification of the non-target nucleic acid is inhibited, the ratio of the amplification product of the target nucleic acid in the amplification product increases, and the detection accuracy of the target nucleic acid is improved.

[0021] Specifically, the method for detecting a target nucleic acid according to the present invention is a method for identifying and detecting a target nucleic acid from a non-target nucleic acid. Using a test nucleic acid sample as a template, a nucleic acid amplification reaction is carried out in the presence of a target region-specific binding molecule using a primer that hybridizes with both the target nucleic acid and the non-target nucleic acid, and the target nucleic acid is detected based on the presence or absence of an amplification product. Since the target nucleic acid is amplified by the nucleic acid amplification reaction, when the test nucleic acid sample contains the target nucleic acid, an amplification product is obtained by the nucleic acid amplification reaction. That is, the target nucleic acid in the test nucleic acid sample can be detected as the amplification product of the nucleic acid amplification reaction.

[0022] In the present invention, the target nucleic acid and the non-target nucleic acid are not particularly limited as long as they have regions with different structures within a common structure. Since the method for detecting a target nucleic acid according to the present invention can detect even a small difference in nucleic acid structure, when the difference in the structure between the target nucleic acid and the non-target nucleic acid is a difference in base sequence, the corresponding target region in the target nucleic acid is preferably a region of 20 bases or less, more preferably a region of 10 bases or less, even more preferably a region of 1 to 5 bases, and still more preferably a region of 1 or 2 bases.

[0023] In the present invention and this specification, the "test nucleic acid sample" is a sample containing a nucleic acid, which is to be used for detecting a target nucleic acid. In the present invention, it is detected whether or not the target nucleic acid is contained in the nucleic acid in the test nucleic acid sample. The test nucleic acid sample is not particularly limited as long as it is a sample containing a nucleic acid. For example, a nucleic acid extracted from an animal, a plant, a microorganism, a virus, cultured cells, etc. can be used as the test nucleic acid sample. Also, a chemically synthesized nucleic acid or a nucleic acid that has been intentionally modified can be used as the test nucleic acid sample. Extraction of nucleic acid from cells, etc. can be performed by a known method such as the phenol / chloroform method.

[0024] The nucleic acid contained in the test nucleic acid sample is not particularly limited as long as it can be used as a template for the nucleic acid amplification reaction to be performed. Since a nucleic acid amplification reaction using a commonly used DNA as a template can be utilized, the nucleic acid in the test nucleic acid sample used for the method for detecting a target nucleic acid according to the present invention is preferably DNA, and more preferably double-stranded DNA. The nucleic acid in the test nucleic acid sample used for the method for detecting a target nucleic acid according to the present invention may also be RNA. When the nucleic acid in the test nucleic acid sample is RNA, the reverse transcription reaction using the RNA as a template may be used as the nucleic acid amplification reaction in the method for detecting a target nucleic acid according to the present invention, or cDNA may be synthesized in advance by a reverse transcription reaction and then used for the nucleic acid amplification reaction.

[0025] In the present invention, a primer for amplifying a target nucleic acid used in a nucleic acid amplification reaction hybridizes with both the target nucleic acid and non-target nucleic acids. Therefore, in the absence of a target region-specific binding molecule, non-target nucleic acids contained in a test nucleic acid sample are also amplified by the nucleic acid amplification reaction using the primer. In the present invention, the nucleic acid amplification reaction is carried out in the presence of a target region-specific binding molecule, thereby inhibiting the nucleic acid amplification of non-target nucleic acids. In the present invention, the primer for amplifying the target nucleic acid used in the nucleic acid amplification reaction may be any primer that hybridizes with both the target nucleic acid and non-target nucleic acids under the conditions for carrying out the nucleic acid amplification reaction, and may be a primer that hybridizes with a partial region having a common base sequence and modification state with the non-target nucleic acid in the target nucleic acid, or may be a primer that hybridizes with a partial region having a different base sequence or modification state from the non-target nucleic acid in the target nucleic acid.

[0026] In the present invention, the primers for amplifying a target nucleic acid used in a nucleic acid amplification reaction are oligonucleotides or modified forms thereof in which nucleotides are linked by phosphodiester bonds. These primers may consist only of natural nucleotides such as DNA and RNA (nucleotides that exist in nature), may consist only of artificial nucleotides in which natural nucleotides are modified and can be linked to natural nucleotides by phosphodiester bonds, or may be chimeric molecules containing both natural nucleotides and artificial nucleotides. Examples of artificial nucleotides include those in which the side chain of a natural nucleotide is modified with a functional group such as an amino group, those in which the hydroxyl group at the 2'-position of the ribose backbone is substituted with a methoxy group, a fluoro group, a methoxyethyl group, etc., phosphorothioate-type nucleotides (in which the oxygen atom of the phosphate group is substituted with a sulfur atom), Morpholino-type nucleotides (in which ribose and deoxyribose are substituted with a morpholine ring), BNA (Bridged nucleic acid), HNA (Hexitol Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), TNA (Threose nucleic acid), GNA (Glycerol nucleic acid), CeNA (Cyclohexenyl nucleic acid), and the like. Examples of modified forms of oligonucleotides include those modified with a labeling substance that contributes to the detection of the amplification product. Examples of such a labeling substance include a fluorescent substance, a radioisotope, a chemiluminescent substance, an enzyme, an antibody, and the like. The labeling substance is bound so as not to inhibit the nucleic acid strand extension reaction of the primer.

[0027] In the present invention, the number of primers used in the nucleic acid amplification reaction may be one, two, or three or more. It can be appropriately determined according to the nucleic acid amplification reaction to be carried out.

[0028] For example, a primer set consisting of a forward primer and a reverse primer can be used. For example, the target base sequence is the base sequence of the entire length or a partial region of the target nucleic acid, and regions having the same structure as the non-target nucleic acid are present on both the 5'-side and the 3'-side of the corresponding target region, specifically, regions having the same base sequence and modification state. It can be set so that there is. A forward primer that hybridizes with a region having the same structure as the non-target nucleic acid at the 5'-end of the target base sequence, and a nucleic acid fragment of the target base sequence that is a primer for amplifying the nucleic acid fragment that hybridizes with a region having the same structure as the non-target nucleic acid at the 3'-end of the target base sequence A primer set can be used.

[0029] In the method for detecting a target nucleic acid according to the present invention, the nucleic acid amplification reaction is carried out under temperature conditions under which the target region-specific binding molecule can bind to the non-target nucleic acid. The nucleic acid amplification reaction may be carried out under temperature conditions under which the target region-specific binding molecule can bind to the non-target nucleic acid throughout the reaction process, and may have a temperature cycle such as the PCR method.

[0030] In the method for detecting a target nucleic acid according to the present invention, it is preferable that the nucleic acid amplification reaction is carried out under temperature conditions of 65 ° C. or lower. By carrying out the reaction under temperature conditions of 65 ° C. or lower, a molecule having relatively low heat resistance such as a protein can be used as the target region-specific binding molecule. In addition, by using a target region-specific binding molecule composed of a heat-resistant protein and using a heat-resistant polymerase or the like, a nucleic acid amplification reaction can also be carried out in a temperature range exceeding 65 ° C., for example, exceeding 65 ° C. and 95 ° C. or lower.

[0031] Since a thermal cycler for performing temperature control as required in PCR is not required, the nucleic acid amplification reaction in the method for detecting a target nucleic acid according to the present invention is preferably carried out under isothermal conditions. The term "under isothermal conditions" means maintaining within a temperature range of ± 3 ° C or ± 1 ° C with respect to the temperature set during the reaction.

[0032] The isothermal conditions are not particularly limited as long as the nucleic acid amplification reaction can proceed. For example, they are a certain temperature included in the optimal temperature of DNA polymerase. Examples of the isothermal conditions include a certain temperature of 10°C or higher, 15°C or higher, 20°C or higher, 25°C or higher, or 30°C or higher, and a certain temperature of 65°C or lower, 60°C or lower, 50°C or lower, 45°C or lower, or 40°C or lower. Also, the isothermal conditions can be, for example, a certain temperature included in the range of 10°C to 65°C, a certain temperature included in the range of 15°C to 50°C, a certain temperature included in the range of 20°C to 45°C, a certain temperature included in the range of 20°C to 45°C, or a certain temperature included in the range of 30°C to 45°C. In this specification, terms such as "incubate isothermally", "keep warm under isothermal conditions", and "react isothermally" mean to keep within a temperature range of ±7°C, ±5°C, ±3°C, or ±1°C with respect to the temperature set during the reaction.

[0033] In the method for detecting a target nucleic acid according to the present invention, the nucleic acid amplification reaction carried out under isothermal conditions is not particularly limited, and a known isothermal nucleic acid amplification reaction or its modified reaction can be carried out. Examples of the known isothermal nucleic acid amplification reactions include the RPA method (Patent Document 1), the LAMP (Loop-Mediated Isothermal Amplification) method (Non-Patent Document 3), the MDA (Multiple displacement amplification) method (Non-Patent Document 4), a reverse transcription reaction using reverse transcriptase, the NASBA (Nucleic Acid Sequence-Based amplification) method, and the like.

[0034] The RPA method is a method in which a primer that hybridizes with one terminal region of a target base sequence and a primer that hybridizes with the other terminal region are each formed into a complex with a recombinase, brought into contact with a template nucleic acid, and after forming a replication fork together with a single-strand binding protein (SSB), a double-stranded nucleic acid is synthesized by DNA polymerase. The primers to be used can be designed in the same manner as primers for PCR. The SSB, DNA polymerase, buffer for preparing the reaction solution, etc. to be used can be appropriately selected from those generally used in the RPA method and their modified forms. Also, commercially available RPA kits such as "TwistAmp (registered trademark)" (manufactured by TwistDx) can be used. The reaction conditions can be appropriately carried out under conditions generally used in the RPA method or conditions modified therefrom.

[0035] The LAMP method is a method of amplification using a strand displacement reaction with four types of primers (FIP primer, F3 primer, BIP primer, and B3 primer) selected from six regions of a target base sequence and combined. The F3 primer corresponds to the "forward primer that hybridizes with the 5' terminal region of the target base sequence" in the present invention, and the B3 primer corresponds to the "reverse primer that hybridizes with the 3' terminal region of the target base sequence" in the present invention. These primers can be designed, for example, using the LAMP method primer design support software "PrimerExplorer" (manufactured by Eiken Chemical Co., Ltd.). The strand displacement type DNA polymerase, buffer for preparing the reaction solution, etc. to be used can be appropriately selected from those generally used in the LAMP method and their modified forms, and the reaction conditions can also be appropriately carried out under conditions generally used in the LAMP method or conditions modified therefrom.

[0036] The MDA method generally uses random primers to synthesize single-stranded nucleic acids from the positions where the primers bind by Phi29 DNA Polymerase. Phi29 DNA Polymerase itself has helicase-like activity and can proceed with the DNA synthesis reaction while unwinding the double-stranded nucleic acid (e.g., the complex of template DNA and primer) even when it encounters a double-stranded nucleic acid during nucleic acid synthesis. It is known that more than 70,000 base pairs are synthesized in a reaction at 30°C for 12 hours. By using random primers or primers near the target base sequence, the target base sequence can be amplified. The primers, DNA polymerase, buffer for preparing the reaction solution, etc. used can be appropriately selected from those commonly used in the MDA method and their variants, and the reaction conditions can also be appropriately carried out under the conditions commonly used in the MDA method or modified conditions thereof.

[0037] The NASBA method is an isothermal nucleic acid amplification reaction that uses RNA as a template, AMV reverse transcriptase, RNase H, and T7 RNA polymerase, along with two types of primers (an F primer and an R primer having a T7 promoter sequence on the 5' side). First, the R primer (corresponding to the "reverse primer that hybridizes to the 3' end region of the target base sequence" in the present invention) anneals to the template RNA, and cDNA is synthesized by reverse transcriptase. The RNase H digests the RNA strand portion of the obtained RNA / DNA strand. The F primer (corresponding to the "forward primer that hybridizes to the 5' end region of the target base sequence" in the present invention) anneals to the remaining cDNA, and cDNA is synthesized by reverse transcriptase. Using the obtained double-stranded cDNA as a template, antisense single-stranded RNA is synthesized by the transcription reaction of T7 RNA polymerase. After the F primer anneals to this single-stranded RNA and an RNA / DNA strand is obtained by reverse transcriptase, the RNase H digests the RNA strand portion. The R primer anneals to the remaining cDNA, and double-stranded cDNA is obtained by reverse transcriptase. Using this as a template, antisense single-stranded RNA is synthesized by the transcription reaction of T7 RNA polymerase. By repeating this, the antisense single-stranded RNA of the target nucleic acid, RNA, is amplified. The primers, reverse transcriptase, RNase H, and T7 RNA polymerase used, as well as the buffer for preparing the reaction solution, etc., can be appropriately selected from those commonly used in the NASBA method and their variants, and the reaction conditions can also be appropriately carried out under the conditions commonly used in the NASBA method or conditions modified therefrom.

[0038] The reverse transcription reaction is a reaction in which reverse transcriptase synthesizes the antisense cDNA strand of template RNA using RNA as a template. By performing the RT-PCR method in combination with PCR using the obtained cDNA as a template, the target RNA can be detected with high sensitivity. The primers, reverse transcriptase, DNA polymerase, buffer for preparing the reaction solution, etc., used can be appropriately selected from those commonly used in the reverse transcription reaction and their variants.

[0039] In the RPA method, LAMP method, MDA method, NASBA method, and reverse transcription reaction, the reaction temperature is not particularly limited as long as the enzyme used exhibits enzyme activity within the temperature range. Also, in isothermal nucleic acid amplification reactions, the amplification products obtained are dependent on the reaction time. For this reason, the reaction time can be appropriately set according to the amount of the target amplification product. For example, in the RPA method and the LAMP method, it can be set to 5 minutes to 6 hours, preferably 5 minutes to 1 hour, and more preferably 5 to 30 minutes. In the MDA method, it can be set to 5 minutes to 32 hours, preferably 5 minutes to 24 hours, and more preferably 5 minutes to 16 hours. In the NASBA method, it can be set to 5 minutes to 6 hours, preferably 5 minutes to 3 hours, and more preferably 5 minutes to 1.5 hours. In the reverse transcription reaction, it can be set to 5 minutes to 3 hours, preferably 5 minutes to 1 hour, and more preferably 5 to 30 minutes.

[0040] In the method for detecting a target nucleic acid according to the present invention, the target region-specific binding molecule to be contained in the reaction system of the nucleic acid amplification reaction is not particularly limited as long as it is a molecule that can specifically bind to a nucleic acid other than the target nucleic acid, that is, a non-target nucleic acid, which is not amplifiable by the nucleic acid amplification reaction using the primer used for the amplification of the target nucleic acid, and can inhibit nucleic acid amplification.

[0041] When the difference in the structures of the target nucleic acid and the non-target nucleic acid is a difference in the base sequence, as the target region-specific binding molecule, a molecule that specifically recognizes and binds to the base sequence of the target region of the non-target nucleic acid can be used. Examples of such a molecule when the target nucleic acid is DNA include a complex (CRISPR complex) of a DNA strand cleavage activity-deficient Cas9 (dCas9) protein and a gRNA (guide RNA). Examples of such a molecule when the target nucleic acid is RNA include a complex (CRISPR complex) of a Cas13a (Cas13a) (Non-Patent Document 7) RNA strand cleavage activity-deficient (dCas13a) protein and a gRNA that constitutes a CRISPR complex that binds to RNA. In particular, it has been reported that the Cas13a / gRNA complex can distinguish a difference of one base in RNA (Non-Patent Document 8). Therefore, by using the Cas13a / gRNA complex as the target region-specific binding molecule in the method for detecting a target nucleic acid according to the present invention, a target nucleic acid can be detected by distinguishing between a target nucleic acid and a non-target nucleic acid that differ by only one base.

[0042] Examples of the dCas9 protein include a protein in which a mutation is introduced into the nuclease domain in the Cas9 protein and the nuclease activity is inactivated while maintaining the DNA binding ability. Examples of such a protein include mutants in which at least one of two point mutations, D10A (a point mutation in which the 10th aspartic acid is substituted with alanine) in the RuvC nuclease domain and H840A (a point mutation in which the 840th histidine is substituted with alanine) in the HNH nuclease domain, are introduced into the wild-type Cas9 protein derived from Streptococcus pyogenes. In addition, mutants in which two point mutations corresponding to D10A and H840A of the Cas9 protein derived from Streptococcus pyogenes are introduced into various Cas9 proteins can also be used. As the dCas9 protein, commercially available proteins such as "EnGen Spy dCas9 (SNAP-tag)" (manufactured by New England Biolabs) can also be used.

[0043] Cas13a is a protein that constitutes an RNA-targeting CRISPR complex, also known as C2c2, and has two HEPN domains (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) and one nuclease domain. Examples of dCas13a include proteins in which mutations are introduced into the nuclease domain of the Cas13a protein, and the nuclease activity is inactivated while maintaining the RNA-binding ability. Examples of such proteins include mutants in which at least one of two point mutations, R474A (a point mutation in which the 474th arginine is substituted with alanine) and R1046A (a point mutation in which the 1046th arginine is substituted with alanine) in the nuclease domain of the wild-type Cas13a protein derived from Leptotrichia wadei are introduced (Non-Patent Document 10). In addition, mutants in which two point mutations corresponding to R474A and R1046A of the Cas13a protein derived from Leptotrichia wadei are introduced into various Cas13a proteins can also be used.

[0044] When using Cas9, the gRNA contains a bacterium-derived crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA). The crRNA is a single-stranded RNA that includes a region consisting of a base sequence complementary to a part of the tracrRNA (binding region to tracrRNA) and a region consisting of a base sequence complementary to the base sequence that specifically recognizes and binds (target nucleic acid binding region). The target nucleic acid binding region is a DNA binding region when the target nucleic acid is DNA and an RNA binding region when the target nucleic acid is RNA. The tracrRNA has a region consisting of a base sequence complementary to a part of the crRNA (binding region to crRNA), and is a single-stranded RNA that hybridizes with the crRNA in this region to form a hairpin structure. The crRNA and tracrRNA may each be an independent single-stranded RNA, or may be a single-stranded RNA (sgRNA) in which both are linked via an appropriate RNA linker. These can be designed in the same manner as commonly used in genome editing. On the other hand, Cas13a does not require tracrRNA and functions as a gRNA with only crRNA. Therefore, a single-stranded RNA may be used as the gRNA.

[0045] The target nucleic acid binding region (DNA binding region or RNA binding region) in the gRNA used in the present invention is a region that binds to the target region in the non-target nucleic acid, and is a region consisting of a base sequence complementary to the base sequence of the target region. When a CRISPR complex, that is, a complex of dCas9 protein and gRNA when the target nucleic acid is DNA, or a complex of dCas13a protein and gRNA when the target nucleic acid is RNA, binds to the target region of the non-target nucleic acid, the annealing of the primer to the template nucleic acid and the nucleic acid strand elongation reaction by the polymerase are inhibited, and the amplification of the non-target nucleic acid is suppressed.

[0046] When using Cas9, the target nucleic acid binding region (DNA binding region) in the gRNA usually selects the base sequence of the region where the PAM sequence comes immediately after it. The PAM sequence is a sequence recognized by the dCas9 protein and is determined depending on the Cas9 protein or the like used. The base length of the DNA binding region to which the dCas9 protein binds is not particularly limited, and for example, it can be about 15 to 30 bases in length, and preferably 18 to 23 bases in length.

[0047] When using Cas13a, the target nucleic acid binding region (RNA binding region) in the gRNA usually selects the base sequence of the region where the PFS sequence comes immediately after it. The PFS sequence is a sequence recognized by the dCas13a protein and is determined depending on the Cas13a protein or the like used. The base length of the RNA binding region to which the dCas13a protein binds is not particularly limited, and for example, it can be about 15 to 40 bases in length, and preferably 20 to 30 bases in length.

[0048] The amount of the dCas9 protein or Cas13a protein in the reaction solution is not particularly limited, and for example, it can be 200 ng or less per 20 μL of the reaction solution, and preferably 10 to 80 ng. Also, the amount of the gRNA in the reaction solution is not particularly limited, and for example, it can be 50 nM or less, and preferably 2.5 to 20 nM.

[0049] As target region-specific binding molecules, in addition to the CRISPR complex, molecules such as proteins with DNA sequence-specific binding ability or proteins with RNA sequence-specific binding ability that do not bind to the corresponding target region in the target nucleic acid but specifically bind to the target region in the non-target nucleic acid can be used. Examples of such proteins include transcription regulators such as zinc finger proteins that recognize and specifically bind to a specific DNA sequence, transcription-activator like effector (TALE), and LexA protein. Also, for example, PPR (pentatricopeptide repeat) proteins that recognize and specifically bind to a specific RNA sequence can be mentioned.

[0050] Among the cases where the non-target nucleic acid is a nucleic acid with a different modification state from the target nucleic acid, when the target region in the non-target nucleic acid is in a state with a specific modification and the corresponding target region in the target nucleic acid is in a state without the modification, a molecule that specifically binds to the modified nucleic acid can be used as the target region-specific binding molecule. For example, when the target region in the non-target nucleic acid is CpG-methylated and the corresponding target region in the target nucleic acid is not CpG-methylated, a CpG-methylated DNA-binding protein can be used as the target region-specific binding molecule. As the CpG-methylated DNA-binding protein, known proteins such as proteins having MBD (Methyl-CpG-binding domain) (MBD proteins) and antibodies that recognize methylated CpG can be appropriately used. The amount of MBD2 protein in the reaction solution is not particularly limited, and for example, it can be 10 μg or less per 20 μL of the reaction solution, and 0.05 - 2 μg is preferable.

[0051] When the non-target nucleic acid is a nucleic acid with a modification state different from that of the target nucleic acid, if the target region in the non-target nucleic acid is in a state without a specific modification and the corresponding target region in the target nucleic acid is in a state with the modification, a molecule that specifically binds to the nucleic acid without the modification and does not bind to the nucleic acid with the modification can be used as a target region-specific binding molecule. For example, when the target region in the non-target nucleic acid is not CpG-methylated and the corresponding target region in the target nucleic acid is CpG-methylated, a protein that specifically binds to non-CpG-methylated DNA and does not bind to CpG-methylated nucleic acids can be used as a target region-specific binding molecule. Examples of proteins that specifically bind to non-CpG-methylated DNA include, for example, CpG methyltransferase mutants with inactivated enzyme activity. Also, if there is no substance that can be a methyl group donor in the reaction solution, the wild-type protein of CpG methyltransferase can also be used as such a protein. The amount of the protein that specifically binds to non-CpG-methylated DNA in the reaction solution is not particularly limited and can be, for example, 10 μg or less per 20 μL of the reaction solution, and 0.05 - 2 μg is preferred.

[0052] When there are two or more non-target nucleic acids, target region-specific binding molecules for each of them can be added to the reaction system of the nucleic acid amplification reaction.

[0053] In the method for detecting a target nucleic acid according to the present invention, when an amplification product is obtained by a nucleic acid amplification reaction, the target nucleic acid is detected and it is determined that the test nucleic acid sample contains the target nucleic acid. On the other hand, when no amplification product is obtained, the target nucleic acid is not detected and it is determined that the test nucleic acid sample does not contain the target nucleic acid. Since the target nucleic acid is detected as a positive signal of the amplification product, the target nucleic acid can be detected with sufficient detection sensitivity.

[0054] The method for detecting a target nucleic acid according to the present invention is suitable for, for example, detecting gene mutations. A corresponding target region is set as a mutation site to be detected, and primers used in a nucleic acid amplification reaction are designed such that a nucleic acid amplification product of a region containing the mutation site can be obtained when the target nucleic acid or non-target nucleic acid is used as a template. For example, a nucleic acid with a mutant type at the mutation site (mutant nucleic acid) is used as the target nucleic acid, a nucleic acid with a wild type at the mutation site (wild type nucleic acid) is used as the non-target nucleic acid, and a molecule that specifically binds to a nucleic acid with a base sequence where the mutation site is wild type and does not bind to a nucleic acid with a base sequence where the mutation site is mutant type (excluding molecules consisting only of nucleic acids) is used as a target region-specific binding molecule. In the presence of the target region-specific binding molecule and under temperature conditions that do not lose the binding activity of the target region-specific binding molecule to the wild type nucleic acid, a nucleic acid amplification reaction is performed. When the tested nucleic acid sample contains mutant nucleic acid, an amplification product is obtained; when the tested nucleic acid sample does not contain mutant nucleic acid, no amplification product is obtained. The presence or absence of the amplification product can be used to detect the mutant nucleic acid, which is the target nucleic acid. Therefore, with this method, not only homozygous mutations but also heterozygous mutations can be detected.

[0055] Figure 1 is a schematic diagram showing a method for detecting a gene mutation using a dCas9 protein and gRNA as target region-specific binding molecules. In the figure, the left side shows an amplification reaction using wild type nucleic acid as a template, and the right side shows an amplification reaction using mutant nucleic acid as a template.

[0056] Figure 2 is a schematic diagram showing a method for detecting a gene mutation by RT-PCR using RNA as a target nucleic acid. A dCas13a protein / gRNA complex that binds to wild type RNA was used as the target region-specific binding molecule. In the figure, the left side shows an amplification reaction using mutant RNA as a template, and the right side shows an amplification reaction using wild type RNA as a template. Since the dCas13a protein / gRNA complex binds to wild type RNA, the elongation of the cDNA strand by reverse transcriptase is inhibited, and as a result, no amplification product of PCR is obtained. An amplification product can be obtained from mutant RNA by RT-PCR.

[0057] The method for detecting a target nucleic acid according to the present invention is also suitable for, for example, detecting gene polymorphisms. A target region is set as a polymorphic site for detection purposes, and primers used in a nucleic acid amplification reaction are designed such that a nucleic acid amplification product of a region containing the polymorphic site can be obtained when the target nucleic acid or non-target nucleic acid is used as a template. For example, a nucleic acid having a genotype of the polymorphic site as the target nucleic acid, a nucleic acid having a genotype other than the target genotype as the non-target nucleic acid, and a molecule that specifically binds to a nucleic acid having a genotype other than the target genotype (excluding molecules consisting only of nucleic acids) is used as a target region-specific binding molecule. In the presence of the target region-specific binding molecule and under temperature conditions that do not lose the binding activity of the target region-specific binding molecule to the non-target nucleic acid, a nucleic acid amplification reaction is performed. If the test nucleic acid sample to be tested contains the nucleic acid of the genotype to be detected, an amplification product is obtained; if the test nucleic acid sample does not contain the nucleic acid of the genotype, no amplification product is obtained. The presence or absence of the amplification product can be used to detect the gene polymorphism of a specific genotype that is the target nucleic acid. Similarly, with this method, not only homozygous polymorphisms but also heterozygous polymorphisms can be detected.

[0058] For DNA methylation analysis, there is a method using bisulfite treatment. After performing bisulfite treatment on the DNA to be analyzed for an appropriate reaction time in which methylated bases are not deaminated and non-methylated bases are deaminated, when a nucleic acid amplification reaction is performed by a polymerase, an amplification product is obtained in which methylated bases are the original bases and non-methylated bases are deaminated and converted to other bases. For example, since cytosine is converted to uracil by deamination, in the amplification product, methylated cytosine is cytosine and non-methylated cytosine is thymine.

[0059] When there are differences in the methylation states between the corresponding target region and the target region, base differences occur between the corresponding target region and the target region by bisulfite treatment. Therefore, by using the nucleic acid treated with bisulfite as the test nucleic acid sample, the presence or absence of methylation of a specific base in the nucleic acid can be analyzed by the method for detecting a target nucleic acid according to the present invention. A nucleic acid in which the base to be analyzed is methylated may be used as the target nucleic acid, or a nucleic acid in which the base to be analyzed is not methylated may be used as the target nucleic acid.

[0060] For example, when analyzing the presence or absence of methylation of a specific cytosine, if the nucleic acid obtained by bisulfite treatment of the nucleic acid in which the cytosine to be analyzed is methylated is used as the target nucleic acid, the partial region containing the cytosine (methylated cytosine) to be analyzed in the target nucleic acid is used as the corresponding target region, and the nucleic acid obtained by bisulfite treatment of the nucleic acid in which the cytosine to be analyzed is not methylated is used as the non-target nucleic acid. In this case, the cytosine to be analyzed in the non-target nucleic acid is converted to uracil by bisulfite treatment. The partial region containing this uracil is used as the target region. When the test nucleic acid sample contains a nucleic acid in which the cytosine to be analyzed is methylated cytosine, a nucleic acid amplification product in which the base is cytosine can be obtained by performing a nucleic acid amplification reaction in the presence of a target region-specific binding molecule. When all of the nucleic acids contained in the test nucleic acid sample are such that the cytosine to be analyzed is non-methylated cytosine, no amplification product is obtained in the nucleic acid amplification reaction in the presence of a target region-specific binding molecule.

[0061] In addition, when a bisulfite-treated nucleic acid in which the cytosine to be analyzed is not methylated is used as the target nucleic acid, a partial region containing the cytosine to be analyzed (unmethylated cytosine) in the target nucleic acid is defined as the corresponding target region, and a bisulfite-treated nucleic acid in which the cytosine to be analyzed is methylated is defined as the non-target nucleic acid. In this case, since the cytosine to be analyzed in the non-target nucleic acid is methylated, it remains as methylated cytosine even after bisulfite treatment. The partial region containing this methylated cytosine is defined as the target region. When the test nucleic acid sample contains a nucleic acid in which the cytosine to be analyzed is unmethylated cytosine, a nucleic acid amplification product in which the base is thymine can be obtained by performing a nucleic acid amplification reaction in the presence of a target region-specific binding molecule. When all of the nucleic acids contained in the test nucleic acid sample have the cytosine to be analyzed as methylated cytosine, no amplification product can be obtained in the nucleic acid amplification reaction in the presence of a target region-specific binding molecule.

[0062] In each of these methods, as the target region-specific binding molecule that specifically binds to the target region and does not bind to the corresponding target region, for example, a CRISPR complex or the like in which the target nucleic acid binding region (DNA binding region or RNA binding region) of gRNA has a base sequence complementary to the target region can be used.

[0063] The amplification product of the target nucleic acid obtained by nucleic acid amplification reaction can be detected by various methods commonly used for detecting the amplification products of nucleic acid amplification reactions such as PCR. Examples of such detection methods include a method of staining a band fractionated by agarose gel electrophoresis with ethidium bromide or the like, an intercalator method, and a melting curve analysis method. The intercalator method is a method that utilizes the fact that a fluorescent intercalator such as SYBR Green binds to the generated double-stranded DNA and emits fluorescence, and the fluorescence intensity increases as the amplification product increases. By irradiating the reaction solution with excitation light and measuring the fluorescence intensity, the production amount of the amplification product can be monitored. In addition, when a primer modified with a labeling substance is used, the labeling substance can be used as an index for detection. When a primer labeled with a fluorescent substance is used, the amplification product can be detected by removing the unreacted primer from the reaction solution after the nucleic acid amplification reaction by column chromatography or the like and then measuring the fluorescence intensity.

[0064] By kitifying the reagents and the like used in the method for detecting a target nucleic acid according to the present invention, the method can be carried out more simply. Among the methods for detecting a target nucleic acid according to the present invention, as a kit for the method using a dCas9 protein and a gRNA as a target region-specific binding molecule, it preferably has a primer that hybridizes with the target nucleic acid and the non-target nucleic acid, a dCas9 protein, and a gRNA. Among the methods for detecting a target nucleic acid according to the present invention, as a kit for the method using a dCas13a protein and a gRNA as a target region-specific binding molecule, it preferably has a primer that hybridizes with the target nucleic acid and the non-target nucleic acid, a dCas13a protein, and a gRNA. In addition, as a kit for the method using a CpG-methylated DNA-binding protein as a target region-specific binding molecule, it preferably has a primer that hybridizes with both the target nucleic acid and the non-target nucleic acid and a CpG-methylated DNA-binding protein. In addition, as a kit for the method using a transcription regulator as a target region-specific binding molecule, it preferably has a primer that hybridizes with both the target nucleic acid and the non-target nucleic acid and a transcription regulator.

[0065] The kit for detecting a target nucleic acid according to the present invention preferably also contains an enzyme for nucleic acid amplification reaction, a concentrated buffer for preparing a reaction solution, dNTP, etc. For example, in the case of a kit used for a method of performing a nucleic acid amplification reaction by the RPA method, it preferably further contains recombinase, SSB, and DNA polymerase.

[0066] The kit for detecting a target nucleic acid according to the present invention preferably further includes a document on which a protocol for performing the method for detecting a target nucleic acid according to the present invention using the kit is described. The protocol may be described on the surface of the container containing the kit.

[0067] <Method for Detecting Nucleic Acid-Binding Molecule> By binding a substance to the nucleic acid serving as a template, a reaction that inhibits the annealing of a primer to the template nucleic acid and the nucleic acid strand elongation reaction by polymerase can be utilized to detect a nucleic acid-binding molecule (a molecule having nucleic acid-binding ability).

[0068] That is, in the method for detecting a nucleic acid-binding molecule according to the present invention, a nucleic acid amplification reaction is performed using a test sample, a nucleic acid, and a primer that hybridizes with the nucleic acid. When a nucleic acid-binding molecule that binds to the nucleic acid used as a template is contained in the test sample, an amplification product cannot be obtained by the nucleic acid amplification reaction. On the other hand, when the test sample does not contain a nucleic acid-binding molecule that binds to the nucleic acid, an amplification product can be obtained by the nucleic acid amplification reaction. That is, based on the amount of the nucleic acid amplification product after the nucleic acid amplification reaction, the nucleic acid-binding molecule present in the test sample can be detected.

[0069] The test sample used in the method for detecting a nucleic acid-binding molecule according to the present invention is not particularly limited as long as it is a sample in which the presence of a nucleic acid-binding molecule is expected. For example, extracts (lysates) of tissues and cells of animals and plants, cell extracts of cultured cells, samples collected from nature such as soil, and their crude purification products can be used. The extracts of tissues and cells can be prepared by conventional methods.

[0070] In the method for detecting a nucleic acid-binding molecule according to the present invention, the nucleic acid-binding molecule to be detected is not particularly limited, but is preferably a peptide, protein, or low molecular compound, and may be a complex of a nucleic acid and another molecule such as a protein. Further, the nucleic acid-binding molecule may be an unidentified substance. For example, by using a test sample containing various substances, such as a cell extract or nuclear extract of various cells, it is possible to examine whether the test sample contains a nucleic acid-binding molecule. In the method for detecting a nucleic acid-binding molecule according to the present invention, the nucleic acid-binding ability of the nucleic acid-binding molecule to be detected is not particularly limited, and may be a base sequence-specific nucleic acid-binding molecule that recognizes and specifically binds to a specific base sequence, or may be a nucleic acid-binding molecule that recognizes and binds to a specific modified state, or may be a nucleic acid-binding molecule that binds generally to nucleic acids without being affected by the base sequence or modified state.

[0071] The nucleic acid used in the method for detecting a nucleic acid-binding molecule according to the present invention is used as a template for a nucleic acid amplification reaction, and is not particularly limited as long as it can be a template for a nucleic acid amplification reaction, but DNA or RNA is preferred. The nucleic acid may have a specified base sequence or may not have a specified base sequence. Further, the nucleic acid may consist of only one type of nucleic acid, or may be a mixture of various nucleic acids, such as DNA extracted and purified from cells. For example, when detecting a nucleic acid-binding molecule that binds to a nucleic acid without recognizing a base sequence, the nucleic acid used as a template may have an unknown base sequence or may be a mixture of various nucleic acids.

[0072] For example, when detecting a sequence-specific nucleic acid-binding molecule that recognizes and binds to a specific base sequence, a nucleic acid having the specific base sequence is used as a template, and in a nucleic acid amplification reaction, a primer designed to hybridize with the upstream (5' side) of the specific base sequence in the template nucleic acid is used so that the region containing the specific base sequence in the template nucleic acid is amplified. The primer can be designed and synthesized by a conventional method based on the base sequence of the nucleic acid used as a template.

[0073] When detecting a nucleic acid-binding molecule that specifically binds to a nucleic acid in a specific modification state, the nucleic acid in the specific modification state is used as a template. Examples of such modification states include methylation, fluorination, phosphorothioation, phosphorodithioation, sugar addition, PEG (polyethylene glycol) addition, peptide addition, and the like.

[0074] Examples of the nucleic acid amplification reaction performed in the method for detecting a nucleic acid-binding molecule according to the present invention include the same ones as those performed in the method for detecting a target nucleic acid according to the present invention described above. When the nucleic acid-binding molecule to be detected is a molecule with relatively low heat resistance such as a protein, the nucleic acid amplification reaction is preferably carried out in a temperature range of 65°C or lower. Conversely, when the detection of a heat-resistant nucleic acid-binding molecule is intended, a nucleic acid amplification reaction is carried out in a temperature range exceeding 65°C and 100°C or lower using a heat-resistant polymerase. In addition, as the nucleic acid amplification reaction performed in the method for detecting a nucleic acid-binding molecule according to the present invention, since a thermal cycler for performing temperature control as required in PCR is not necessary, a nucleic acid amplification reaction carried out under isothermal conditions such as the RPA method, the LAMP method, the MDA method, the NASBA method, etc. is preferable.

[0075] For example, a nucleic acid amplification reaction is carried out using a test sample, a nucleic acid, and a primer that hybridizes with the nucleic acid, and the amount of the obtained nucleic acid amplification product is compared with the amount of the nucleic acid amplification product obtained by carrying out the nucleic acid amplification reaction under the same conditions except that the test sample is not added. When the test sample contains a nucleic acid-binding molecule that binds to the nucleic acid used as a template, the amount of the nucleic acid amplification product obtained in the presence of the test sample is less than the amount of the nucleic acid amplification product obtained in the absence of the test sample. Conversely, when the amount of the nucleic acid amplification product obtained in the presence of the test sample is equal to or more than the amount of the nucleic acid amplification product obtained in the absence of the test sample, the test sample does not contain a nucleic acid-binding molecule that binds to the nucleic acid used as a template.

[0076] By kitifying reagents and the like used in the method for detecting a nucleic acid-binding molecule according to the present invention, the method can be carried out more simply. For example, as a kit for detecting a nucleic acid-binding molecule for using the method, it is preferable to contain a nucleic acid as a template and a primer that hybridizes with the nucleic acid. In addition, it is also preferable that it contains an enzyme for nucleic acid amplification reaction, a concentrated buffer for preparing a reaction solution, dNTP, etc. For example, in the case of a kit used for a method of performing a nucleic acid amplification reaction by the RPA method, it is further preferable to contain recombinase, SSB, and DNA polymerase.

[0077] The kit for detecting a nucleic acid-binding molecule according to the present invention preferably further includes a written document in which a protocol for performing the method for detecting a nucleic acid-binding molecule according to the present invention using the kit is described. The protocol may be described on the surface of the container containing the kit.

[0078] <Method for evaluating nucleic acid-binding ability> By binding a certain substance to the nucleic acid serving as a template, the nucleic acid-binding ability of the nucleic acid-binding molecule can be evaluated by utilizing a reaction that inhibits the annealing of the primer to the template nucleic acid and the nucleic acid strand elongation reaction by polymerase.

[0079] That is, in the method for evaluating nucleic acid-binding ability according to the present invention, a nucleic acid amplification reaction is carried out using a test substance, a nucleic acid whose nucleic acid-binding ability is to be evaluated as the test substance, and a primer that hybridizes with the nucleic acid. When the test substance has the ability to bind to the nucleic acid used as a template, no amplification product can be obtained by the nucleic acid amplification reaction. On the other hand, when the test substance does not have the ability to bind to the nucleic acid, an amplification product can be obtained by the nucleic acid amplification reaction. That is, based on the amount of the nucleic acid amplification product after the nucleic acid amplification reaction, the binding ability of the test substance to the nucleic acid used as a template can be evaluated.

[0080] In the method for evaluating nucleic acid binding ability according to the present invention, the test substance to be evaluated is not particularly limited, but is preferably a peptide, a protein, or a low molecular compound. In addition, it may be a complex of a nucleic acid and other molecules such as a protein. Further, the test substance may be a composition containing substances other than the test substance in addition to the purified one. Furthermore, the test substance may be an unidentified substance. For example, substances containing various substances such as cell extracts and nuclear extracts of various cells can also be used as the test substance.

[0081] In the method for evaluating nucleic acid binding ability according to the present invention, the nucleic acid binding ability to be evaluated is not particularly limited, and may be a sequence-specific nucleic acid binding ability that recognizes and binds to a specific base sequence, a nucleic acid binding ability that recognizes and binds to a specific modification state, or a nucleic acid binding ability that binds generally to nucleic acids without being affected by the base sequence or modification state.

[0082] In the method for evaluating nucleic acid binding ability according to the present invention, the nucleic acid used is a nucleic acid for which it is evaluated whether or not the test substance binds, and is not particularly limited as long as it can be a template for a nucleic acid amplification reaction, but DNA or RNA is preferred. The nucleic acid may have a specified base sequence or may not have a specified base sequence. In addition, the nucleic acid may consist of only one type of nucleic acid or may be a mixture of various nucleic acids.

[0083] For example, when evaluating the sequence-specific nucleic acid binding ability to recognize and bind to a specific base sequence, a nucleic acid having the specific base sequence is used as a template, and in a nucleic acid amplification reaction, a primer designed to hybridize with the upstream (5' side) of the specific base sequence in the template nucleic acid is used so that the region containing the specific base sequence in the template nucleic acid is amplified. The primer can be designed and synthesized by a conventional method based on the base sequence of the nucleic acid used as a template.

[0084] When evaluating the nucleic acid-binding ability to specifically bind to a nucleic acid in a specific modification state, a nucleic acid in the specific modification state is used as a template. Examples of such modification states include methylation, fluorination, phosphorothioation, phosphorodithioation, sugar addition, PEG (polyethylene glycol) addition, peptide addition, and the like.

[0085] As the nucleic acid amplification reaction performed in the method for evaluating nucleic acid-binding ability according to the present invention, those similar to the nucleic acid amplification reaction performed in the method for detecting a target nucleic acid according to the present invention described above can be mentioned. When the test substance is a molecule with relatively low heat resistance such as a protein, the nucleic acid amplification reaction is preferably carried out in a temperature range of 65°C or lower. Conversely, when the test substance is a molecule with high heat resistance and the nucleic acid-binding ability under a high-temperature environment is to be evaluated, a heat-resistant polymerase is used to carry out the nucleic acid amplification reaction in a temperature range exceeding 65°C and not exceeding 100°C. In addition, as the nucleic acid amplification reaction performed in the method for evaluating nucleic acid-binding ability according to the present invention, since a thermal cycler for performing temperature control as required in PCR is not required, a nucleic acid amplification reaction carried out under isothermal conditions such as the RPA method, LAMP method, MDA method, NASBA method, etc. is preferable.

[0086] For example, a nucleic acid amplification reaction is carried out using a test substance, a nucleic acid, and a primer that hybridizes with the nucleic acid, and the amount of the obtained nucleic acid amplification product is compared with the amount of the nucleic acid amplification product obtained by carrying out the nucleic acid amplification reaction under the same conditions except that the test substance is not added. When the test substance has a nucleic acid-binding ability to the template nucleic acid under the temperature conditions and the like under which the nucleic acid amplification reaction is carried out, the amount of the nucleic acid amplification product obtained in the presence of the test substance is less than the amount of the nucleic acid amplification product obtained in the absence of the test substance. That is, when the amount of the nucleic acid amplification product obtained in the presence of the test substance is less than the amount of the nucleic acid amplification product obtained in the absence of the test substance, the test substance is evaluated as having a nucleic acid-binding ability to the nucleic acid used as a template under the temperature conditions and the like under which the nucleic acid amplification reaction is carried out. Conversely, when the amount of the nucleic acid amplification product obtained in the presence of the test substance is equal to or more than the amount of the nucleic acid amplification product obtained in the absence of the test substance, the test substance is evaluated as not having a nucleic acid-binding ability to the nucleic acid used as a template.

[0087] By making a reagent or the like used in the method for evaluating nucleic acid binding ability according to the present invention into a kit, the method can be carried out more simply. For example, as a kit for evaluating nucleic acid binding ability for using the method, it preferably contains a nucleic acid as an object for evaluating the nucleic acid binding ability of a test substance and a primer that hybridizes with the nucleic acid. In addition, it is also preferable that it contains an enzyme for nucleic acid amplification reaction, a concentrated buffer for preparing a reaction solution, dNTP, etc. For example, in the case of a kit used for a method of performing a nucleic acid amplification reaction by the RPA method, it is further preferable to contain recombinase, SSB, and DNA polymerase.

[0088] The kit for evaluating nucleic acid binding ability according to the present invention preferably further includes a written document on which a protocol for performing the method for evaluating nucleic acid binding ability according to the present invention using the kit is described. The protocol may be described on the surface of the container containing the kit.

Examples

[0089] Next, the present invention will be described in more detail by way of examples and the like, but the present invention is not limited by these examples.

[0090] [RNA and Primer] The RNA used in the following experiments was chemically synthesized by commissioning FASMAC Co., Ltd. The used RNA is shown in Table 1.

[0091]

Table 1

[0092] The primers used in the following experiments were chemically synthesized by commissioning eurofin Co., Ltd. The used primers are shown in Table 2.

[0093]

Table 2

[0094] [Preparation of gRNA] 1 μL of 10 μM crRNA, 1 μL of 10 μM tracrRNA, and 2 μL of nuclease-free water were mixed and incubated at 98 °C for 2 minutes to form gRNA (crRNA / tracrRNA complex).

[0095] [RPA reaction] The RPA reaction was carried out as follows. First, 29.5 μL of rehydration buffer, 2.5 μL of 10 μM forward primer, and 2.5 μL of 10 μM reverse primer were added to one tube of RPA reagent (product name "TwistAmp® Basic kit", manufactured by TwistDx) (containing lyophilized reagent) and mixed. Then, 13.6 μL of the prepared solution was aliquoted into another tube, and 20 ng of template DNA and nuclease-free water were added to prepare 19 μL of RPA reaction preparation solution. After adding 1 μL of 280 mM MgOAc solution to this RPA reaction preparation solution, it was incubated at 37 °C for 30 minutes to carry out the RPA reaction.

[0096] The RPA reaction using dCas9 protein and gRNA was carried out as follows. Note that dCas9 used was synthesized by commissioning to Sysmex Corporation (ProCube, manufacturing number 14M_029), which was obtained by introducing point mutations of D10A and H840A into Cas9 derived from Streptococcus pyogenes. First, 0.8 μL of gRNA, 0.4 μg of dCas9 protein, and nuclease-free water were mixed to make 10 μL, and this was used as the CRISPR solution. During the RPA reaction, 1 μL of the CRISPR solution was added to the RPA reaction preparation solution prepared above (in the solution after addition, the dCas9 protein was 40 ng and the gRNA was 10 nM), and it was incubated at 37 °C for 5 minutes. Then, 1 μL of 280 mM MgOAc solution was added to the reaction solution, and it was incubated at 37 °C for 30 minutes to carry out the RPA reaction.

[0097] After the reaction was completed, nucleic acids were purified from each reaction solution using the "PCR / Gel DNA purification kit" (manufactured by Nippon Genetics Co., Ltd.). The purified products were electrophoresed using an agarose gel containing "SYBR (registered trademark) Safe DNA Gel Stain" (manufactured by ThermoFisher Scientific).

[0098] [Example 1] Two types of gRNAs were prepared for the KRAS gene of the cultured cell line 293T cells, added to the reaction system together with dCas9, and an RPA reaction was performed. Genomic DNA (293T gDNA) extracted and purified from 293T cells was used as a template, and gRNA_KRAS (SEQ ID NOs: 1 and 6) and gRNA_KRAS#2 (SEQ ID NOs: 2 and 6) were used as gRNAs. As a primer set, a forward primer (SEQ ID NO: 7) and a reverse primer (SEQ ID NO: 8) for amplifying the region containing the target regions of gRNA_KRAS and gRNA_KRAS#2 in the genomic DNA of the KRAS gene were used. The partial region of the KRAS gene of 293T cells and the complementary sequence portions of the genomic DNA of the two types of gRNAs used are shown in Figure 3. Furthermore, as a control, a gRNA for the CDKN2A (p16) gene (gRNA_p16_Gx5#2, SEQ ID NOs: 3 and 6) was used.

[0099] The results of the electrophoresis are shown in Figure 4. In the reaction solution to which gRNA_p16_Gx5#2 was added, amplification product bands were detected in the same manner as in the reaction solution to which neither gRNA nor dCas9 was added. On the other hand, in both reaction solutions to which gRNA_KRAS and gRNA_KRAS#2 were added, amplification product bands were not detected, and nucleic acid amplification of the KRAS gene by the forward and reverse primers was inhibited. From these results, it was confirmed that nucleic acid amplification by the RPA reaction can be inhibited by containing gRNA and dCas9 for the DNA for which nucleic acid amplification is to be inhibited in the reaction solution.

[0100] [Example 2] In the KRAS gene of the cultured cell line HCT116 cells, there is a single nucleotide substitution mutation (GGC→GAC) only on one allele, and the 13th glycine is substituted with aspartic acid. Due to this single nucleotide substitution, the PAM sequence (TGG) in gRNA_KRAS#2 shown in Figure 3 becomes TGA, and it is no longer the PAM sequence (NGG).

[0101] An RPA reaction was performed in the same manner as in Example 1, except that genomic DNA (HCT116 gDNA) extracted and purified from HCT116 cells was used as the template nucleic acid, and after purification, electrophoresis was carried out.

[0102] The results of electrophoresis are shown in Figure 5. Similar to Example 1 using 293T gDNA having only the wild-type KRAS gene as the template, nucleic acid amplification products were confirmed in the reaction solution to which gRNA_p16_Gx5#2 was added, and no amplification products were confirmed in the reaction solution to which gRNA_KRAS was added. That is, gRNA_KRAS functioned as a gRNA even when there was a single nucleotide mismatch between the gRNA and the template nucleic acid. On the other hand, different from Example 1, nucleic acid amplification products were confirmed in the reaction solution to which gRNA_KRAS#2 was added.

[0103] By sequence analysis, when the nucleotide sequences of the amplification products obtained in each reaction solution were confirmed, both the wild-type KRAS and the mutant KRAS (G13D) originally contained in HCT116 gDNA were included in the amplification products of the reaction solution without added gRNA and the reaction solution added with gRNA_p16_Gx5#2. On the other hand, only the mutant KRAS (G13D) was included in the amplification product of the reaction solution added with gRNA_KRAS#2, and nucleic acid amplification of the wild-type KRAS could not be confirmed. Fig. 6 schematically shows the RPA reaction performed in the reaction solution added with gRNA_KRAS#2. In wild-type KRAS, since gRNA_KRAS#2 binds near the PAM sequence together with dCas9, the amplification reaction by DNA polymerase was inhibited. However, in mutant KRAS, since there is no PAM sequence near the region where gRNA_KRAS#2 is supposed to bind, dCas9 was not recruited and the amplification reaction proceeded normally. That is, it was confirmed that by designing gRNA so that the mutation site comes to the PAM sequence, a single-base mutation can be detected with very high accuracy.

[0104] Next, as shown in Fig. 7, a single-base substitution mutation was introduced into gRNA_KRAS. This gRNA_KRAS_mut (SEQ ID NOs: 4 and 6) differed from wild-type KRAS by one base and from mutant KRAS (G13D) by two bases. Using HCT116 gDNA or 293T gDNA as the template nucleic acid, an RPA reaction was performed in the same manner as in Example 1 except that gRNA_KRAS_mut was used as the gRNA, and after purification, electrophoresis was performed.

[0105] The results of electrophoresis are shown in Fig. 8. As a result, in the reaction solution to which 293T gDNA was added, nucleic acid amplification was inhibited by gRNA_KRAS_mut. In the reaction solution to which HCT116 gDNA was added, even when gRNA_KRAS_mut was added, a band of the amplification product was detected. When the nucleotide sequence of the amplification product in the reaction solution to which HCT116 gDNA was added was confirmed, both wild-type KRAS and mutant KRAS (G13D) were included when gRNA_KRAS_mut was not added. On the other hand, when gRNA_KRAS_mut was added, only mutant KRAS (G13D) was included. That is, even if there was a single-base mismatch between the gRNA and the template nucleic acid, it could function as a gRNA and inhibit nucleic acid amplification by the RPA reaction. However, when there were two-base mismatches between the gRNA and the template nucleic acid, it could not function as a gRNA, and nucleic acid amplification by the RPA reaction proceeded normally. From these results, it was confirmed that even when the mutation did not exist on the PAM sequence, by artificially introducing a mutation into the gRNA and increasing the mismatch with the target base sequence, a single-base mutation could be distinguished.

[0106] [Example 3] As shown in Fig. 9, in the CDKN2A (p16) gene of HCT116 cells, only one G is inserted into one allele (Gx5). Therefore, gRNA_p16_Gx5#2 has the same nucleotide sequence as the Gx5 allele but does not match the other allele (Gx4).

[0107] Using genomic DNA (HCT116 gDNA) extracted and purified from HCT116 cells as the template nucleic acid, and using a forward primer (SEQ ID NO: 9) and a reverse primer (SEQ ID NO: 10) for amplifying the region containing the target region of gRNA_p16_Gx5#2 in the genomic DNA of the CDKN2A (p16) gene as the primer set, an RPA reaction was performed in the same manner as in Example 1 except that gRNA_p16_Gx5#2 or gRNA_KRAS was used as the gRNA, and after purification, electrophoresis was performed.

[0108] The results of electrophoresis are shown in Fig. 10. Although the amplification products in the reaction solution added with gRNA_p16_Gx5#2 were less than those in the reaction solution added with gRNA_KRAS or the reaction solution without added gRNA, amplification products were confirmed.

[0109] By sequence analysis, when the base sequences of the amplification products obtained in each reaction solution were confirmed, both Gx5 type p16 and Gx4 type p16 originally contained in HCT116 gDNA were included in the amplification products of the reaction solution without added gRNA and the reaction solution added with gRNA_KRAS. On the other hand, only Gx4 type p16 was included in the amplification products of the reaction solution added with gRNA_p16_Gx5#2, and nucleic acid amplification of Gx5 type p16 could not be confirmed. This is because gRNA_p16_Gx5#2 was recruited together with dCas9 to the Gx5 type p16 sequence, inhibiting the amplification reaction by DNA polymerase, but dCas9 was not recruited to the Gx4 type p16 sequence, and the amplification reaction proceeded normally. That is, it was confirmed that the method for detecting a target nucleic acid according to the present invention can detect a mutation with one base insertion or deletion with very high accuracy.

[0110] [Example 4] Genome editing was performed on the CDKN2A (p16) gene of 293T cells, and an RPA reaction was performed in the presence of gRNA (gRNA_mid2, SEQ ID NOs: 5 and 6) targeting wild-type nucleic acid and dCas9 on the genome after editing, and only the nucleic acid with genome editing was amplified and detected. Fig. 11 schematically shows the genome editing performed on the CDKN2A (p16) gene.

[0111] When genome editing has not been performed, since the base sequence remains wild-type, RPA amplification is suppressed by gRNA_mid2 and dCas9. On the other hand, when genome editing has been performed and the base sequence has changed, the amplification inhibitory effect by gRNA_mid2 and dCas9 does not occur, and RPA amplification is observed.

[0112] First, genome editing was performed as follows. 2 μg of Cas9 expression plasmid (manufactured by Addgene, #41815) and 2 μg of sgRNA expression plasmid (sgRNA-mid2) targeting the human CDKN2A (p16) gene (Non-Patent Document 5) were transfected into 293T cells (4 × 105 cells) using the transfection reagent "Lipofectamine 3000" (manufactured by ThermoFisher Scienticif). After culturing for 2 days, the cells were collected, and genomic DNA was extracted and purified.

[0113] Next, using the obtained genomic DNA as a template at a total amount of 100 ng, an RPA reaction was performed in the same manner as in Example 1 except that a forward primer (SEQ ID NO: 9) and a reverse primer (SEQ ID NO: 10) for RPA amplification of the region containing the base sequence targeted by gRNA_mid2 in the genomic DNA of the CDKN2A (p16) gene of 293T cells were used. After purification, electrophoresis was performed. Note that the ratio of the genome-edited genomic DNA was decreased by mixing a small amount of 293T gDNA subjected to genome editing with 293T gDNA not subjected to genome editing.

[0114] The results of electrophoresis are shown in Fig. 12. No amplification product was confirmed in the reaction solution using only 293T gDNA without genome editing as a template, indicating that the nucleic acid amplification reaction was inhibited by gRNA_mid2. On the other hand, an amplification product was confirmed in the reaction solution using a mixture of 293T gDNA (80 ng) without genome editing and 293T gDNA (20 ng) with genome editing as a template. When the nucleotide sequences of each amplification product were confirmed, the amplification product of the reaction solution using 293T gDNA without genome editing as a template was the same as the wild-type nucleic acid sequence of the CDKN2A (p16) gene. The amplification product of the reaction solution using a mixture of 293T gDNA without genome editing and 293T gDNA with genome editing as a template was mostly the wild-type nucleic acid sequence, but also slightly contained a nucleotide sequence rich in diversity that was considered to be the genome-edited sequence. On the other hand, only a nucleotide sequence rich in diversity was detected in the amplification product of the reaction solution using a mixture of 293T gDNA without genome editing and 293T gDNA with genome editing as a template and adding dCas9 and gRNA_mid2, and the wild-type nucleic acid sequence was not amplified.

[0115] [Example 5] A small amount of HCT116 gDNA was mixed with 293T gDNA, and whether the RPA amplification of wild-type KRAS can be suppressed by gRNA_KRAS#2 and dCas9 was examined using as a template a nucleic acid with a reduced abundance ratio of mutant KRAS (G13D) ([content of gDNA derived from cells with mutant KRAS (G13D)] / ([content of gDNA derived from cells with mutant KRAS (G13D)] + [content of gDNA derived from cells with only wild-type KRAS]) × 100%).

[0116] With HCT116 gDNA fixed at 20 ng, an RPA reaction was performed in the same manner as in Example 1 except that a mixture of 293T gDNA and HCT116 gDNA was used as a template so that the abundance ratio of mutant KRAS (G13D) was as shown in Fig. 13, and gRNA_KRAS#2 was used as the gRNA. After purification, electrophoresis was carried out.

[0117] The results of electrophoresis are shown in Fig. 14. Nucleic acid amplification products were confirmed even in a reaction solution in which the abundance ratio of gDNA derived from cells having mutant KRAS (G13D) was only 2%. When the nucleotide sequences of the respective amplification products were confirmed, only mutant KRAS (G13D) was detected in the amplification products of reaction solutions in which the abundance ratio of gDNA derived from cells having mutant KRAS (G13D) was 5 to 100%, but both mutant KRAS (G13D) and wild-type KRAS were confirmed in the amplification products of a reaction solution in which the abundance ratio of gDNA derived from cells having mutant KRAS (G13D) was 2%.

[0118] Next, with 293T gDNA fixed at 400 ng, an RPA reaction was carried out in the same manner as in Example 1 except that a mixture of 293T gDNA and HCT116 gDNA was used as a template so that the abundance ratio of mutant KRAS (G13D) was as shown in Fig. 14, and after purification, electrophoresis was performed.

[0119] The results of electrophoresis are shown in Fig. 14. Nucleic acid amplification products were confirmed even in a reaction solution in which the abundance ratio of gDNA derived from cells having mutant KRAS (G13D) was only 1%. When the nucleotide sequences of the respective amplification products were confirmed, only mutant KRAS (G13D) was detected in the amplification products of reaction solutions in which the abundance ratio of gDNA derived from cells having mutant KRAS (G13D) was 1 to 100%. From these results, it was shown that the target nucleic acid (mutant KRAS (G13D) in this experiment) can be detected even when the abundance ratio thereof is as low as 1%.

[0120] [Example 6] In the CDKN2A (p14ARF) gene of HCT116 cells, only one G is deleted at one allele (Gx4). Among the CDKN2A (p14ARF) genes of HCT116 cells, the cytosine around the Gx4 position in the Gx4 allele is not methylated, while the cytosine around the Gx5 position in the Gx5 allele is methylated. Therefore, a CpG methylated DNA binding protein was added to the reaction solution of the RPA reaction to suppress the RPA amplification using the Gx5 allele as a template and detect the Gx4 allele. As the CpG methylated DNA binding protein, the MBD2 protein (「EpiXplore (registered trademark) Methylated DNA Enrichment Kit」, manufactured by Takara Bio Inc.) was used.

[0121] An RPA reaction was performed in the same manner as in Example 1 except that 0.25 μg of MBD2 protein was used instead of gRNA and dCas9, and after purification, electrophoresis was performed. As the primer set, a forward primer (SEQ ID NO: 11) and a reverse primer (SEQ ID NO: 12) for amplifying the Gx4 and Gx5 positions of the CDKN2A (p14ARF) gene were used. Neither of the two primers contained CpG.

[0122] The results of electrophoresis are shown in Fig. 15. Nucleic acid amplification products were confirmed in the reaction solution without the addition of MBD2. Amplification products were also seen when MBD2 was added. When the nucleotide sequences of each amplification product were confirmed, both Gx4-type p14ARF and Gx5-type p14ARF were included in the amplification product of the reaction solution without the addition of MBD2. On the other hand, only Gx4-type p14ARF was included in the amplification product of the reaction solution with the addition of MBD2, and nucleic acid amplification of Gx5-type p14ARF could not be confirmed. From these results, it was shown that by having a CpG methylated DNA binding protein present in the reaction solution of the RPA reaction, nucleic acid amplification using a CpG methylated nucleic acid as a template can be inhibited and a non-CpG methylated target nucleic acid can be detected.

[0123] Among the CDKN2A (p16) genes of HCT116 cells, the cytosine around the Gx5 position in the Gx5 allele is not methylated, while the cytosine around the Gx4 position in the Gx4 allele is methylated. Therefore, a CpG-methylated DNA-binding protein was added to the reaction solution of the RPA reaction to suppress the RPA amplification using the Gx4 allele as a template and detect the Gx5 allele. MBD2 was used as the CpG-methylated DNA-binding protein.

[0124] An RPA reaction was performed in the same manner as in Example 3, except that 0.5 μg of MBD2 protein was used instead of gRNA and dCas9, and after purification, electrophoresis was performed. As the primer set, a forward primer (SEQ ID NO: 13) and a reverse primer (SEQ ID NO: 14) for amplifying the Gx4 and Gx5 positions of the CDKN2A (p16) gene were used. The reverse primer (SEQ ID NO: 14) contained one CpG.

[0125] The results of the electrophoresis are shown in Fig. 16. Nucleic acid amplification products were confirmed in the reaction solution without the addition of MBD2. Amplification products were also seen when MBD2 was added. When the nucleotide sequences of each amplification product were confirmed, both Gx5-type p16 and Gx4-type p16 were included in the amplification product of the reaction solution without the addition of MBD2. On the other hand, only Gx5-type p16 was included in the amplification product of the reaction solution with the addition of MBD2, and nucleic acid amplification of Gx4-type p16 could not be confirmed. These results also showed that by having a CpG-methylated DNA-binding protein present in the reaction solution of the RPA reaction, nucleic acid amplification using CpG-methylated nucleic acid as a template can be inhibited and non-CpG-methylated target nucleic acid can be detected.

[0126] Next, it was examined whether nucleic acid amplification was suppressed by this method also for DNA artificially CpG-methylated in vitro. First, using a forward primer (SEQ ID NO: 15) and a reverse primer (SEQ ID NO: 16) that sandwich the Gx4·Gx5 positions of the CDKN2A (p14ARF) gene, the nucleotide sequence sandwiched between both primers was amplified by PCR. For the PCR, a PCR reaction solution containing 10 ng of HCT116 genomic DNA and 0.5 μM of each primer in 10 μL was prepared using "AmpliTaq Gold (registered trademark) 360 Master Mix" (manufactured by ThermoFisher Scientific). The reaction was first denatured at 95°C for 10 minutes, followed by 30 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and then treated at 72°C for 1 minute. The amplification product was purified using a "PCR / Gel DNA purification kit" (manufactured by Nippon Genetics Co., Ltd.), cloned into T-Vector pMD20 (manufactured by Takara Bio Inc.), and amplified with Competent Quick DH5a (manufactured by Toyobo Co., Ltd.). The amplified plasmid was purified using "NucleoBond (registered trademark) Xtra Midi Plus" (manufactured by Takara Bio Inc.). Two types of purified plasmids were prepared: one containing Gx4 (p14_Gx4 plasmid) and one containing Gx5 (p14_Gx5 plasmid).

[0127] In HCT116 cells, the cytosine around the Gx4 position of the CDKN2A (p14ARF) gene is not methylated, while the cytosine around the Gx5 position is methylated. However, the plasmid purified from Escherichia coli is not subjected to these modifications. Therefore, intentionally, the cytosine around the Gx4 position rather than the Gx5 position was methylated in vitro. Specifically, 1 μg of purified p14_Gx4 plasmid was methylated by reacting it with 6 units of CpG methyltransferase M.SssI (manufactured by New England BioLabs) and 160 μM of S-adenosylmethionine at 37°C for 1 hour. The methylated p14_Gx4 plasmid and the non-methylated p14_Gx5 plasmid were purified using the "PCR / Gel DNA purification kit" (manufactured by Nippon Genetics Co., Ltd.).

[0128] Instead of genomic DNA, 1 pg of plasmid DNA was used, and an RPA reaction preparation solution was prepared in the same manner as in Example 1. After adding 0.5 μg of MBD2 protein to the RPA reaction preparation solution, it was incubated at 37°C for 10 minutes, and then 1 μL of 280 mM MgOAc solution was added, followed by incubation at 37°C for 10 minutes to conduct the RPA reaction. As the primer set, a forward primer (SEQ ID NO: 11) and a reverse primer (SEQ ID NO: 12) for amplifying the Gx4 and Gx5 positions of the CDKN2A (p14ARF) gene were used. After the reaction, the reaction solution was not purified, and 2 μL was electrophoresed.

[0129] The results of the electrophoresis are shown in Fig. 17. Nucleic acid amplification products were confirmed in the reaction solution without the addition of MBD2. When MBD2 was added, nucleic acid amplification was observed from the non-methylated p14_Gx5 plasmid, but nucleic acid amplification from the methylated p14_Gx4 plasmid was suppressed. From these results, it was shown that artificially CpG-methylated DNA can also be used as a template, and that CpG methylation is directly involved in nucleic acid amplification inhibition.

[0130] [Example 7] We examined whether the RPA reaction is suppressed by DNA-binding proteins other than dCas9 / gRNA and MBD2. The chicken DT40 #205-2 cell line has a LexA binding sequence to which the LexA protein, a bacterial DNA-binding protein, binds in the promoter region of the Pax5 gene (Non-Patent Document 6). Therefore, using the DT40 #205-2 cell genomic DNA as a template, we examined whether the RPA amplification of the LexA binding sequence is suppressed when the LexA protein is added to the reaction solution of the RPA reaction.

[0131] An RPA reaction was performed in the same manner as in Example 1, except that 20 ng of LexA protein was used instead of gRNA and dCas9, and after purification, electrophoresis was performed. As the LexA protein, a product synthesized by commissioning the DNA-binding domain of the LexA protein to Sisumex Co., Ltd. (ProCube, manufacturing number 13T_0170) was used. As the negative control protein, 20 ng of dCas9 protein was used. As the primer set, a forward primer (SEQ ID NO: 17) and a reverse primer (SEQ ID NO: 18) for amplifying the region containing the LexA binding sequence were used.

[0132] The results of the electrophoresis are shown in Fig. 18. Nucleic acid amplification was observed in the reaction solution to which no LexA protein was added. When the LexA protein was added, nucleic acid amplification from genomic DNA was suppressed. When dCas9 was added as the negative control protein, nucleic acid amplification from genomic DNA was not suppressed. From these results, it was shown that any DNA-binding protein other than the CRISPR complex and MBD2 protein can inhibit the target nucleic acid amplification. By using this technique, it is possible to evaluate whether a specific DNA-binding molecule has the ability to bind to a specific base sequence. It can also be used to detect whether there is a molecule having the ability to bind to a specific base sequence in a molecular population.

[0133] [Example 8] Single-stranded RNA in the test nucleic acid sample was detected as a target nucleic acid by RT-PCR. The mRNA of the human NEAT1 gene (NEAT1-RNA) was used as the target nucleic acid.

[0134] [gRNA and primers] In this experiment, single-stranded RNA consisting of the nucleotide sequences shown in Table 3 was used as gRNA. Among the nucleotide sequences of the gRNA in Table 3, the regions in capital letters are regions (RNA-binding regions) complementary to the partial region of the target nucleic acid NEAT1-RNA. The gRNA used was chemically synthesized by commissioning to Gene Design Inc. The gRNA was prepared by mixing 1 μL of 10 μM gRNA and 3 μL of nuclease-free water in advance, incubating at 100 °C for 2 minutes, and then cooling to room temperature before use in the experiment.

[0135]

Table 3

[0136] In this experiment, primers consisting of the nucleotide sequences shown in Table 4 were used. The forward primer (Human NEAT1-F2) and the reverse primer (Human NEAT1-R2) are primers designed to sandwich the cDNA sequence complementary to the RNA sequence targeted by dCas13a / gRNA_NEAT1. The forward primer (MY-0119) and the reverse primer (MY-0129) are primers designed to sandwich the cDNA sequence complementary to the RNA sequence targeted by dCas13a / gRNA_NEAT1_2. As a control, forward primer (hGAPDH-dCas13a-F3) and reverse primer (hGAPDH-dCas13a-R3) were used to amplify cDNA using the mRNA of the human GAPDH gene as a template. The primers used were chemically synthesized by commissioning to eurofin.

[0137]

Table 4

[0138] [Preparation of test nucleic acid samples] Total RNA extracted from human-derived cultured MRC-5 cells was used as the test nucleic acid sample. MRC-5 cells were cultured in E-MEM (Wako) medium containing 10% FBS (fetal bovine serum) and penicillin-streptomycin (Sigma). RNA from MRC-5 cells was extracted and purified using the commercially available RNA extraction reagent "Isogen II" (Nippon Gene).

[0139] [dCas13a] The dCas13a protein used was a mutant protein in which the point mutations R474A and R1046A were introduced into the wild-type Cas13a derived from Leptotrichia wadii, and the protein was synthesized by Sysmex Corporation (ProCube, serial number 17T_042).

[0140] [RT-PCR using gRNA_NEAT1] 0.46 μL of gRNA, 0.24 μg of dCas13a protein, and nuclease-free water were mixed to make 5 μL, which was used as the dCas13a / gRNA solution.

[0141] First, the reverse transcription reaction (RT) was performed using "ReverTraAce qPCR RT Master Mix with gDNA Remover" (TOYOBO). First, 2 μL of "4 × DN Master Mix" (gDNA Remover added), 1 ng of RNA, 5 μL of dCas13a / gRNA solution, and Nuclease-free water were mixed to make 8 μL, and incubated at 37 ° C for 5 minutes. Then, 2 μL of "5 × RT Master Mix II" was added to the reaction solution, and the reaction solution was incubated at 37 ° C for 30 minutes, then at 50 ° C for 5 minutes, and then at 98 ° C for 5 minutes to perform the reverse transcription reaction. After the reverse transcription reaction, 10 μL of Nuclease-free water was mixed with the solution to make 20 μL of cDNA solution.

[0142] Next, PCR was performed using "EmeraldAmp (registered trademark) MAX PCR Master Mix" (manufactured by Takara Bio Inc.). A PCR reaction solution containing 1 μL of cDNA, 0.5 μM of Human NEAT1-F2 primer and Human NEAT1-R2 primer in 10 μL was prepared. The reaction first involved denaturation at 94°C for 1 minute, followed by 35 cycles of 94°C for 15 seconds, 60°C for 15 seconds, and 72°C for 1 minute, and then treatment at 72°C for 1 minute.

[0143] [RT-PCR using gRNA_NEAT1_2] The preparation of the dCas13a / gRNA solution and RT were performed in the same manner as the RT-PCR using gRNA_NEAT1.

[0144] Next, PCR was performed using "AmpliTaq Gold (registered trademark) 360 Master Mix" (manufactured by ThermoFisher Scientific). A PCR reaction solution containing 1 μL of cDNA, 0.5 μM of MY-0119 primer and MY-0129 primer in 10 μL was prepared. The reaction first involved denaturation at 95°C for 10 minutes, followed by 38 cycles of 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, and then treatment at 72°C for 1 minute.

[0145] [RT-PCR of GAPDH] The amplification of GAPDH was performed using "EmeraldAmp (registered trademark) MAX PCR Master Mix" (manufactured by Takara Bio Inc.). A PCR reaction solution containing 1 μL of cDNA, 0.5 μM of hGAPDH-dCas13a-F3 primer and hGAPDH-dCas13a-R3 primer in 10 μL was prepared. The reaction first involved denaturation at 94°C for 1 minute, followed by 28 or 32 cycles of 94°C for 15 seconds, 60°C for 15 seconds, and 72°C for 1 minute, and then treatment at 72°C for 1 minute.

[0146] [Electrophoresis] The amplification products obtained in each amplification reaction were electrophoresed using an agarose gel containing "SYBR (registered trademark) Safe DNA Gel Stain" (manufactured by ThermoFisher Scientific).

[0147] The results of the amplification products of RT-PCR using gRNA_NEAT1 are shown in Fig. 19, and the results of the amplification products of RT-PCR using gRNA_NEAT1_2 are shown in Fig. 20, respectively. As shown in Fig. 19, compared with the reaction solutions without addition of dCas13a and with addition of dCas13a only, the amplification of NEAT1 by PCR was attenuated in the reaction solution added with the dCas13a / gRNA_NEAT1 complex solution. On the other hand, when the dCas13a / gRNA_NEAT1 complex amplified GAPDH which was not the target, such attenuation of DNA amplification was not observed. The same was confirmed in Fig. 20. From these results, it was shown that the dCas13a / gRNA_NEAT1 complex specifically inhibited the reverse transcription reaction of NEAT1, and it was confirmed that the sequence-specific reverse transcription reaction could be inhibited by the presence of the dCas13a / gRNA complex in the reaction solution.

[0148] [Example 9] It has already been reported that Stat5 binds to the Cis promoter 30 minutes after IL-3 stimulation (10 ng / mL) in Ba / F3 cells (Non-Patent Document 9). Therefore, Stat5 in the nuclear extract of Ba / F3 cells was detected using a double-stranded DNA having a Stat5 binding site (TTCNNNGAA) and a primer set for RPA amplification of the region containing the Stat5 binding site of the double-stranded DNA.

[0149] [Template Nucleic Acid] The region flanking the Stat5 binding site within the mouse Cis gene promoter (Cis region in Table 5: SEQ ID NO: 27) was used as the template nucleic acid, and a plasmid (Cis plasmid) into which this was inserted was prepared. This region contains four Stat5 binding sites. The white portions in SEQ ID NO: 27 in Table 5 are the Stat5 binding sites. Also, as a negative control, a mutant sequence (CisM region in Table 5: SEQ ID NO: 28) with a mutation introduced into the Stat5 binding site to abolish Stat5 binding ability was used as the template nucleic acid, and a plasmid (CisM plasmid) into which this was inserted was prepared. The white portions in SEQ ID NO: 28 in Table 5 are the Stat5 binding sites with mutations introduced.

[0150]

Table 5

[0151] First, using the genomic DNA of Ba / F3 cells as a template, a forward primer (mCis_-259 / -199_F) and a reverse primer (mCis_-188 / -104_R) flanking the Cis region within the mouse Cis gene promoter were used to amplify the nucleotide sequence flanked by both primers by PCR. For PCR, a PCR reaction solution containing 10 ng of Ba / F3 genomic DNA and 0.5 μM of each primer in 10 μL was prepared using "EmeraldAmp (registered trademark) MAX PCR Master Mix" (manufactured by Takara Bio Inc.). The reaction first involved denaturation at 94°C for 1 minute, followed by 35 cycles of 94°C for 15 seconds, 60°C for 15 seconds, and 72°C for 1 minute, and then treatment at 72°C for 1 minute.

[0152] The amplified product obtained was purified using "PCR / Gel DNA purification kit" (manufactured by Nippon Genetics Co., Ltd.), then cloned into T-Vector pMD20 (manufactured by Takara Bio Inc.), and amplified using "Competent Quick DH5a" (manufactured by Toyobo Co., Ltd.). The amplified plasmid was purified using "NucleoSpin (registered trademark) Plasmid QuickPure" (manufactured by Takara Bio Inc.) (Cis plasmid).

[0153] The CisM plasmid was prepared as follows. Using the Cis plasmid as a template, a forward primer (mCis_-259 / -199_mut_F) and a reverse primer (mCis_-188 / -104_mut_R) sandwiching the Stat5 binding site were used to amplify the nucleotide sequence sandwiched between the two primers by PCR. For PCR, a PCR reaction solution containing 1 pg of the Cis plasmid and 0.5 μM of each primer in 10 μL was prepared using "EmeraldAmp (registered trademark) MAX PCR Master Mix" (manufactured by Takara Bio Inc.). The reaction was first denatured at 94°C for 1 minute, followed by 30 cycles of 94°C for 15 seconds, 55°C for 15 seconds, and 72°C for 1 minute, and then treated at 72°C for 1 minute. The amplification product was purified in the same manner as the amplification product of the Cis region, incorporated into T-Vector pMD20 after purification, and further purified after amplification to obtain the CisM plasmid. As a result of DNA sequence analysis, although unexpected mutations were observed on the reverse primer side in the CisM plasmid, it was confirmed that the mutations shown in Table 5 were introduced into the Stat5 binding site.

[0154]

Table 6

[0155] [Preparation of nuclear extract of Ba / F3 cells] The nuclear extracts of Ba / F3 cells without IL-3 stimulation and Ba / F3 cells after IL-3 stimulation treatment were prepared as follows. Ba / F3 cells were cultured in RPMI-1640 medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% FBS, 10 mM HEPES buffer (pH 7.2) (manufactured by Nacalai Tesque), 1× non-essential amino acids, 1 mM sodium pyruvate (manufactured by Nacalai Tesque), 5 μM 2-mercaptoethanol (manufactured by Sigma-Aldrich), 1 ng / mL IL-3 (manufactured by ThermoFisher Scientific), and penicillin-streptomycin (manufactured by Nacalai Tesque).

[0156] Preparation of nuclear extracts without IL-3 stimulation and after IL-3 stimulation treatment: First, Ba / F3 cells were washed three times with PBS and then cultured in medium without IL-3 for 6 hours (without IL-3 stimulation). Then, IL-3 was added to the medium to a concentration of 10 ng / mL and cultured at 37°C for 30 minutes (IL-3 stimulation treatment). Ba / F3 cells without IL-3 stimulation and after IL-3 stimulation treatment were collected, and nuclear extracts were prepared using "NE-PER® Nuclear and Cytoplasmic Extraction Reagents" (manufactured by ThermoFisher Scientific).

[0157] [RPA reaction] The RPA reaction was performed as follows. First, to one tube of RPA reagent (product name "TwistAmp® Basic kit", manufactured by TwistDx), 29.5 μL of rehydration buffer, 2.5 μL of 10 μM forward primer (M13 Primer RV), 2.5 μL of 10 μM reverse primer (M13 Primer M4), and nuclease-free water were added to make 50 μL and mixed. Then, 10 μL of the prepared solution was aliquoted into another tube, 1 pg of plasmid DNA (Cis plasmid or CisM plasmid) and 3 ng of nuclear extract were added, and then 10 mM Tris (pH 8.0) was added to prepare 11.3 μL of reaction solution. The reaction solution was incubated at 37°C for 5 minutes, then 1 μL of 280 mM MgOAc solution was added, and incubated at 37°C for 30 minutes to perform the RPA reaction. After the reaction, the reaction solution was not purified and 2 μL was electrophoresed.

[0158]

Table 7

[0159] The results of electrophoresis are shown in Fig. 21. In the RPA reaction using the Cis plasmid as a template, in the reaction solution to which the nuclear extract after IL-3 stimulation treatment was added, DNA amplification by RPA was attenuated compared to the reaction solution to which the nuclear extract without IL-3 stimulation was added. This is because Stat5 present in the nuclear extract after IL-3 stimulation treatment binds to the Stat5 binding site in the Cis plasmid, inhibiting DNA amplification. However, Stat5 was not present in the nuclear extract without IL-3 stimulation, and the RPA reaction was not inhibited. On the other hand, in the RPA reaction using the CisM plasmid as a template, similar levels of DNA amplification were observed regardless of the presence or absence of IL-3 stimulation treatment. From the above results, it was possible to evaluate whether a protein that binds to the target DNA is present in a solution such as a cell lysate by nucleic acid amplification by RPA.

[0160] [Example 10] Using a genomic DNA library as a template, an RPA reaction was performed in the presence of a CpG methylated DNA binding protein to examine whether DNA that is not CpG methylated is amplified.

[0161] [Genomic DNA Library] The DNA library was prepared by requesting Promega Corporation using genomic DNA extracted and purified from HCT116 cells (product number: KK0500). The adapter sequence added during DNA library preparation included index15 (ATGTCA) for next-generation sequencing by Illumina. The addition of the adapter was performed without using DNA amplification operations.

[0162] [RPA Reaction] The RPA reaction was performed as follows using primers complementary to the adapter sequence. First, 29.5 μL of rehydration buffer, 2.5 μL of 10 μM forward primer (P5-NGS-Lib-Promega-RPA-F), 2.5 μL of 10 μM reverse primer (P7-NGS-Lib-Promega-RPA-R), and 9.5 μL of nuclease-free water were added to and mixed with 1 tube (containing lyophilized reagent) of RPA reagent (product name "TwistAmp® Basic kit", manufactured by TwistDx). Next, 17.5 μL of the prepared solution was aliquoted into another tube, and 0.5 μL of DNA library, 0.5 μg of MBD2 protein ("EpiXplore® Methylated DNA Enrichment Kit", manufactured by Takara Bio Inc.), and nuclease-free water were added to prepare 19 μL of RPA reaction preparation solution. This RPA reaction preparation solution was incubated at 37°C for 10 minutes, and then 1 μL of 280 mM MgOAc solution was added and incubated at 37°C for 10 minutes to perform the RPA reaction. After the reaction was completed, nucleic acids were purified from the reaction solution using "PCR / Gel DNA purification kit" (manufactured by Nippon Genetics Co., Ltd.).

[0163] [Analysis of Amplification Products of RPA Reaction] In the CDKN2A (p14ARF) gene of HCT116 cells, only one G is deleted at one allele (Gx4). Also, among the CDKN2A (p14ARF) genes of HCT116 cells, the cytosine around the Gx4 position in the Gx4 allele is not methylated, while the cytosine around the Gx5 position in the Gx5 allele is methylated. Therefore, when MBD2 protein was added to the reaction solution of the RPA reaction, it was confirmed by PCR whether the RPA amplification using the Gx5 allele as a template was suppressed. Specifically, a forward primer (HP14ARF-Ex1-F) and a reverse primer (HP14ARF-Ex1-R) were used to amplify the nucleotide sequence sandwiched between both primers by PCR so as to amplify the CDKN2A (p14ARF) gene. PCR was performed using "AmpliTaq Gold (registered trademark) 360 Master Mix" (manufactured by ThermoFisher Scientific). A PCR reaction solution containing 0.5 μL of DNA after the RPA reaction, 0.5 μM of HP14ARF-Ex1-F primer and HP14ARF-Ex1-R primer in 10 μL was prepared. The PCR reaction was first denatured at 95°C for 10 minutes, followed by 30 cycles of 15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C, and then treated at 72°C for 1 minute.

[0164]

Table 8

[0165] After the PCR reaction, 2 μL of the reaction solution was electrophoresed without purification. As a result, PCR amplification products were confirmed regardless of the presence or absence of the addition of MBD2 protein in the RPA reaction. When the nucleotide sequences of the respective amplification products were confirmed, when PCR was performed using as a template the DNA that was the amplification product of the RPA reaction without the addition of MBD2 protein, both Gx4-type p14ARF and Gx5-type p14ARF were amplified. On the other hand, when the RPA reaction was performed with the addition of MBD2 protein and PCR was performed using as a template the DNA that was the amplification product of the obtained RPA reaction, Gx4-type p14ARF was predominantly amplified and the amplification of Gx5-type p14ARF was decreased. From these results, when using a primer set that amplifies the entire library with genomic DNA library as a template, by having a CpG methylation DNA-binding protein present in the reaction solution of the RPA reaction, nucleic acid amplification using CpG-methylated DNA as a template is inhibited, and it was shown that only non-CpG-methylated DNA can be amplified.

[0166] Using the amplification product after the RPA reaction as a template, the presence or absence of amplification of CpG-methylated DNA was confirmed by PCR amplification and DNA sequence analysis (Sanger sequencing), but next-generation sequencing analysis can also be used instead of DNA sequence analysis. That is, by comprehensively analyzing the nucleotide sequence of the amplification product of the RPA reaction performed in the presence of MBD2 protein with genomic DNA library as a template by next-generation sequencing and comparing it with the nucleotide sequence of the amplification product of the RPA reaction performed in the absence of MBD2 protein with the same genomic DNA library as a template, CpG methylation sites on the genome can be comprehensively identified.

Claims

1. A method for identifying and detecting a target nucleic acid from a non-target nucleic acid having a base sequence different from a part of the target nucleic acid, comprising: designating, as a target region, a region in the non-target nucleic acid that has a base sequence different from the target nucleic acid; designating, as a corresponding target region, a region in the target nucleic acid that has a base sequence different from the non-target nucleic acid; using a test nucleic acid sample as a template; in the presence of a molecule (excluding a molecule consisting only of nucleic acids) that specifically binds to the target region in the non-target nucleic acid; using a primer that hybridizes to both the target nucleic acid and the non-target nucleic acid; performing a nucleic acid amplification reaction under temperature conditions under which the molecule can bind to the non-target nucleic acid, and detecting the target nucleic acid based on the presence or absence of an amplification product; wherein the molecule is a complex of a DNA strand cleavage activity-deficient Cas9 protein and a gRNA; A method for detecting a target nucleic acid, wherein the nucleic acid amplification reaction is performed under temperature conditions of 65° C. or lower.

2. The method for detecting a target nucleic acid according to claim 1, wherein the gRNA specifically recognizes and binds to DNA consisting of a base sequence complementary to the target region in the non-target nucleic acid.

3. A method for identifying and detecting a target nucleic acid from a non-target nucleic acid having a base sequence different from a part of the target nucleic acid, comprising: designating, as a target region, a region in the non-target nucleic acid that has a base sequence different from the target nucleic acid; designating, as a corresponding target region, a region in the target nucleic acid that has a base sequence different from the non-target nucleic acid; using a test nucleic acid sample as a template; in the presence of a molecule (excluding a molecule consisting only of nucleic acids) that specifically binds to the target region in the non-target nucleic acid; using a primer that hybridizes to both the target nucleic acid and the non-target nucleic acid; performing a nucleic acid amplification reaction under temperature conditions under which the molecule can bind to the non-target nucleic acid, and detecting the target nucleic acid based on the presence or absence of an amplification product; wherein the molecule is a complex of an RNA strand cleavage activity-deficient Cas13a protein and a gRNA; A method for detecting a target nucleic acid, wherein the nucleic acid amplification reaction is performed under temperature conditions of 65° C. or lower.

4. The method for detecting a target nucleic acid according to claim 3, wherein the gRNA specifically recognizes and binds to RNA consisting of a base sequence complementary to the target region in the non-target nucleic acid.

5. The method for detecting a target nucleic acid according to any one of claims 1 to 4, wherein when an amplification product is obtained by the nucleic acid amplification reaction, the test nucleic acid sample contains the target nucleic acid.

6. The method for detecting a target nucleic acid according to any one of claims 1 to 5, wherein the nucleic acid amplification reaction is an isothermal nucleic acid amplification reaction.

7. The method for detecting a target nucleic acid according to any one of claims 1 to 6, wherein the nucleic acid amplification reaction is a Recombinase Polymerase Amplification method.

8. The method for detecting a target nucleic acid according to any one of claims 1 to 7, wherein the target region in the target nucleic acid and the target region in the non-target nucleic acid have different nucleotide sequences.

9. The method for detecting a target nucleic acid according to claim 8, wherein the target region is a mutation site of a gene mutation or a polymorphism site of a gene polymorphism.

10. A kit for use in the method for detecting a target nucleic acid according to claim 1 or 2, comprising: a primer that hybridizes to both the target nucleic acid and the non-target nucleic acid; a DNA strand cleavage activity-deficient Cas9 protein; a gRNA; A kit for detecting a target nucleic acid.

11. The kit for detecting a target nucleic acid according to claim 10, further comprising a recombinase, a single-stranded DNA binding protein, and a DNA polymerase.

12. A kit for use in the method for detecting a target nucleic acid according to claim 3 or 4, comprising: a primer that hybridizes to both the target nucleic acid and the non-target nucleic acid; an RNA strand cleavage activity-deficient Cas13a protein; a gRNA; A kit for detecting a target nucleic acid.

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