Primer, DNA detection method and DNA detection kit

Abstract

The invention relates to a nucleic acid amplification reaction, in particular to a primer, a DNA (deoxyribonucleic acid) detection method and a DNA detection kit in an asymmetric nucleic acid amplification reaction. Specifically, the present invention relates to a primer for introducing and amplifying a mutation into a nucleic acid to be detected, and a DNA detection method and a DNA detection kit using the primer, the primer comprising a mutation in the sequence thereof for introducing a mutation into the nucleic acid to be detected, the ratio of a single strand of a binding region of a probe to a nucleic acid obtained by amplifying the nucleic acid to be detected is increased at a temperature at which the nucleic acid obtained by amplifying the nucleic acid to be detected binds to the probe.

Description

Technical Field

[0001] The invention relates to a nucleic acid amplification reaction, in particular to primers in an asymmetric nucleic acid amplification reaction, a DNA detection method and a DNA detection kit. Background Art

[0002] There are methods such as polymerase chain reaction (PCR), real-time PCR, and digital PCR in gene detection. In real-time PCR and digital PCR, which can perform quantitative analysis with higher accuracy than PCR, DNA is detected using intercalators or fluorescently labeled probes. Hydrolysis probes and molecular beacons are often used in the detection of DNA using fluorescently labeled probes. Hydrolysis probes use the nuclease activity of DNA polymerase to degrade the probe, so that the fluorescent pigment is released from the probe and emits fluorescence. Unlike such hydrolysis probes, molecular beacons form stem loops in a free state and bind to the DNA of the detection object in the loop portion. They have the characteristic of not being degraded in PCR. After PCR, not only can the fluorescence intensity be used to determine whether amplification has occurred, but also melting curve analysis can be performed.

[0003] Among them, the following method has been reported: in DNA detection using molecular beacons, in order to increase the amount of binding between the molecular beacon and the amplified DNA of the detection object and to increase the fluorescence intensity, the concentrations of the forward primer and the reverse primer are made asymmetric in such a way that the chain complementary to the molecular beacon is over-amplified, thereby asymmetric amplification of the DNA of the detection object is performed (non-patent document 1).

[0004] The inventors of the present invention have developed a technology that applies an asymmetric nucleic acid amplification reaction using a molecular beacon to digital PCR and identifies the genotype of a target gene with high sensitivity and high multiplex by melting curve analysis after amplification (Patent Document 1, Non-Patent Document 2).

[0005] The following is an example of a digital PCR detection method. First, DNA polymerase, primers, and fluorescently labeled probes required for PCR are added to the limiting dilution test body to prepare a PCR reaction solution. The PCR reaction solution is divided into micro-partitions such as wells or droplets, so that each partition is either loaded with one molecule of the target gene or does not contain the target gene.

[0006] Next, the target gene in the micropartition is amplified by PCR. After PCR, the fluorescence intensity of each micropartition is measured, and the micropartitions having a fluorescence intensity exceeding a threshold value are counted, thereby enabling the target gene to be quantified.

[0007] When an asymmetric nucleic acid amplification reaction using a molecular beacon is applied, melting curve analysis of the target gene amplified in micropartitions and the molecular beacon is performed after PCR, and the different melting temperatures (Tm) of the target gene for each genotype can be observed, thereby enabling discrimination.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: Japanese Patent Application Publication No. 2018-108063

[0011] Non-patent literature

[0012] Non-patent document 1: BMC Microbiol., 13, pp295, 2013

[0013] Non-patent literature 2: Anal. Chem., 92, pp11705-11713, 2020 Summary of the invention

[0014] Technical problem to be solved by the invention

[0015] However, the inventors of the present invention first recognized that there are problems in asymmetric nucleic acid amplification reactions: one strand of the over-amplified DNA forms a secondary structure in the molecule, and the molecular beacon designed in the region that becomes a single-stranded structure binds to the amplified detection target DNA, while the molecular beacon designed in the region that becomes a double-stranded structure has a reduced binding ratio, and the fluorescence intensity decreases. In particular, in the case of PCR performed in micro-partitions such as digital PCR instead of PCR performed in a test tube, once the fluorescence intensity decreases, it will become less than the detectable intensity and cannot be detected. In melting curve analysis, the change in fluorescence intensity is small relative to the temperature change, and the melting temperature cannot be calculated, or it cannot be easily measured, and the measurement sensitivity and accuracy decrease.

[0016] Therefore, the purpose of the present invention is to provide novel primers, DNA detection methods and DNA detection kits, which can reduce the proportion of double-stranded structure formed by the molecular beacon binding region of one chain of over-amplified DNA in an asymmetric nucleic acid amplification reaction, increase the proportion of single-stranded structure formed, inhibit the reduction of the binding ratio between the molecular beacon and the amplified detection object DNA, and detect the detection object gene with good accuracy and high sensitivity to perform genotype discrimination.

[0017] Technical solutions for solving technical problems

[0018] The inventors of the present invention have found that even when the same primer set is used for amplification in an asymmetric nucleic acid amplification reaction, the fluorescence intensity varies greatly depending on the binding region of the molecular beacon, and the fluorescence intensity of the molecular beacon is low in the region where one strand after amplification is designed to form a double-stranded structure in the molecule. Based on this, the inventors have found that by intentionally introducing a mutation different from the sequence of the target gene into the primers used in the asymmetric nucleic acid amplification reaction and performing the asymmetric nucleic acid amplification reaction, the ratio of the binding region of the molecular beacon forming a double-stranded structure is reduced and the fluorescence intensity is increased, thereby completing the present invention.

[0019] One embodiment of the present invention is a method for detecting a target nucleic acid, comprising:

[0020] A step of amplifying the test nucleic acid in the presence of a primer pair including a forward primer and a reverse primer and a probe; and

[0021] a step of measuring the binding between the nucleic acid amplified by the forward primer and the reverse primer and the probe,

[0022] Either or both of the forward primer or the reverse primer contains a mutation in its sequence, and the mutation is used to introduce a mutation into the amplified nucleic acid, so that the proportion of single-stranded bases in the probe binding region in the amplified nucleic acid is increased at a temperature at which the probe binds to the amplified nucleic acid.

[0023] Another embodiment of the present invention is a primer for introducing a mutation into a test nucleic acid and performing amplification, wherein the primer contains a mutation in its sequence, and the mutation is used to introduce a mutation into the test nucleic acid so as to increase the proportion of single-stranded bases in the binding region of the probe in the nucleic acid amplified from the test nucleic acid at a temperature at which the nucleic acid amplified from the test nucleic acid binds to the probe.

[0024] Another embodiment of the present invention is a kit for detecting a target nucleic acid, comprising:

[0025] a primer pair comprising a forward primer and a reverse primer; and

[0026] a probe capable of binding to a nucleic acid amplified using a forward primer and a reverse primer,

[0027] Either or both of the forward primer or the reverse primer contains a mutation in its sequence, which is used to introduce a mutation into the amplified nucleic acid so as to increase the proportion of single-stranded bases in the probe binding region of the amplified nucleic acid at a temperature at which the probe binds to the amplified nucleic acid.

[0028] Effects of the Invention

[0029] According to the present invention, novel primers, DNA detection methods and DNA detection kits can be provided, which can detect or quantify target genes with higher accuracy and high sensitivity in asymmetric nucleic acid amplification reactions. Therefore, the present invention is useful in the fields of basic research, inspection, drug development, etc., such as gene detection and genotyping. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram showing an example of a method for measuring the melting temperature of DNA using a fluorescently labeled probe.

[0031] Figure 2 This is a schematic diagram showing an example of the behavior of a fluorescent-labeled probe in a symmetric nucleic acid amplification reaction and an asymmetric nucleic acid amplification reaction.

[0032] Figure 3 This is a schematic diagram showing the binding between the secondary structure of the DNA to be detected and the fluorescent labeled probe obtained by amplification in an asymmetric nucleic acid amplification reaction using a primer into which a mutation is introduced in one embodiment of the present invention.

[0033] Figure 4 This is a schematic diagram showing the binding between the secondary structure of the DNA to be detected and the fluorescent labeled probe obtained by amplification in an asymmetric nucleic acid amplification reaction using a primer for introducing a mutation having a tag in one embodiment of the present invention.

[0034] Figure 5 This is a diagram showing an example of a melting curve when a mutation-introducing primer is used in one embodiment of the present invention.

[0035] Figure 6 This is a flow chart showing one embodiment of a DNA detection method using a mutation-introducing primer to perform an asymmetric nucleic acid amplification reaction.

[0036] Fig. 7A This is a diagram showing the positional relationship between the secondary structure of the single-stranded DNA to be detected amplified in the example of the present invention and the binding region of the fluorescent labeled probe corresponding to the wild type.

[0037] Figure 7B This is a diagram showing the positional relationship between the secondary structure of the single-stranded DNA to be detected amplified in the example of the present invention and the binding region of the fluorescent labeled probe corresponding to the mutant type.

[0038] Figure 7C It is a graph showing the measurement results (differential curve of melting curve) when no mutation-introducing primer is used in the Examples of the present invention.

[0039] Fig. 8AThis is a diagram showing the positional relationship between the secondary structure of the amplified single-stranded DNA to be detected, the binding region of the fluorescent-labeled probe corresponding to the wild type, and the planned position of the mutation introduced by the primer.

[0040] Figure 8B This is a diagram showing the positional relationship between the secondary structure of the single-stranded DNA to be detected amplified in the examples of the present invention, the binding region of the fluorescent labeled probe corresponding to the mutant type, and the position of the mutation introduced by the primer.

[0041] Fig. 9A This is a diagram showing the positional relationship between the secondary structure of the amplified single-stranded DNA to be detected, the binding region of the fluorescent-labeled probe corresponding to the wild type, and the planned position of the mutation introduced by the primer.

[0042] Fig. 9B This is a diagram showing the positional relationship between the secondary structure of the single-stranded DNA to be detected amplified in the examples of the present invention, the binding region of the fluorescent labeled probe corresponding to the mutant type, and the position of the mutation introduced by the primer. DETAILED DESCRIPTION

[0043] According to the description of this specification, those skilled in the art can understand the purpose, features, advantages and ideas involved in the present invention. As long as they are skilled in the art, they can easily reproduce the present invention according to the description of this specification. The following embodiments of the invention and specific examples etc. represent preferred embodiments of the present invention, which are shown for the purpose of illustration or description, and the present invention is not limited thereto. Those skilled in the art can make various changes and modifications based on the description of this specification without departing from the intention and scope of the present invention disclosed in this specification.

[0044] (1) Primers

[0045] In one embodiment, the present invention provides a primer for introducing a mutation into a test nucleic acid and amplifying the primer, wherein the primer includes a mutation in its sequence, and the mutation is used to introduce a mutation into the test nucleic acid so that the ratio of single-stranded bases in the binding region of the probe in the nucleic acid amplified from the test nucleic acid is increased at a temperature at which the nucleic acid amplified from the test nucleic acid binds to the probe. The primer can also be used as a reagent for introducing a mutation into a test nucleic acid and amplifying the primer.

[0046] In the present invention, the nucleic acid to be tested is not particularly limited as long as it contains the target nucleic acid to be detected or has the possibility of containing the target nucleic acid, for example, it contains messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, genomic DNA, and their fragments, hybrid nucleic acids of DNA and RNA, etc., and can be single-stranded or double-stranded. In the case where the target nucleic acid to be detected is RNA such as mRNA, the nucleic acid to be tested can be DNA (cDNA, etc.) obtained by reverse transcription of its RNA.

[0047] The primer can be a forward primer or a reverse primer, and both primers can contain mutations. Preferably, the primer is a reverse primer.

[0048] The primer contains a mutation for introducing a mutation into the test nucleic acid and amplifying it. The mutation is introduced in a manner that increases the ratio of single-stranded bases (the ratio of single-stranded bases to double-stranded bases) in the binding region of the probe in the nucleic acid amplified from the test nucleic acid at a temperature at which the nucleic acid amplified from the test nucleic acid binds to the probe. The nucleic acid amplified from the test nucleic acid has a single-stranded structure, and sometimes forms a double-stranded structure inside due to the presence of complementary bases. What kind of structure the amplified nucleic acid forms varies depending on its sequence composition, and programs for predicting such structures are commonly used in this technical field (for example, OligoAnalyzer TM Tool (IDT), RNAfold (University of Vienna)). Therefore, using such a program, the structure of the amplified nucleic acid is predicted, and the ratio of single-stranded bases in the probe binding region in the amplified nucleic acid is determined. When the ratio of single-stranded bases is low, for example, less than 50%, a mutation is introduced into the amplified nucleic acid so that the ratio of single-stranded bases is increased, for example, to at least 50%, preferably 60% or more.

[0049] In addition to including the above-mentioned mutations, the primers can be designed by conventional primer design methods and can be designed to have a length, sequence composition, and melting temperature suitable for specific binding to the target nucleic acid.

[0050] In order to increase the ratio of the above-mentioned single-stranded bases, the primer may also include a tag sequence. Such a tag sequence is set to a length and composition that does not affect the detection of the target nucleic acid. The tag sequence may be included in either the forward primer or the reverse primer, or both may further include a tag sequence. The nucleic acid amplified using such a primer includes the tag sequence.

[0051] One embodiment disclosed in this specification is a primer for asymmetric nucleic acid amplification reaction, in which a mutation is introduced so that the fluorescent labeled probe binding region of the DNA to be detected has a lower proportion of double-stranded structure and an increased proportion of single-stranded structure at a temperature at which the fluorescent labeled probe such as a molecular beacon binds. Figures 1 to 5 The primers are described in detail with reference to the schematic diagram of FIG.

[0052] Figure 1 Schematic diagram showing the behavior of the fluorescently labeled probe when the temperature of the reaction solution is changed. Examples of fluorescently labeled probes include molecular beacons and Taqman probes, but molecular beacons are used as an example here. Figure 1 Provide explanation.

[0053] The molecular beacon is composed of an oligonucleotide and has a sequence complementary to a sequence between a primer pair used in a nucleic acid amplification reaction for amplifying a target gene. In addition, the molecular beacon has sequences complementary to each other at both ends, and a fluorescent dye 103 is provided at one end and an extinction dye (quencher) 104 is provided at the other end. In the nucleic acid amplification reaction, in the initial state, Figure 1 As shown in B, the molecular beacon 102 exists alone and free. At this time, the molecular beacon 102 forms a stem loop, and the fluorescent dye 103 is close to the quencher 104, so it does not emit fluorescence. In the initial denaturation step, if the sample solution is heated, as shown in Figure 1 The structure is highly free as shown in C, but since the fluorescent pigment and the extinction pigment are always inseparable, the fluorescence remains in the extinction state. In the annealing process, when the temperature is lowered to room temperature, as shown in Figure 1 As shown in A, the loop portion of the molecular beacon 102 anneals with the DNA 101 amplified in the sample solution. As a result, the fluorescent dye 103 and the quencher 104 are separated, so that the fluorescent labeled probe 102 emits strong fluorescence. In the next extension step, the molecular beacon 102 is freed and becomes Figure 1 The fluorescence is extinguished in the state shown in B. In the next denaturation step, it becomes Figure 1 The fluorescence remains in the extinction state. In the nucleic acid amplification reaction, since the process is repeated, the fluorescence intensity can be measured at a certain stage during heating or cooling. When the fluorescence intensity is measured after the nucleic acid amplification reaction is completed, the measurement can be performed in the same manner. After the nucleic acid amplification reaction is completed, the fluorescence intensity can also be measured by heating or cooling only for the purpose of measuring the fluorescence intensity.

[0054] When a DNA intercalator is used instead of a combination of a fluorescent dye and a quencher, when the test DNA is double-stranded, the DNA intercalator intercalates between the double strands to emit fluorescence, but when the test DNA is single-stranded, the DNA intercalator is free and the fluorescence is extinguished. Therefore, in the nucleic acid amplification reaction, as with the molecular beacon 102, at a certain stage, the fluorescence intensity can be measured during heating or cooling. After the nucleic acid amplification reaction is completed, heating or cooling can also be performed to measure the fluorescence intensity only for the purpose of measuring the fluorescence intensity.

[0055] In the molecular beacon 102 used here, the combination of the fluorescent dye 103 and the quencher 104 is not particularly limited as long as it is a combination generally used in real-time PCR. For example, examples of the fluorescent dye 103 include FAM, VIC, ROX, Cy3, Cy5, etc., and examples of the quencher 104 include TAMRA, BHQ1, BHQ2, BHQ3, etc. All of them can be conventionally used and can be obtained as commercial products.

[0056] When two genes having different sequences are used as target genes, the sequences of the molecular beacon 102 are prepared so as to specifically bind to the target genes respectively, and by binding them to different fluorescent dyes, the two target genes can be distinguishably detected in one reaction system.

[0057] As a DNA intercalator, there is no particular limitation as long as the fluorescence intensity increases by binding to double-stranded DNA and the substance can be used in the detection of double-stranded DNA. Specifically, SYBR (registered trademark) Green I, SYBR Gold, PicoGreen (registered trademark), SYTO (registered trademark) Blue, SYTO Green, SYTO Orange, SYTORed, POPO (registered trademark) -1, BOBO (registered trademark) -1, YOYO (registered trademark) -1, TOTO (registered trademark) -1, JOJO (registered trademark) -1, POPO-3, LOLO (registered trademark) -1, BOBO-3, YOYO-3, TOTO-3, PO-Pro (registered trademark) -1, YO-Pro (registered trademark) -1, TO-Pro (registered trademark) -1, JO-Pro (registered trademark) -1, PO-Pro-3, YO-Pro-3, TO-Pro-3, TO-Pro-5, ethidium bromide, etc. can be used, and all of them can be obtained as commercial products. When the DNA intercalator is heat-resistant, it can be added to the reaction solution before the nucleic acid amplification reaction.

[0058] Figure 2 Schematic diagram showing the behavior of fluorescent labeled probes in symmetric nucleic acid amplification reactions and asymmetric nucleic acid amplification reactions. Figure 2As shown in A, when the concentrations of the forward primer 201 and the reverse primer 202 are the same, the DNA 204 to be detected is amplified equally on both complementary chains, so a large number of molecules 205 formed by the complementary chains forming double chains are generated, and a small number of molecules 206 formed by the fluorescent labeled probe 203 being bound to the single-chain molecules are generated. Figure 2 As shown in B, in the case where the concentrations of the forward primer 201 and the reverse primer 202 are asymmetric ( Figure 2 In the case of increasing the reverse primer 202 in B), since one strand 207 of the target DNA 204 that becomes the complementary strand of the molecular beacon is excessively amplified, a large amount of molecules 206 are generated in which the fluorescent labeled probe 203 is bound to the single-stranded molecules. By performing the asymmetric nucleic acid amplification reaction in this way, the amount of the fluorescent labeled probe bound to the amplified target DNA increases, and the fluorescence intensity increases.

[0059] Figure 3 Schematic diagram showing the binding of the secondary structure of the DNA to be detected obtained by amplification using a primer into which a mutation is introduced and the fluorescently labeled probe. Figure 3 As shown in A, a forward primer 301, a reverse primer 302 into which a mutation 309 is introduced, and a fluorescent labeled probe 303 are prepared for a DNA 304 to be detected. At this time, the concentration of the reverse primer 302 is excessive relative to the concentration of the forward primer 301, and an asymmetric nucleic acid amplification reaction is performed. Figure 3 B is a schematic diagram showing the binding of the secondary structure 305 of the single-stranded target DNA obtained by amplification through an asymmetric nucleic acid amplification reaction using a reverse primer that does not introduce mutation 309 and the fluorescent labeled probe 303 (existing method). Since mutation 309 is not introduced, position 306 on the secondary structure 305 of the amplified DNA maintains the original base state of the target DNA. In the secondary structure 305 of the single-stranded target DNA obtained by amplification, the binding region of the fluorescent labeled probe 303 forms a double-stranded structure, and the binding amount of the fluorescent labeled probe 303 is reduced. On the other hand, Figure 3 C is a schematic diagram showing the binding of the secondary structure 307 of the single-stranded detection target DNA amplified by an asymmetric nucleic acid amplification reaction using a mutation-introducing reverse primer 302 and the fluorescent-labeled probe 303 (the present invention). The secondary structure 307 of the amplified single-stranded detection target DNA forms a fluorescent-labeled probe 303 by the mutation 308 introduced by the mutation-introducing reverse primer 302. Figure 3 The secondary structure 305 of the single-stranded DNA to be detected obtained by amplification of B is different from that of the secondary structure 305 of the single-stranded DNA to be detected. Figure 3 Compared with B, Figure 3The proportion of double-stranded structures in C is reduced, and the proportion of single-stranded structures is increased, thereby increasing the binding amount of the fluorescent labeled probe 303.

[0060] Figure 4 Schematic diagram showing the binding of the secondary structure of the DNA to be detected and the fluorescent labeled probe when the amplified DNA is subjected to an asymmetric nucleic acid amplification reaction using a primer for introducing a mutation with a label. Figure 4 As shown in A, for the DNA 404 to be detected, a forward primer 401, a mutation-introducing reverse primer 402 to which a tag sequence 405 is added and a mutation 411 is introduced, and a fluorescently labeled probe 403 are prepared. At this time, the concentration of the reverse primer 402 becomes excessive relative to the concentration of the forward primer 401, and an asymmetric nucleic acid amplification reaction is performed. Figure 4 B is a schematic diagram showing the binding of the secondary structure 406 of the single-stranded target DNA obtained by amplification by an asymmetric nucleic acid amplification reaction using a reverse primer (not shown) without a tag sequence and without introducing a mutation 411, and the fluorescent labeled probe 403 (with Figure 3 Since the mutation 411 is not introduced, the position 407 on the secondary structure 406 of the amplified DNA maintains the original base state of the DNA to be detected. In the secondary structure 406 of the amplified single-stranded DNA to be detected, the binding region of the fluorescent labeled probe 403 forms a double-stranded structure, and the binding amount of the fluorescent labeled probe 403 is reduced. On the other hand, Figure 4 FIG. 4 is a schematic diagram showing the binding of the secondary structure 408 of the single-stranded detection target DNA amplified by an asymmetric nucleic acid amplification reaction using the labeled mutation introduction reverse primer 402 and the fluorescent labeling probe 403. The secondary structure 408 of the amplified single-stranded detection target DNA is formed by the mutation 409 introduced by the labeled mutation introduction reverse primer 402 and the tag sequence addition 410. Figure 4 The secondary structure 406 of the single-stranded DNA to be detected obtained by amplification of B is different from that of the secondary structure 406 of the single-stranded DNA to be detected. Figure 4 Compared with B, Figure 4 The proportion of double-stranded structures in C is reduced, and the proportion of single-stranded structures is increased, thereby increasing the binding amount of the fluorescent labeled probe 403.

[0061] Figure 5 The melting curves of the case where the asymmetric nucleic acid amplification reaction is performed using the mutation-introducing primer and the case where the asymmetric nucleic acid amplification reaction is performed without the mutation-introducing primer are shown. When the asymmetric nucleic acid amplification reaction is performed using the mutation-introducing primer, the change in the fluorescence intensity of the solution accompanying the temperature change is measured. Figure 5 As shown in A, the melting curve with large changes in fluorescence intensity is obtained. When the differential curve is obtained, the following is shown: Figure 5 The peak temperature is the melting temperature (Tm) 501. However, when an asymmetric nucleic acid amplification reaction is performed without using a mutation-introducing primer, when the change in the fluorescence intensity of the solution accompanying the temperature change is measured, the result is as follows: Figure 5 As shown in C, the fluorescence intensity is compared with Figure 5 A decreases. Therefore, when finding its differential curve, we get Figure 5 As shown in D, it has a low peak and the peak shape becomes smooth, so it may become an erroneous value different from the melting temperature of the target gene in the solution. In addition, if the degree of reduction in fluorescence intensity is large, the fluorescence intensity caused by the amplification of the target gene will be lower than the detectable intensity and cannot be detected.

[0062] (2) DNA detection method

[0063] In another embodiment, the present invention provides a method for detecting a target nucleic acid, comprising:

[0064] A step of amplifying a test nucleic acid in the presence of a primer pair comprising a forward primer and a reverse primer and a probe;

[0065] a step of measuring the binding between the nucleic acid amplified by the forward primer and the reverse primer and the probe,

[0066] Either or both of the forward primer or the reverse primer contains a mutation in its sequence, and the mutation is used to introduce a mutation into the amplified nucleic acid, so that the proportion of single-stranded bases in the probe binding region in the amplified nucleic acid is increased at a temperature at which the probe binds to the amplified nucleic acid.

[0067] In this specification, detection of a target nucleic acid refers to examining the presence or absence of a nucleic acid to be detected, measuring the concentration or abundance ratio of a nucleic acid to be detected, identifying a nucleic acid to be detected, determining a genotype, and the like.

[0068] Either or both of the forward primer and the reverse primer are the primers described in the preceding item.

[0069] The probe contains a fluorescent pigment, or contains a fluorescent pigment and an extinction pigment. When the probe contains a fluorescent pigment, the binding of the amplified nucleic acid to the probe is determined using the fluorescent pigment. In one embodiment, the 3' terminal sequence and the 5' terminal sequence of the probe have complementary sequences, and when not binding to the amplified nucleic acid, the 3' terminal sequence of the probe binds to the 5' terminal sequence to form a stem structure. When the probe does not contain a fluorescent pigment, for example, a DNA intercalator can also be used.

[0070] The amplification step can be performed by any amplification reaction as long as it is an amplification reaction known in the art. Preferably, the amplification step is performed by an asymmetric nucleic acid amplification reaction. In this case, the amount of addition of either the forward primer or the reverse primer (e.g., a primer containing a mutation) is increased to perform the amplification reaction. In a preferred embodiment, a reverse primer containing a mutation is added in larger amounts than the forward primer to perform the amplification reaction. For asymmetric nucleic acid amplification reactions, reference may be made to, for example, the description of Anal.Chem., 92, pp11705-11713, 2020 (non-patent document 2).

[0071] In one embodiment, the binding between the amplified nucleic acid and the probe is measured with temperature changes. The binding changes according to the melting temperature, and therefore the melting temperature of the double strand of the amplified nucleic acid and the probe can be calculated from the change in binding with temperature changes.

[0072] In one embodiment, the binding between the amplified nucleic acid and the probe can be measured for each cycle of temperature change, and the target nucleic acid can be detected based on the relationship between the number of cycles and the change in binding.

[0073] By using a plurality of primers containing mutations, a plurality of target nucleic acids can be detected. In this case, the binding of the amplified nucleic acid to the probe can be measured for each cycle of temperature change, and the ratio of the initial concentrations of the plurality of target nucleic acids in the nucleic acid under test can be calculated from the relationship between the number of cycles and the change in binding.

[0074] In one embodiment, the binding of the amplified nucleic acid to the probe can be measured for each cycle of temperature change, the relationship between the number of cycles and the change in binding can be compared with the change in binding of a sample containing a target nucleic acid of a known concentration, and the initial concentration of the target nucleic acid of unknown concentration in the test nucleic acid can be calculated.

[0075] The DNA detection method disclosed in the present specification includes the following steps: a step of performing an asymmetric nucleic acid amplification reaction on a solution containing the DNA of the detection object using a primer that introduces a mutation in a manner that reduces the proportion of double-stranded structure and increases the proportion of single-stranded structure in the fluorescent probe binding region of the DNA of the detection object at a temperature that allows the fluorescent probe to bind, a fluorescent probe that can bind to the DNA of the detection object, a primer that forms a pair with the mutation-introducing primer for amplifying the fluorescent probe binding region of the DNA of the detection object, and a DNA polymerase for amplifying the DNA of the detection object; a step of measuring the fluorescence intensity as the temperature of the DNA solution changes; and a step of calculating the melting temperature of the DNA double helix from the change in the intensity of the fluorescence of the above-mentioned DNA solution as the temperature changes. Below, refer to Figure 6 Schematic diagram of the DNA detection method is specifically described.

[0076] First, primers that introduce mutations are designed in a manner that can reduce the proportion of double-stranded structures in the fluorescent probe binding region of the target DNA obtained by amplification and increase the proportion of single-stranded structures (S601). At this time, the mutation-introducing primer may or may not have a tag sequence. Then, the mutation-introducing primer is used to amplify the DNA of the detection object through an asymmetric nucleic acid amplification reaction (S602). The DNA of the detection object obtained by the asymmetric nucleic acid amplification reaction forms a secondary structure in which the proportion of double-stranded structures formed in the probe binding region is reduced and the proportion of single-stranded structures formed is increased (S603). The change in fluorescence intensity of the solution of the DNA of the detection object obtained by the asymmetric nucleic acid amplification reaction with temperature changes is measured, and a melting curve is prepared, and the melting temperature is calculated from the peak temperature of the differential curve of the melting curve (S604). Finally, the type of DNA of the detection object contained in the solution is determined based on the color and melting temperature of the fluorescence of the solution of the DNA of the detection object obtained by the asymmetric nucleic acid amplification reaction (S605).

[0077] In the case where there are multiple types of DNA of the detection object, multiple primers and multiple probes are prepared corresponding to the sequence of the detection object, and an asymmetric nucleic acid amplification reaction can be performed on a DNA solution containing multiple DNAs of the detection objects at the same time. Multiple probes are designed by changing the melting temperature of the DNA of each detection object or changing the type of fluorescent pigment. Thus, after the asymmetric nucleic acid amplification reaction, the type of DNA of the detection object contained in the solution can be distinguished according to the color and melting temperature of the fluorescence of the solution. For example, a case where multiple types of wild-type alleles and mutant alleles are included as the target gene can be exemplified, but multiple types of target genes are not particularly limited to this.

[0078] The process of asymmetric nucleic acid amplification reaction using mutation-introducing primers can also be monitored in real time using fluorescently labeled probes. For example, in an asymmetric nucleic acid amplification reaction, the fluorescence intensity is measured for each cycle of temperature change, and the relationship between the cycle number and the change in fluorescence intensity can be used to calculate the ratio of the initial concentrations of the DNA of the detection object contained in multiple solutions. In addition, at this time, the DNA of the detection object with a known concentration is prepared as a control and measured at the same time, thereby measuring the fluorescence intensity for each cycle of temperature change, comparing the relationship between the cycle number and the change in fluorescence intensity with the change in fluorescence intensity of the sample with a known concentration, and the initial concentration of the DNA of the detection object of the sample with an unknown concentration can also be calculated.

[0079] Asymmetric nucleic acid amplification using mutation-introducing primers can also be applied to digital PCR. The reaction solution containing the mutation-introducing primers is divided into micropartitions, and melting curve analysis is performed after the amplification reaction, and the target gene contained in the micropartition can also be detected (for example, genotype determination).

[0080] For example, when the DNA solution of the inspection object contains the target gene P and the target gene Q, in the micropartition containing the target gene P, the fluorescent labeled probe corresponding to the target gene P hybridizes with the DNA obtained by PCR amplification and emits fluorescence. By analyzing the generated fluorescence, the melting temperature corresponding to the fluorescent labeled probe of the target gene P can be calculated. In addition, in the micropartition containing the target gene Q, the fluorescent labeled probe corresponding to the target gene Q hybridizes with the DNA obtained by PCR amplification and emits fluorescence. By analyzing the generated fluorescence, the melting temperature corresponding to the fluorescent labeled probe of the target gene Q can be calculated. In this way, the presence or absence of the target gene P and the presence or absence of the target gene Q can be judged based on the fluorescence intensity, the type of fluorescence (e.g., color) and the melting temperature.

[0081] Since the melting temperature of DNA is not affected by the reaction efficiency of PCR and the in-plane measurement deviation during fluorescence measurement, the type of DNA in the micropartition (e.g., genotype) can be determined with high accuracy by using the melting temperature of DNA. For example, the sequence of the fluorescent labeled probe is predetermined so that the melting temperature (Tm) of each fluorescent labeled probe for the target gene is different, and for the DNA in the micropartition, the change in fluorescence intensity accompanying the temperature change is measured, and the melting curve analysis is performed, and the melting temperature is compared, thereby enabling the detection of the DNA in the micropartition (e.g., genotype determination).

[0082] When multiple target genes are detected simultaneously, they can be distinguished based on the reference melting temperature and the measured melting temperature, preferably based on the reference melting temperature range. For example, if the measured melting temperature of a certain well is within a specified range including the reference melting temperature recorded in the database for a certain target gene (e.g., within ±1°C of the reference melting temperature), it is determined that the target gene is arranged in the well. Using such a reference melting temperature range, a more accurate determination can be made that appropriately considers the allowable range.

[0083] Alternatively, as information on fluorescence intensity, a ratio or difference of fluorescence intensity at different temperatures may be used. For example, by using a ratio or difference of fluorescence intensity at a temperature lower than the reference melting temperature to fluorescence intensity at a temperature higher than the reference melting temperature, the fluorescence intensity can be standardized. For example, if the ratio or difference is within a specified range for a certain well, the well is determined to be positive, otherwise the well is determined to be negative.

[0084] For example, by subtracting the fluorescence intensity at 85° C. from the fluorescence intensity at 50° C., the influence of the fluorescence of the fluorescent-labeled probe itself, that is, the influence of the background, can be removed.

[0085] In addition, the method for determining the range of fluorescence intensity and the range of reference melting temperature can be arbitrarily selected. For example, a pilot experiment can be performed in advance, and the operator can statistically determine the range based on the results, or the DNA detection system can automatically determine the range. In addition, the threshold of fluorescence intensity and the prescribed range of reference melting temperature can be statistically determined using the measurement data of each well in the box during each digital PCR measurement.

[0086] The data for statistically distinguishing the DNA in the well may include any or all of the following items, and may include items other than these.

[0087] - Fluorescence intensity at temperatures lower than the reference melting temperature

[0088] - Fluorescence intensity at temperatures higher than the baseline melting temperature

[0089] -Ratio of the fluorescence intensity at a temperature lower than the reference melting temperature to the fluorescence intensity at a temperature higher than the reference melting temperature

[0090] -The difference between the fluorescence intensity at a temperature lower than the reference melting temperature and the fluorescence intensity at a temperature higher than the reference melting temperature

[0091] - Characteristic quantity indicating the reference melting temperature

[0092] - Characteristic quantity indicating the shape of the melting curve

[0093] By changing the secondary structure of the amplified nucleic acid as described above and increasing the ratio of single-stranded bases in the probe binding region, the amount of the amplified nucleic acid bound to the probe binding region increases, thereby improving measurement sensitivity and measurement accuracy, such as genotype discrimination accuracy.

[0094] (3) DNA detection kit

[0095] The method of the present invention can be performed more easily and conveniently by using a kit including at least primers for introducing mutations. That is, in one embodiment, the present invention provides a kit for detecting a target nucleic acid, the kit comprising:

[0096] a primer pair comprising a forward primer and a reverse primer; and

[0097] a probe capable of binding to a nucleic acid amplified using a forward primer and a reverse primer,

[0098] Either or both of the forward primer or the reverse primer contains a mutation in its sequence, which is used to introduce a mutation into the amplified nucleic acid so as to increase the proportion of single-stranded bases in the probe binding region of the amplified nucleic acid at a temperature at which the probe binds to the amplified nucleic acid.

[0099] Forward Primer and Reverse Primer As described in the previous item, in one embodiment, the forward primer and reverse primer contained in the kit of the present invention have different concentrations, and the concentration of one of them (preferably the primer for introducing mutation) is higher.

[0100] In one embodiment, the kit of the present invention may include a plurality of primer pairs including forward primers and reverse primers for a plurality of target nucleic acids. In another embodiment, the kit of the present invention may include a plurality of probes for a plurality of target nucleic acids.

[0101] The kit of the present invention may further include other components required for carrying out an amplification reaction, such as a DNA polymerase, a substrate, etc. In addition, it may also include instructions describing the steps and procedures for detecting a target nucleic acid.

[0102] In one embodiment, the DNA detection kit disclosed in the present specification includes: a first primer for introducing mutations at a first concentration in such a manner that the proportion of double-stranded structures in the fluorescent probe binding region of the DNA of the detection object is reduced and the proportion of single-stranded structures is increased at a temperature at which the fluorescent probe binds; a fluorescent probe capable of binding to the DNA of the detection object; a second primer different from the first concentration and paired with the first primer in order to amplify the above-mentioned fluorescent probe binding region of the DNA of the detection object; and a DNA polymerase for amplifying the DNA of the detection object.

[0103] In order to perform an asymmetric nucleic acid amplification reaction, the concentrations of the first primer for introducing a mutation and the second primer paired therewith are different, and the higher concentration may be either the first primer for introducing a mutation or the second primer. In addition, the first primer for introducing a mutation may contain not only a mutation but also a tag sequence.

[0104] In the case where there are multiple DNAs of the detection object, multiple primers and multiple probes can be prepared according to the sequence of the detection object, and asymmetric nucleic acid amplification reaction can be performed on a DNA solution containing multiple DNAs of the detection object at the same time. Multiple probes are designed by changing the melting temperature of the DNA of each detection object or changing the type of fluorescent pigment. Thus, after the asymmetric nucleic acid amplification reaction, the type of DNA of the detection object contained in the solution can be distinguished according to the color of the solution's fluorescence and the melting temperature.

[0105] (4) Primer design method

[0106] The present invention also provides a method for designing primers for detecting a target nucleic acid based on amplification of a test nucleic acid and determination of binding of the amplification product to a probe. The method includes: a step of designing a primer pair for amplifying the test nucleic acid; a step of determining the ratio of single-stranded bases in the probe binding region in the nucleic acid amplified by the designed primer pair; and a step of introducing a mutation into one or both primers of the primer pair so that the ratio of single-stranded bases is at least 50%. The method may also include: a step of determining the ratio of single-stranded bases in the probe binding region in the nucleic acid amplified by the primer pair including the primer into which the mutation is introduced; and a step of confirming whether the ratio of single-stranded bases is at least 50%.

[0107] Example

[0108] [Example 1]

[0109] In this example, an example of performing an asymmetric nucleic acid amplification reaction using a mutation-introducing primer is shown.

[0110] First, the results of melting curve analysis of asymmetric nucleic acid amplification reaction without using mutation-introducing primers are described. Prepare genomic DNA of the G13D mutant of the KRAS gene (final concentration 133 molecules / μL), add the forward primer (final concentration 0.25μM), reverse primer (final concentration 2.0μM), fluorescently labeled probe corresponding to the wild type (final concentration 0.5μM), fluorescently labeled probe corresponding to the G13D mutant (final concentration 0.5μM), and 1x Master Mix (containing DNA polymerase, dNTP) required for PCR, and prepare the PCR reaction solution. At this time, the primer pair is added in an asymmetric manner so that the complementary DNA chain of the fluorescently labeled probe is over-amplified. The sequences of the primers and probes are shown below. In addition, the fluorescently labeled probes all have complementary sequences near both ends and are designed in such a way that they form double strands within the molecule. In addition, the fluorescent labeling probe corresponding to the wild type has HEX as a fluorescent dye bound to the 5' end and BHQ-1 as a quencher bound to the 3' end, and the fluorescent labeling probe corresponding to the mutant type has FAM as a fluorescent dye bound to the 5' end and BHQ-1 as a quencher bound to the 3' end.

[0111] Forward primer: 5'-GTCACATTTTCATTATTTTTATTATAAGG-3' (SEQ ID NO: 1)

[0112] Reverse primer: 5'-GTATCGTCAAGGCACTCTTGCC-3' (SEQ ID NO: 2)

[0113] Fluorescently labeled probe corresponding to the wild type: 5'-TTGGAGCTGGTGGCGT-3' (SEQ ID NO: 3)

[0114] Fluorescently labeled probe corresponding to the mutant type: 5'-CTGGTGACGTAGGCA-3' (SEQ ID NO: 4)

[0115] Fig. 7A The diagram shows the positional relationship between the secondary structure 701 (SEQ ID NO: 5) of the amplified G13D mutant single-stranded DNA and the binding region 702 of the fluorescent-labeled probe corresponding to the wild-type. Figure 7B The diagram shows the positional relationship between the secondary structure 701 (SEQ ID NO: 5) of the amplified G13D mutant single-stranded DNA and the binding region 703 of the fluorescent labeled probe corresponding to the mutant. Fig. 7A The single-stranded formation ratio of the binding region 702 of the fluorescent labeled probe in the embodiment is 69%. Figure 7B The single-stranded formation ratio of the fluorescent-labeled probe binding region 703 is 47%.

[0116] Figure 7C It is the result of melting curve analysis after asymmetric nucleic acid amplification reaction, and is the differential curve of the obtained melting curve. Compared with the peak 704 of the differential curve of the melting curve of the amplified G13D mutant DNA 701 and the fluorescently labeled probe 702 corresponding to the wild type, the peak 705 of the differential curve of the melting curve of the amplified G13D mutant DNA 701 and the fluorescently labeled probe 703 corresponding to the mutant is smaller. From this result, it can be seen that if the single-stranded formation ratio of the binding region of the fluorescently labeled probe is low, the binding amount of the amplified DNA and the fluorescently labeled probe is reduced. In such a case, the mutant sometimes exists only in a small amount, so the fluorescence amount will be further reduced and difficult to detect.

[0117] Next, in Fig. 8A and Figure 8B The results of asymmetric nucleic acid amplification reactions using mutation-introducing primers are shown in FIG. Fig. 8A The diagram does not use Fig. 7A The positional relationship between the secondary structure 801 (SEQ ID NO: 5) of the single-stranded DNA of the G13D mutant type amplified by the mutation-introducing primers shown and the binding region 802 of the fluorescent-labeled probe corresponding to the mutant type shows a position 803 where the mutation is to be introduced by the primers. Figure 8B The diagram shows a mutation introduction primer designed to change a base sequence such as CC at a predetermined position 803 where the mutation is introduced by the primer to AA, and a position 805 where the mutation is introduced by the primer in the positional relationship between the secondary structure 804 (sequence number 6) of the single-stranded DNA of the G13D mutant amplified by an asymmetric nucleic acid amplification reaction using the primer and the binding region 802 of the fluorescent labeled probe corresponding to the mutant. Figure 8B In the example, the single-stranded formation ratio of the fluorescent-labeled probe binding region 802 corresponding to the mutant type increases, and the binding amount of the fluorescent-labeled probe increases.

[0118] Thus, by using mutation-introducing primers to perform an asymmetric nucleic acid amplification reaction, the secondary structure of the gene to be amplified is changed, the single-stranded formation ratio is increased, and thereby the binding amount of the fluorescent-labeled probe is increased, thereby improving the measurement sensitivity and measurement accuracy.

[0119] [Example 2]

[0120] This example shows an example of performing an asymmetric nucleic acid amplification reaction using a mutation-introducing primer with a tag.

[0121] Fig. 9A and Fig. 9B The results of an asymmetric nucleic acid amplification reaction using a tagged mutation-introducing primer are shown in FIG. Fig. 9A The diagram does not use Fig. 7A The positional relationship between the secondary structure 901 (SEQ ID NO: 5) of the single-stranded DNA of the G13D mutant type amplified by the mutation-introducing primers shown and the binding region 902 of the fluorescent-labeled probe corresponding to the mutant type shows a position 903 where the mutation is to be introduced by the primers. Fig. 9B The diagram shows a mutation introduction primer designed to change the base sequence CC at the predetermined position 903 where the mutation is introduced by the primer to AA, and further to add a tag sequence to the 3′ end. When an asymmetric nucleic acid amplification reaction is carried out using the primer, the secondary structure 904 (sequence number 7) of the G13D mutant single-stranded DNA amplified and the positional relationship between the binding region 902 of the fluorescent labeled probe corresponding to the mutant, the position 905 where the mutation is introduced by the primer, and the position 906 of the tag added by the primer are shown. Fig. 9B In the example, the single-stranded formation ratio of the fluorescent-labeled probe binding region 902 corresponding to the mutant type increases, and the binding amount of the fluorescent-labeled probe increases. Fig. 9A and 9B As shown, the secondary structure of single-stranded DNA may change significantly before and after the introduction of a mutation.

[0122] Thus, by performing an asymmetric nucleic acid amplification reaction using a tagged mutation-introducing primer, the secondary structure of the amplified target gene is changed, the single-stranded formation ratio is increased, and thereby the binding amount of the fluorescent-labeled probe is increased, thereby improving the measurement sensitivity and measurement accuracy.

[0123] Explanation of symbols

[0124] 101…DNA

[0125] 102…Fluorescently labeled probe

[0126] 103…Fluorescent pigment

[0127] 104…Quencher

[0128] 201…Forward primer

[0129] 202…Reverse primer

[0130] 203…Fluorescently labeled probe

[0131] 204…DNA of the test subject

[0132] 205…The complementary strands of the DNA to be detected form a double-stranded structure.

[0133] 206… a molecule in which a fluorescent labeled probe is bound to a single-stranded molecule of the DNA to be detected

[0134] 207…a strand of the DNA of the test subject

[0135] 301…Forward primer

[0136] 302…Reverse primer for mutation introduction

[0137] 303…Fluorescent labeled probe

[0138] 304…DNA of the test subject

[0139] 305…Secondary structure of the single-stranded DNA to be detected amplified by the reverse primer without mutation

[0140] 306…Introducing mutation into the intended site

[0141] 307…Secondary structure of the single-stranded DNA to be detected obtained by amplification with the reverse primer for mutation introduction

[0142] 308…Introduced mutations

[0143] 309…Mutation

[0144] 401…Forward primer

[0145] 402…Tagged mutation introduction reverse primer

[0146] 403…Fluorescent labeled probe

[0147] 404…DNA of the test subject

[0148] 405...Tag Sequence

[0149] 406…Secondary structure of the single-stranded DNA to be detected amplified by the non-mutation reverse primer

[0150] 407…Mutation introduction into the intended site

[0151] 408 ... the secondary structure of the single-stranded DNA to be detected obtained by amplification of the reverse primer for introducing a mutation carrying a tag

[0152] 409…Introduced mutations

[0153] 410 ... Tag sequence added

[0154] 411…Mutation

[0155] 501…Melting temperature

[0156] 701…Secondary structure of the single-stranded DNA to be detected amplified by the reverse primer without mutation

[0157] 702…binding region of fluorescently labeled probe corresponding to wild type

[0158] 703…Binding region of fluorescent labeled probe corresponding to the mutant type

[0159] 704…The peaks of the amplified G13D mutant DNA and the fluorescent labeled probe corresponding to the wild type

[0160] 705…The peak of the amplified G13D mutant DNA and the fluorescent labeled probe corresponding to the mutant

[0161] 801…Secondary structure of single-stranded DNA of G13D mutant amplified without using mutation-introducing primers

[0162] 802…Binding region of fluorescent labeled probe

[0163] 803…Predetermined location for mutation introduction

[0164] 804…Secondary structure of single-stranded DNA of G13D mutant amplified using mutation-introducing primers

[0165] 805…Mutation introduction position

[0166] 901…Secondary structure of single-stranded DNA of G13D mutant amplified without using mutation-introducing primers

[0167] 902…Binding region of fluorescent labeled probe

[0168] 903…Predetermined position for mutation introduction

[0169] 904…Secondary structure of single-stranded DNA of G13D mutant amplified using mutation-introducing primers

[0170] 905… mutation introduction position

[0171] 906…Tag sequence.

[0172] Sequence Listing Free Text

[0173] SEQ ID NOs. 1 to 7: DNA (artificial sequence, synthetic polynucleotide). Sequence Listing <110> Hitachi High-tech Co., Ltd. <120> Primers, DNA detection methods and DNA detection kits <130> PH-9445-PCT <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 1 gtcacatttt cattattttt attataagg 29 <210> 2 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 2 gtatcgtcaa ggcactcttg cc 22 <210> 3 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 3 ttggagctgg tggcgt 16 <210> 4 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 4 ctggtgacgt aggca 15 <210> 5 <211> 103 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 5 gtcacatttt cattattttt attataaggc ctgctgaaaa tgactgaata taaacttgtg 60 gtagttggag ctggtgacgt aggcaagagt gccttgacga tac 103 <210> 6 <211> 103 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 6 gtcacatttt cattattttt attataaggc ctgctgaaaa tgactgaata taaacttgtg 60 gtagttggag ctggtgacgt aggcaagagt gaattgacga tac 103 <210> 7 <211> 109 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 7 gtcacatttt cattattttt attataaggc ctgctgaaaa tgactgaata taaacttgtg 60 gtagttggag ctggtgacgt aggcaagagt gaattgacga taccgtcaa 109

Claims

1. A method for detecting a target nucleic acid, characterized in that: include: A step of amplifying a test nucleic acid in the presence of a primer pair comprising a forward primer and a reverse primer and a probe; and a step of measuring binding between the nucleic acid amplified by the forward primer and the reverse primer and the probe, Either or both of the forward primer and the reverse primer contain a mutation in their sequence, and the mutation is used to introduce a mutation into the amplified nucleic acid so as to increase the proportion of single-stranded bases in the probe binding region in the amplified nucleic acid at a temperature at which the probe binds to the amplified nucleic acid.

2. The method according to claim 1, characterized in that: The reverse primer comprises the mutation.

3. The method according to claim 1, characterized in that: The probe contains a fluorescent dye or contains a fluorescent dye and an extinction dye, and the binding between the amplified nucleic acid and the probe is measured using the fluorescent dye.

4. The method according to claim 1, characterized in that: The 3' terminal sequence and the 5' terminal sequence of the probe have complementary sequences.

5. The method according to claim 1, characterized in that: The amplification step is performed by an asymmetric nucleic acid amplification reaction.

6. The method according to claim 1, characterized in that: Either or both of the forward primer and the reverse primer further include a tag sequence, so that the amplified nucleic acid includes the tag sequence.

7. The method according to claim 1, characterized in that: The ratio of single-stranded bases in the probe binding region in the amplified nucleic acid is 50% or more.

8. The method according to claim 1, characterized in that: The binding between the amplified nucleic acid and the probe is measured along with the temperature change, and the melting temperature of the double strand of the amplified nucleic acid and the probe is calculated from the change in the binding along with the temperature change.

9. The method according to claim 1, characterized in that include: The step of measuring the binding between the amplified nucleic acid and the probe for each cycle of temperature change, and detecting the target nucleic acid based on the relationship between the number of cycles and the change in the binding.

10. The method according to claim 1, characterized in that include: The step of detecting a plurality of target nucleic acids using a plurality of primers comprising the mutation.

11. The method according to claim 10, characterized in that include: The step of measuring the binding of the amplified nucleic acid to the probe for each cycle of temperature change, and calculating the ratio of the initial concentrations of the plurality of target nucleic acids in the test nucleic acid from the relationship between the number of cycles and the change in the binding.

12. The method according to claim 1, characterized in that include: The step of measuring the binding of the amplified nucleic acid to the probe for each cycle of temperature change, comparing the relationship between the number of cycles and the change in binding with the change in binding of a sample containing a target nucleic acid of known concentration, and calculating the initial concentration of the target nucleic acid of unknown concentration in the test nucleic acid.

13. The method of claim 1, wherein: The detection of the target nucleic acid includes the determination of the genotype of the target nucleic acid.

14. A primer for introducing a mutation into a nucleic acid to be tested and for amplification, characterized in that: Its sequence contains a mutation, which is used to introduce a mutation into the test nucleic acid so that the proportion of single-stranded bases in the binding region of the probe in the nucleic acid amplified from the test nucleic acid is increased at a temperature at which the nucleic acid amplified from the test nucleic acid is bound to the probe.

15. The primer according to claim 14, characterized in that: This is the reverse primer.

16. The primer according to claim 14, characterized in that: Also contains the tag sequence.

17. A kit for detecting a target nucleic acid, characterized in that: include: a primer pair comprising a forward primer and a reverse primer; and a probe capable of binding to a nucleic acid amplified using a forward primer and a reverse primer, Either or both of the forward primer and the reverse primer contain a mutation in their sequence, which is used to introduce a mutation into the amplified nucleic acid so as to increase the proportion of single-stranded bases in the probe binding region in the amplified nucleic acid at a temperature at which the probe binds to the amplified nucleic acid.

18. The kit according to claim 17, characterized in that: Either or both of the forward primer and the reverse primer further include a tag sequence.

19. The kit according to claim 17, wherein: The concentration of the forward primer is different from the concentration of the reverse primer.

20. The kit according to claim 17, wherein: The probe comprises a fluorescent pigment, or comprises a fluorescent pigment and an extinction pigment.

21. The kit according to claim 17, characterized in that: The 3' terminal sequence and the 5' terminal sequence of the probe have complementary sequences.

22. The kit according to claim 17, wherein: The ratio of single-stranded bases in the probe binding region in the amplified nucleic acid is 50% or more.

23. The kit according to claim 17, wherein: for a plurality of target nucleic acids, respectively comprising a plurality of primer pairs including the forward primer and the reverse primer, and / or A plurality of the probes are included for a plurality of target nucleic acids, respectively.

24. The kit according to claim 17, wherein: Also contains DNA polymerase.

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