Primer, DNA detection method, and DNA detection kit

Abstract

The present invention relates to: a primer for a nucleic acid amplification reaction, in particular, an asymmetric nucleic acid amplification reaction; a DNA detection method; and a DNA detection kit. Specifically, the present invention relates to: a primer for introducing a mutation into a subject nucleic acid and amplifying the nucleic acid, the primer comprising, in the sequence thereof, a mutation for introducing a mutation in the subject nucleic acid that results in, at the temperature at which a probe and a nucleic acid amplified from the subject nucleic acid are bonded, an increased ratio of the single-strand formation bases in the probe bonding region of the nucleic acid amplified from the subject nucleic acid; and a DNA detection method and a DNA detection kit which use the primer.

Description

Primer, DNA detection method and DNA detection kit

[0001] The present invention relates to a primer for a nucleic acid amplification reaction, particularly an asymmetric nucleic acid amplification reaction, a DNA detection method, and a DNA detection kit.

[0002] Genetic testing techniques include polymerase chain reaction (PCR), real-time PCR, and digital PCR. Real-time PCR and digital PCR, which enable more accurate quantification than PCR, use intercalators or fluorescently labeled probes to detect DNA. Two types of DNA detection methods using fluorescently labeled probes are commonly used: hydrolysis probes and molecular beacons. Unlike hydrolysis probes, which utilize the nuclease activity of DNA polymerase to decompose the probe, releasing a fluorescent dye and emitting fluorescence. Molecular beacons form stem-loops in their free state and bind to the target DNA at the loop. These beacons are not decomposed during PCR, allowing for post-PCR amplification determination based on fluorescence intensity and melting curve analysis.

[0003] Here, in DNA detection using molecular beacons, a method has been reported in which the concentrations of the forward primer and reverse primer are asymmetrically adjusted to amplify the target DNA asymmetrically, so that the strand complementary to the molecular beacon is excessively amplified, in order to increase the amount of binding between the molecular beacon and the amplified target DNA and thereby increase the fluorescence intensity (Non-Patent Document 1).

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

[0005] An example of a digital PCR detection method is shown below. First, DNA polymerase, primers, and fluorescently labeled probes required for PCR are added to a limiting diluted sample to prepare a PCR reaction solution. The PCR reaction solution is divided into microcompartments such as wells or droplets. Each compartment is designed to either contain one molecule of the target gene or not.

[0006] The gene of interest in the microcompartments is then amplified by PCR, and the fluorescence intensity of each microcompartment is measured after PCR, and the number of microcompartments with fluorescence intensity above a threshold is counted, thereby quantifying the gene of interest.

[0007] When an asymmetric nucleic acid amplification reaction using a molecular beacon is applied, after PCR, a melting curve analysis of the target gene amplified in the microcompartment and the molecular beacon is performed, and different melting temperatures (Tm) are observed for each genotype of the target gene, making it possible to distinguish them.

[0008] JP 2018-108063 A

[0009] BMC Microbiol. , 13, pp295, 2013 Anal. Chem. , 92, pp11705-11713, 2020

[0010] However, the present inventors were the first to recognize that in asymmetric nucleic acid amplification reactions, one strand of excessively amplified DNA forms a secondary structure within the molecule, and molecular beacons designed in a region that forms a single-stranded structure bind to the amplified target DNA, while molecular beacons designed in a region that forms a double-stranded structure bind at a lower rate, resulting in a decrease in fluorescence intensity. In particular, in PCR that is performed in a microcompartment such as digital PCR, rather than in PCR that is performed in a tube such as real-time PCR, a decrease in fluorescence intensity can result in the fluorescence intensity falling below the detectable level and becoming undetectable, or the change in fluorescence intensity relative to temperature change in melting curve analysis is so small that it becomes impossible to calculate the melting temperature, or it cannot be easily measured, resulting in a decrease in measurement sensitivity and accuracy.

[0011] Therefore, an object of the present invention is to provide novel primers, a DNA detection method, and a DNA detection kit that, in an asymmetric nucleic acid amplification reaction, suppress a decrease in the binding rate between the molecular beacon and the amplified DNA to be detected by reducing the rate at which the molecular beacon binding region of one strand of over-amplified DNA forms a double-stranded structure and increasing the rate at which it forms a single-stranded structure, thereby enabling accurate and highly sensitive detection of the target gene and discrimination of its genotype.

[0012] The present inventors have clarified that in asymmetric nucleic acid amplification reactions, even when amplification is performed with the same primer set, the fluorescence intensity varies greatly depending on the binding region of the molecular beacon, and that the fluorescence intensity of a molecular beacon designed in a region where one of the amplified strands forms a double-stranded structure within the molecule is low. Therefore, the present inventors have discovered that by intentionally introducing a mutation different from the sequence of the target gene into the primer used in the asymmetric nucleic acid amplification reaction and then performing the asymmetric nucleic acid amplification reaction, the proportion of the binding region of the molecular beacon that forms a double-stranded structure decreases, thereby increasing the fluorescence intensity, leading to the completion of the present invention.

[0013] One embodiment of the present invention is a method for detecting a target nucleic acid, comprising: amplifying a test nucleic acid in the presence of a primer pair including a forward primer and a reverse primer, and a probe; and measuring binding between the nucleic acid amplified by the forward primer and the reverse primer and the probe, wherein either or both of the forward primer or the reverse primer contain a mutation in their sequence for introducing 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.

[0014] Another embodiment of the present invention is a primer for introducing a mutation into a test nucleic acid and amplifying the nucleic acid, the primer comprising a mutation in its sequence for introducing a mutation into the test nucleic acid so as to increase the proportion of single-stranded bases in the probe binding region in the nucleic acid amplified from the test nucleic acid at a temperature at which the nucleic acid amplified from the test nucleic acid is bound to the probe.

[0015] Another embodiment of the present invention is a kit for detecting a target nucleic acid, comprising: a primer pair comprising a forward primer and a reverse primer; and a probe that binds to a nucleic acid to be amplified using the forward primer and the reverse primer, wherein either the forward primer or the reverse primer, or both, contain a mutation in their sequence 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.

[0016] The present invention provides novel primers, a DNA detection method, and a DNA detection kit that can detect or quantify a target gene with high accuracy and sensitivity in an asymmetric nucleic acid amplification reaction. Therefore, the present invention is useful in fields such as basic research, testing, and drug discovery that involve gene detection and genotyping.

[0017] FIG. 1 is a schematic diagram showing an example of a method for measuring the melting temperature of DNA using a fluorescently labeled probe. FIG. 1 is a schematic diagram showing an example of the behavior of a fluorescently labeled probe in a symmetric nucleic acid amplification reaction and an asymmetric nucleic acid amplification reaction. FIG. 2 is a schematic diagram showing the binding of a fluorescently labeled probe to the secondary structure of amplified DNA to be detected when an asymmetric nucleic acid amplification reaction is performed using a primer that introduces a mutation, according to one embodiment of the present invention. FIG. 3 is a schematic diagram showing the binding of a fluorescently labeled probe to the secondary structure of amplified DNA to be detected when an asymmetric nucleic acid amplification reaction is performed using a tagged primer that introduces a mutation, according to one embodiment of the present invention. FIG. 4 is a diagram showing an example of a melting curve when a mutation-introducing primer is used, according to one embodiment of the present invention. FIG. 5 is a flowchart showing one embodiment of a DNA detection method that performs an asymmetric nucleic acid amplification reaction using a mutation-introducing primer. FIG. 6 is a diagram showing the positional relationship between the secondary structure of amplified single-stranded DNA to be detected and the binding region of a fluorescently labeled probe corresponding to the wild type, according to an example of the present invention. FIG. 7 is a diagram showing the positional relationship between the secondary structure of amplified single-stranded DNA to be detected and the binding region of a fluorescently labeled probe corresponding to the mutant type, according to an example of the present invention. FIG. 8 is a diagram showing the measurement results (differential curve of the melting curve) when a mutation-introducing primer is not used, according to an example of the present invention. FIG. 1 is a diagram showing the positional relationship between the secondary structure of the amplified single-stranded DNA to be detected and the binding region of a fluorescently labeled probe corresponding to the wild type, and the position where a mutation is to be introduced by a primer. FIG. 2 is a diagram showing the positional relationship between the secondary structure of the amplified single-stranded DNA to be detected and the binding region of a fluorescently labeled probe corresponding to the mutant type, and the position where a mutation is to be introduced by a primer, in an example of the present invention. FIG. 3 is a diagram showing the positional relationship between the secondary structure of the amplified single-stranded DNA to be detected and the binding region of a fluorescently labeled probe corresponding to the wild type, and the position where a mutation is to be introduced by a primer, in an example of the present invention.

[0018] The objectives, features, advantages, and ideas relating to the present invention will be apparent to those skilled in the art from the description in this specification. Those skilled in the art can easily reproduce the present invention from the description in this specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are presented for illustration or explanation purposes, and are not intended to limit the present invention thereto. It will be apparent to those skilled in the art that various changes and modifications can be made based on the description in this specification without departing from the spirit and scope of the present invention disclosed herein.

[0019] (1) Primer In one aspect, the present invention provides a primer for introducing a mutation into a test nucleic acid and amplifying the test nucleic acid, the primer comprising a mutation in its sequence for introducing a mutation into the test nucleic acid so as to increase the proportion of single-stranded bases in the probe-binding region in the nucleic acid amplified from the test nucleic acid at a temperature at which the nucleic acid amplified from the test nucleic acid is bound to the probe. Such a primer may be used as a reagent for introducing a mutation into the test nucleic acid and amplifying the test nucleic acid.

[0020] In the present invention, the test nucleic acid is not particularly limited as long as it is a nucleic acid that contains or may contain the target nucleic acid to be detected, and includes, for example, messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, genomic DNA, and fragments thereof, hybrid nucleic acids of DNA and RNA, etc., and may be single-stranded or double-stranded. When the target nucleic acid to be detected is RNA such as mRNA, the test nucleic acid may be DNA (e.g., cDNA) obtained by reverse transcribing that RNA.

[0021] The primer may be a forward primer or a reverse primer, or both primers may contain the mutation. Preferably, the primer is a reverse primer.

[0022] The primer contains a mutation for introducing a mutation into the test nucleic acid to amplify it. The mutation is introduced so as to increase the ratio of single-stranded bases (the ratio of single-stranded bases to double-stranded bases) in the probe-binding region of the nucleic acid amplified from the test nucleic acid at the temperature at which the nucleic acid amplified from the test nucleic acid and the probe bind. The nucleic acid amplified from the test nucleic acid has a single-stranded structure, but may form a double-stranded structure internally due to the presence of complementary bases. The type of structure formed by the amplified nucleic acid depends on its sequence composition, and in the art, there are programs for predicting such structures (e.g., OligoAnalyzer ＴＭ The following programs are commonly used: RNAFold (University of Vienna), RNAFold (IDT), and RNAFold (University of Vienna). Therefore, such programs are used to predict the structure of the nucleic acid to be amplified, and to determine the proportion of single-stranded bases in the probe binding region of the amplified nucleic acid. If the proportion of single-stranded bases is low, for example, less than 50%, mutations are introduced into the nucleic acid to be amplified, so that the proportion of single-stranded bases increases, for example, to at least 50%, preferably 60% or more.

[0023] The primers can be designed by conventional primer design methods, except for containing the above-mentioned mutations, and can be designed to have a length, sequence composition, and melting temperature suitable for specifically binding to the target nucleic acid.

[0024] The primer may further comprise a tag sequence to increase the proportion of single-stranded bases. Such a tag sequence has 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 comprise the tag sequence. The nucleic acid amplified using such primers will contain the tag sequence.

[0025] One embodiment disclosed herein is a primer for asymmetric nucleic acid amplification reactions, which has been mutated so that the proportion of double-stranded structures in the region of the DNA to be detected that binds to a fluorescently labeled probe such as a molecular beacon is reduced and the proportion of single-stranded structures is increased at a temperature at which the fluorescently labeled probe binds. This primer will be specifically described below with reference to the schematic diagrams of Figures 1 to 5.

[0026] 1 is a schematic diagram showing the behavior of a fluorescently labeled probe when the temperature of a reaction solution is changed. Examples of fluorescently labeled probes include molecular beacons and Taqman probes, but FIG. 1 will be explained using a molecular beacon as an example.

[0027] A molecular beacon is configured as an oligonucleotide and has a sequence complementary to the sequence between a primer pair used in a nucleic acid amplification reaction to amplify a target gene. The molecular beacon also has complementary sequences at both ends, with a fluorescent dye 103 at one end and a quencher dye 104 at the other end. In a nucleic acid amplification reaction, in the initial state, as shown in FIG. 1B, the molecular beacon 102 exists alone and free. At this time, the molecular beacon 102 forms a stem-loop, and the fluorescent dye 103 and quencher 104 are in close proximity, so no fluorescence is emitted. In the initial denaturation step, when the sample solution is heated, a highly flexible structure is formed as shown in FIG. 1C, but the fluorescent dye and quencher dye are not always separated, so the fluorescence remains quenched. In the annealing step, when the temperature is lowered to approximately room temperature, the loop portion of the molecular beacon 102 anneals to the DNA 101 amplified in the sample solution as shown in FIG. 1A. As a result, the fluorescent dye 103 and the quencher 104 are constantly separated, causing the fluorescently labeled probe 102 to emit strong fluorescence. In the next extension step, the molecular beacon 102 is released, and the state returns to that shown in FIG. 1B, and the fluorescence is quenched. In the next denaturation step, the state returns to that shown in FIG. 1C, and the fluorescence remains quenched. Since this process is repeated in the nucleic acid amplification reaction, it is sufficient to measure the fluorescence intensity at some stage during heating or cooling. The same method can be used to measure the fluorescence intensity after the completion of the nucleic acid amplification reaction. After the completion of the nucleic acid amplification reaction, the reaction may be heated or cooled just to measure the fluorescence intensity, and then the fluorescence intensity may be measured.

[0028] When a DNA intercalator is used instead of a combination of a fluorescent dye and a quencher, when the sample DNA is double-stranded, the DNA intercalator intercalates between the double strands and emits fluorescence, but when the sample DNA is single-stranded, the DNA intercalator is released and the fluorescence is quenched. Therefore, in the nucleic acid amplification reaction, the fluorescence intensity may be measured at some stage during heating or cooling, as with the molecular beacon 102. After the nucleic acid amplification reaction is completed, the sample may be heated or cooled and the fluorescence intensity may be measured just to measure the fluorescence intensity.

[0029] 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, and Cy5, and examples of the quencher 104 include TAMRA, BHQ1, BHQ2, and BHQ3. All of these are commonly used and commercially available.

[0030] When two types of target genes with different sequences are used, the molecular beacon 102 sequences are prepared so that they specifically bind to each of the target genes, and by binding different fluorescent dyes to them, the two types of target genes can be distinguished and detected in a single reaction system.

[0031] The DNA intercalator is not particularly limited as long as it increases the fluorescence intensity by binding to double-stranded DNA and can be used to detect double-stranded DNA. Specific examples include SYBR (registered trademark) Green I, SYBR Gold, PicoGreen (registered trademark), SYTO (registered trademark) Blue, SYTO Green, SYTO Orange, and SYTO Red, 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., are applicable, all of which are commercially available. When the DNA intercalator is heat-resistant, it can be added to the reaction solution before the nucleic acid amplification reaction is performed.

[0032] FIG. 2 is a schematic diagram showing the behavior of a fluorescently labeled probe in a symmetric nucleic acid amplification reaction and an asymmetric nucleic acid amplification reaction. As shown in FIG. 2A, when the concentrations of forward primer 201 and reverse primer 202 are the same, both complementary strands of target DNA 204 are amplified in equal amounts, resulting in a large number of molecules 205 in which the complementary strands form double strands, and a small number of molecules 206 in which the fluorescently labeled probe 203 binds to the single-stranded molecules. As shown in FIG. 2B, when the concentrations of forward primer 201 and reverse primer 202 are asymmetric (in FIG. 2B, the reverse primer 202 is increased), one strand 207 of target DNA 204, which is the complementary strand of the molecular beacon, is over-amplified, resulting in a large number of molecules 206 in which the fluorescently labeled probe 203 binds to the single-stranded molecules. By performing an asymmetric nucleic acid amplification reaction in this way, the amount of binding between the fluorescently labeled probe and the amplified target DNA increases, increasing the fluorescence intensity.

[0033] FIG. 3 is a schematic diagram showing the binding of a fluorescently labeled probe to the secondary structure of amplified target DNA when an asymmetric nucleic acid amplification reaction is performed using a primer that introduces a mutation. As shown in FIG. 3A, a forward primer 301, a mutation-introduced reverse primer 302 with a mutation 309 introduced, and a fluorescently labeled probe 303 are prepared for target DNA 304. The concentration of reverse primer 302 is in excess of the concentration of forward primer 301, and an asymmetric nucleic acid amplification reaction is performed. FIG. 3B is a schematic diagram showing the binding of a fluorescently labeled probe 303 to a secondary structure 305 of single-stranded target DNA amplified by an asymmetric nucleic acid amplification reaction using a reverse primer that does not introduce mutation 309 (conventional method). Because mutation 309 is not introduced, position 306 on the secondary structure 305 of the amplified DNA remains the original base of the target DNA. In the secondary structure 305 of the amplified single-stranded target DNA, the binding region of the fluorescently labeled probe 303 forms a double-stranded structure, resulting in a reduced binding amount of the fluorescently labeled probe 303. On the other hand, Figure 3C is a schematic diagram (present invention) showing binding of a fluorescently labeled probe 303 to a secondary structure 307 of single-stranded target DNA amplified by an asymmetric nucleic acid amplification reaction using a mutation-introducing reverse primer 302. Due to mutation 308 introduced by the mutation-introducing reverse primer 302, the secondary structure 307 of the amplified single-stranded target DNA forms a secondary structure different from the secondary structure 305 of the amplified single-stranded target DNA in Figure 3B. Therefore, the proportion of double-stranded structures formed in the binding region of the fluorescently labeled probe 303 in Figure 3C is reduced compared to Figure 3B, and the proportion of single-stranded structures formed is increased, resulting in an increased amount of binding of the fluorescently labeled probe 303.

[0034] FIG. 4 is a schematic diagram showing the binding of a fluorescently labeled probe to the secondary structure of amplified target DNA when an asymmetric nucleic acid amplification reaction is performed using a tagged primer that introduces a mutation. As shown in FIG. 4A, a forward primer 401, a mutation-introducing reverse primer 402 with a tag sequence 405 and a mutation 411 introduced therein, and a fluorescently labeled probe 403 are prepared for the target DNA 404. The concentration of the reverse primer 402 is in excess of the concentration of the forward primer 401, and an asymmetric nucleic acid amplification reaction is performed. FIG. 4B is a schematic diagram (similar to FIG. 3B) showing the binding of a fluorescently labeled probe 403 to a secondary structure 406 of a single-stranded target DNA amplified by an asymmetric nucleic acid amplification reaction using a reverse primer (not shown) that does not have a tag sequence and does not introduce a mutation 411. Because no mutation 411 is introduced, position 407 on the secondary structure 406 of the amplified DNA remains the original base of the target DNA. In the secondary structure 406 of the amplified single-stranded target DNA, the binding region of the fluorescently labeled probe 403 forms a double-stranded structure, resulting in a reduced amount of binding of the fluorescently labeled probe 403. Meanwhile, FIG. 4C is a schematic diagram showing the binding of the fluorescently labeled probe 403 to the secondary structure 408 of the single-stranded target DNA amplified by an asymmetric nucleic acid amplification reaction using the tagged mutagenesis reverse primer 402. Due to the mutation 409 introduced by the tagged mutagenesis reverse primer 402 and the tag sequence addition 410, the secondary structure 408 of the amplified single-stranded target DNA forms a secondary structure different from the secondary structure 406 of the amplified single-stranded target DNA in FIG. 4B. Therefore, the binding region of the fluorescently labeled probe 403 forms a lower proportion of double-stranded structures in FIG. 4C compared to FIG. 4B, and the proportion of single-stranded structures increases, resulting in an increased amount of binding of the fluorescently labeled probe 403.

[0035] FIG. 5 shows examples of melting curves obtained when an asymmetric nucleic acid amplification reaction is performed using a mutagenesis primer and when an asymmetric nucleic acid amplification reaction is performed without a mutagenesis primer. When an asymmetric nucleic acid amplification reaction is performed using a mutagenesis primer, measuring the change in fluorescence intensity of the solution with temperature results in a melting curve with a large change in fluorescence intensity, as shown in FIG. 5A. The differential curve shows a high peak, as shown in FIG. 5B, with the temperature of this peak representing the melting temperature (Tm) 501. However, when an asymmetric nucleic acid amplification reaction is performed without a mutagenesis primer, measuring the change in fluorescence intensity of the solution with temperature results in a decrease in fluorescence intensity, as shown in FIG. 5C, compared to FIG. 5A. Therefore, when a differential curve is obtained, it has a low peak, as shown in FIG. 5D, and the peak shape is gentle, which may result in an erroneous value that differs from the melting temperature of the target gene in the solution. Furthermore, if the degree of decrease in fluorescence intensity is large, the fluorescence intensity due to amplification of the target gene becomes lower than the detectable intensity and becomes undetectable.

[0036] (2) DNA Detection Method In another aspect, the present invention provides a method for detecting a target nucleic acid, comprising: amplifying a test nucleic acid in the presence of a probe and a primer pair comprising a forward primer and a reverse primer; and measuring binding of the nucleic acid amplified by the forward primer and the reverse primer to the probe, wherein either or both of the forward primer and the reverse primer contain a mutation in their sequences for introducing 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.

[0037] As used herein, detection of a target nucleic acid means examining the presence or absence of the nucleic acid to be detected, measuring the concentration or abundance ratio of the nucleic acid to be detected, identifying the nucleic acid to be detected, determining the genotype, etc.

[0038] Either or both of the forward primer and the reverse primer are the primers described in the previous section.

[0039] The probe contains a fluorescent dye or a fluorescent dye and a quencher dye. When the probe contains a fluorescent dye, the fluorescent dye is used to measure the binding of the amplified nucleic acid to the probe. In one embodiment, the 3'-end sequence and the 5'-end sequence of the probe have complementary sequences, and when the probe does not bind to the amplified nucleic acid, the 3'-end sequence and the 5'-end sequence of the probe bind to form a stem structure. When the probe does not contain a fluorescent dye, for example, a DNA intercalator can be used.

[0040] The amplification step can be performed by any 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 either the forward primer or the reverse primer (e.g., a primer containing a mutation) added is increased to perform the amplification reaction. In a preferred embodiment, the amplification reaction is performed by adding more reverse primer containing a mutation than the forward primer. For details of the asymmetric nucleic acid amplification reaction, see, for example, Anal. Chem., 92, pp. 11705-11713, 2020 (Non-Patent Document 2).

[0041] In one embodiment, the binding between the amplified nucleic acid and the probe is measured with a change in temperature. Since the binding changes depending on the melting temperature, the melting temperature of the duplex of the amplified nucleic acid and the probe can be calculated from the change in binding with a change in temperature.

[0042] In one embodiment, the binding between the amplified nucleic acid and the probe is measured for each cycle of temperature change, and the target nucleic acid can be detected from the relationship between the number of cycles and the change in binding.

[0043] By using multiple types of primers containing the above-mentioned mutations, multiple target nucleic acids can be detected. In this case, the binding between the amplified nucleic acid and the probe is measured for each temperature change cycle, and the ratio of the initial concentrations of the multiple target nucleic acids in the test nucleic acid can be calculated from the relationship between the number of cycles and the change in binding.

[0044] In one embodiment, the binding between the amplified nucleic acid and the probe is measured for each cycle of temperature change, and the relationship between the number of cycles and the change in binding is compared with the change in binding in a sample containing a known concentration of target nucleic acid, allowing the initial concentration of the target nucleic acid in the test nucleic acid, whose concentration is unknown, to be calculated.

[0045] The DNA detection method disclosed herein includes the steps of: performing an asymmetric nucleic acid amplification reaction on a solution containing the target DNA using a primer that introduces a mutation so that the proportion of double-stranded structures in the fluorescent probe-binding region of the target DNA is reduced and the proportion of single-stranded structures is increased at the temperature at which the fluorescent probe binds; a fluorescent probe that binds to the target DNA; a primer that pairs with the mutation-introducing primer to amplify the fluorescent probe-binding region of the target DNA; and a DNA polymerase that amplifies the target DNA; measuring the fluorescence intensity of the DNA solution as the temperature changes; and calculating the melting temperature of the DNA double strand from the change in fluorescence intensity as the temperature changes. This DNA detection method will be specifically described below with reference to the schematic diagram of FIG.

[0046] First, a mutated primer is designed to reduce the proportion of double-stranded structures in the fluorescent probe binding region in the amplified target DNA and increase the proportion of single-stranded structures (S601). The mutated primer may or may not have a tag sequence. Next, the target DNA is amplified by an asymmetric nucleic acid amplification reaction using the mutated primer (S602). The target DNA after the asymmetric nucleic acid amplification reaction forms a secondary structure in which the proportion of double-stranded structures in the probe binding region decreases and the proportion of single-stranded structures increases (S603). The change in fluorescence intensity with temperature change of the target DNA solution after the asymmetric nucleic acid amplification reaction is measured, a melting curve is created, and the melting temperature is calculated from the peak temperature of the differential curve of the melting curve (S604). Finally, the type of target DNA contained in the solution after the asymmetric nucleic acid amplification reaction is identified based on the fluorescent color and melting temperature of the target DNA solution after the asymmetric nucleic acid amplification reaction (S605).

[0047] When there are multiple types of DNA to be detected, multiple primers or multiple probes may be prepared according to the sequences of the target DNAs, and added simultaneously to perform an asymmetric nucleic acid amplification reaction on a DNA solution containing the multiple target DNAs.The multiple probes may be designed to have different melting temperatures with the respective target DNAs or different types of fluorescent dyes, so that the type of target DNA contained in the solution can be determined from the fluorescent color and melting temperature of the solution after the asymmetric nucleic acid amplification reaction.For example, a case can be exemplified in which the target genes contain multiple types of wild-type alleles and mutant alleles, but the multiple types of target genes are not particularly limited thereto.

[0048] The process of asymmetric nucleic acid amplification using a mutation-introducing primer can also be monitored in real time using a fluorescently labeled probe. For example, in an asymmetric nucleic acid amplification reaction, the ratio of the initial concentrations of the target DNA contained in multiple solutions can be calculated from the process of measuring the fluorescence intensity for each temperature change cycle and the relationship between the number of cycles and the change in fluorescence intensity. Furthermore, by preparing a target DNA of known concentration as a control and measuring it simultaneously, it is possible to compare the process of measuring the fluorescence intensity for each temperature change cycle and the relationship between the number of cycles and the change in fluorescence intensity with the change in fluorescence intensity of a sample of known concentration, and calculate the initial concentration of the target DNA of a sample of unknown concentration.

[0049] Asymmetric nucleic acid amplification reactions using mutagenesis primers can also be applied to digital PCR. A reaction solution containing mutagenesis primers can be divided into microcompartments, and after the amplification reaction, melting curve analysis can be performed to detect (e.g., determine the genotype of) the target gene contained in the microcompartment.

[0050] For example, if the DNA solution to be tested contains target gene P and target gene Q, in the microcompartment containing target gene P, a fluorescently labeled probe corresponding to target gene P hybridizes to DNA amplified by PCR and emits fluorescence. By analyzing the emitted fluorescence, the melting temperature corresponding to the fluorescently labeled probe for target gene P can be calculated. Furthermore, in the microcompartment containing target gene Q, a fluorescently labeled probe corresponding to target gene Q hybridizes to DNA amplified by PCR and emits fluorescence. By analyzing the emitted fluorescence, the melting temperature corresponding to the fluorescently labeled probe for target gene Q can be calculated. In this way, the presence or absence of target gene P and target gene Q can be determined based on the fluorescence intensity, the type of fluorescence (e.g., color), and the melting temperature.

[0051] Since the DNA melting temperature is not affected by PCR reaction efficiency or in-plane measurement variations during fluorescence measurement, the DNA melting temperature can be used to accurately determine the type of DNA (e.g., genotype) within a microcompartment. For example, by determining the sequence of fluorescently labeled probes so that each fluorescently labeled probe for a target gene has a different melting temperature (Tm), measuring the change in fluorescence intensity of the DNA within the microcompartment with temperature changes, performing melting curve analysis, and comparing the melting temperatures, it becomes possible to detect the DNA within the microcompartment (e.g., determine the genotype).

[0052] When multiple target genes are detected simultaneously, they can be distinguished based on the reference melting temperature and the measured melting temperature, but preferably based on the reference melting temperature range. For example, if the measured melting temperature for a given well is within a predetermined range including the reference melting temperature recorded in the database for a given target gene (e.g., within ±1°C of the reference melting temperature), it is determined that the target gene is located in that well. Using such a reference melting temperature range allows for more accurate determination by appropriately considering the tolerance range.

[0053] Alternatively, the ratio or difference between the fluorescence intensities at different temperatures may be used as information on the fluorescence intensity. For example, the fluorescence intensity can be normalized by using the ratio or 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. For example, if this ratio or difference for a certain well is within a predetermined range, the well is determined to be positive; otherwise, the well is determined to be negative.

[0054] For example, by subtracting the fluorescence intensity at 85°C from the fluorescence intensity at 50°C, the influence of the fluorescence of the fluorescently labeled probe itself, that is, the influence of the background, can be removed.

[0055] The method for determining the range of fluorescence intensity and the range of reference melting temperature can be selected arbitrarily. For example, a pilot experiment or the like may be conducted in advance and the ranges may be statistically determined by an operator based on the results, or the DNA detection system may automatically determine the ranges. Furthermore, the predetermined ranges of the threshold fluorescence intensity and reference melting temperature may be statistically determined using the measurement data of each well in the cartridge each time a digital PCR measurement is performed.

[0056] Data for statistically distinguishing DNA in a well may include any or all of the following items, or may include items other than these: - Fluorescence intensity at a temperature lower than the reference melting temperature - Fluorescence intensity at a temperature higher than the reference melting temperature - Ratio of fluorescence intensity at a temperature lower than the reference melting temperature to fluorescence intensity at a temperature higher than the reference melting temperature - Difference between fluorescence intensity at a temperature lower than the reference melting temperature and fluorescence intensity at a temperature higher than the reference melting temperature - Feature quantity representing the reference melting temperature - Feature quantity representing the shape of the melting curve

[0057] By changing the secondary structure of the amplified nucleic acid in this manner and increasing the proportion of single-stranded bases in the probe binding region, the amount of binding to the probe binding region in the amplified nucleic acid increases, thereby improving measurement sensitivity and accuracy, for example, the accuracy of genotype discrimination.

[0058] (3) DNA Detection Kit The above-described method of the present invention can be carried out more easily and conveniently by using a kit containing at least a primer for introducing a mutation. That is, in one aspect, the present invention provides a kit for detecting a target nucleic acid, the kit comprising: a primer pair containing a forward primer and a reverse primer; and a probe that binds to a nucleic acid to be amplified using the forward primer and the reverse primer, wherein either or both of the forward primer and the reverse primer contain a mutation in their sequences for introducing a mutation into the nucleic acid to be amplified 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.

[0059] The forward primer and reverse primer are as described in the preceding paragraph. In one embodiment, the concentrations of the forward primer and the reverse primer included in the kit of the present invention are different, with one of them (preferably the primer for introducing the mutation) being at a higher concentration.

[0060] In one embodiment, the kit of the present invention may include a plurality of primer pairs each including a forward primer and a reverse primer for a plurality of target nucleic acids, and may also include a plurality of probes for a plurality of target nucleic acids.

[0061] The kit of the present invention may further include other components necessary for carrying out an amplification reaction, such as a DNA polymerase, a substrate, etc. The kit may also include instructions describing procedures and protocols for detecting a target nucleic acid.

[0062] In one embodiment, the DNA detection kit disclosed herein includes: a first primer that introduces a first concentration of mutations so that the proportion of double-stranded structures in the fluorescent probe binding region of the DNA to be detected is reduced and the proportion of single-stranded structures is increased at a temperature at which the fluorescent probe is bound; a fluorescent probe that binds to the DNA to be detected; a second primer that is different in concentration from the first primer and pairs with the first primer to amplify the fluorescent probe binding region of the DNA to be detected; and a DNA polymerase that amplifies the DNA to be detected.

[0063] The first primer that introduces a mutation and its paired second primer have different concentrations to perform an asymmetric nucleic acid amplification reaction, and either the first primer that introduces a mutation or the second primer may be at a higher concentration. Furthermore, the first primer that introduces a mutation may contain not only a mutation but also a tag sequence.

[0064] When there are multiple DNAs to be detected, multiple primers or multiple probes may be prepared according to the sequences of the target DNAs, and added simultaneously to perform an asymmetric nucleic acid amplification reaction on a DNA solution containing the multiple target DNAs.The multiple probes may be designed to have different melting temperatures with the respective target DNAs or different types of fluorescent dyes, so that after the asymmetric nucleic acid amplification reaction, the type of target DNA contained in the solution can be identified from the color of the fluorescence and melting temperature of the solution.

[0065] (4) Primer Design Method The present invention also provides a method for designing primers for detecting a target nucleic acid based on amplifying a test nucleic acid and measuring binding of the amplified product to a probe. This method includes designing a primer pair for amplifying the test nucleic acid, determining the proportion of single-stranded bases in the probe-binding region of the nucleic acid amplified by the designed primer pair, and introducing a mutation into one or both primers of the designed primer pair so that the proportion of single-stranded bases is at least 50%. This method may further include determining the proportion of single-stranded bases in the probe-binding region of the nucleic acid amplified by the primer pair containing the mutated primer, and confirming whether the proportion of single-stranded bases is at least 50%.

[0066] Example 1 In this example, an asymmetric nucleic acid amplification reaction is performed using a mutation-introducing primer.

[0067] First, we will explain the results of asymmetric nucleic acid amplification without using a mutation-introducing primer and melting curve analysis. Genomic DNA of the G13D mutant form of the KRAS gene (final concentration 133 molecules / μL) was prepared, and the forward primer (final concentration 0.25 μM), reverse primer (final concentration 2.0 μM), fluorescently labeled probe corresponding to the wild-type (final concentration 0.5 μM), fluorescently labeled probe corresponding to the G13D mutant (final concentration 0.5 μM), and 1x master mix (containing DNA polymerase and dNTPs) required for PCR were added to prepare a PCR reaction solution. The primer pair was added at an asymmetric concentration to ensure excessive amplification of the complementary DNA strand of the fluorescently labeled probe. The sequences of the primers and probes are as follows. Note that each fluorescently labeled probe has complementary sequences near both ends, designed to form a double strand within the molecule. The fluorescently labeled 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, while the fluorescently labeled 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.

[0068] Forward primer: 5'-GTCACATTTTCATTATTTTTATTATAAGG-3' (SEQ ID NO: 1) Reverse primer: 5'-GTATCGTCAAGGCACTCTTGCC-3' (SEQ ID NO: 2) Fluorescently labeled probe corresponding to the wild type: 5'-TTGGAGCTGGTGGCGT-3' (SEQ ID NO: 3) Fluorescently labeled probe corresponding to the mutant type: 5'-CTGGTGACGTAGGCA-3' (SEQ ID NO: 4)

[0069] Figure 7A shows the positional relationship between the secondary structure 701 (SEQ ID NO: 5) of the amplified single-stranded DNA of the G13D mutant and the binding region 702 of the fluorescently labeled probe corresponding to the wild-type. Figure 7B shows the positional relationship between the secondary structure 701 (SEQ ID NO: 5) of the amplified single-stranded DNA of the G13D mutant and the binding region 703 of the fluorescently labeled probe corresponding to the mutant. The single-strand formation rate of the fluorescently labeled probe binding region 702 in Figure 7A is 69%, and the single-strand formation rate of the fluorescently labeled probe binding region 703 in Figure 7B is 47%.

[0070] Figure 7C shows the differential curve of the melting curve obtained as a result of melting curve analysis after the asymmetric nucleic acid amplification reaction. Compared to 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, 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 was smaller. This result shows that if the rate of single-strand formation in the binding region of the fluorescently labeled probe is low, the amount of binding between the amplified DNA and the fluorescently labeled probe decreases. In such cases, the mutant may be present in small amounts, further reducing the amount of fluorescence and making detection difficult.

[0071] Next, Figures 8A and 8B show the results of asymmetric nucleic acid amplification using a mutation-introducing primer. Figure 8A illustrates the position 803 where a mutation is to be introduced with a primer, along with the positional relationship between the secondary structure 801 (SEQ ID NO: 5) of the amplified single-stranded DNA of the G13D mutant and the binding region 802 of the fluorescently labeled probe corresponding to the mutant, when the mutation-introducing primer shown in Figure 7A was not used. A mutation-introducing primer was designed to change the base sequence CC at position 803 where a mutation is to be introduced with a primer to AA, and the asymmetric nucleic acid amplification reaction was performed using this primer. Figure 8B illustrates the positional relationship between the secondary structure 804 (SEQ ID NO: 6) of the amplified single-stranded DNA of the G13D mutant and the binding region 802 of the fluorescently labeled probe corresponding to the mutant, along with the position 805 where a mutation is introduced with a primer. In Figure 8B, the proportion of single strands formed in the binding region 802 of the fluorescently labeled probe corresponding to the mutant increases, and the amount of fluorescently labeled probe bound increases.

[0072] In this way, by performing an asymmetric nucleic acid amplification reaction using a mutation-introducing primer, the secondary structure of the target gene being amplified is changed, and the rate of single-strand formation increases, thereby increasing the amount of binding of the fluorescently labeled probe, thereby improving the measurement sensitivity and accuracy.

[0073] Example 2 This example illustrates an example of asymmetric nucleic acid amplification reaction using tagged mutation-introducing primers.

[0074] Figures 9A and 9B show the results of asymmetric nucleic acid amplification using a tagged mutation-introducing primer. Figure 9A illustrates the position 903 where a mutation is to be introduced by the primer, along with the secondary structure 901 (SEQ ID NO: 5) of the amplified single-stranded DNA of the G13D mutant when the mutation-introducing primer shown in Figure 7A was not used, and the positional relationship between the secondary structure 901 (SEQ ID NO: 5) of the amplified single-stranded DNA and the binding region 902 of the fluorescently labeled probe corresponding to the mutant was shown. A mutation-introducing primer was designed in which the base sequence CC at position 903 where a mutation is to be introduced by the primer was changed to AA, and a tag sequence was added to the 3' end. When an asymmetric nucleic acid amplification reaction was performed using this primer, the position 905 where a mutation was introduced by the primer and the position 906 of the tag added by the primer are shown in Figure 9B. In Figure 9B, the proportion of single-strand formation in the binding region 902 of the fluorescently labeled probe corresponding to the mutant increases, and the amount of fluorescently labeled probe bound increases. As shown in Figures 9A and 9B, the secondary structure of the single-stranded DNA may change significantly before and after the introduction of the mutation.

[0075] In this way, by performing an asymmetric nucleic acid amplification reaction using a tagged mutation-introducing primer, the secondary structure of the target gene being amplified is changed, and the rate of single-strand formation increases, thereby increasing the amount of binding of the fluorescently labeled probe, thereby improving the measurement sensitivity and accuracy.

[0076] DESCRIPTION OF SYMBOLS 101...DNA 102...Fluorescently labeled probe 103...Fluorescent dye 104...Quencher 201...Forward primer 202...Reverse primer 203...Fluorescently labeled probe 204...DNA to be detected 205...Molecule in which complementary strands of DNA to be detected form a double-stranded structure 206...Molecule in which fluorescently labeled probe is bound to a single-stranded molecule of DNA to be detected 207...One strand of DNA to be detected 301...Forward primer 302...Mutation-introduced reverse primer 303...Fluorescently labeled probe 304...DNA to be detected 305...Secondary structure of single-stranded DNA to be detected amplified with reverse primer without mutation 306...Site to be introduced into mutation 307...Secondary structure of single-stranded DNA to be detected amplified with reverse primer with mutation introduction 308...Introduced mutation 309...Mutation 401...Forward primer 402...Tagged mutation-introduced reverse primer 403...Fluorescently labeled probe 404... DNA to be detected 405... Tag sequence 406... Secondary structure of single-stranded DNA to be detected amplified with a reverse primer without mutation 407... Site to be mutagenized 408... Secondary structure of single-stranded DNA to be detected amplified with a tagged reverse primer for mutagenesis 409... Mutation introduced 410... Added tag sequence 411... Mutation 501... Melting temperature 701... Secondary structure of single-stranded DNA to be detected amplified with a reverse primer without mutation 702... Binding region of fluorescently labeled probe corresponding to wild type 703... Binding region of fluorescently labeled probe corresponding to mutant type 704... Peak of amplified DNA of G13D mutant type and fluorescently labeled probe corresponding to wild type 705... Peak of amplified DNA of G13D mutant type and fluorescently labeled probe corresponding to mutant type 801... Secondary structure of single-stranded DNA of G13D mutant type amplified without using a primer for mutagenesis 802... Binding region of fluorescently labeled probe 803... Position to be mutagenized 804... Secondary structure of single-stranded DNA of G13D mutant type amplified using the mutation-introducing primer 805... Mutation introduction position 901... Secondary structure of single-stranded DNA of G13D mutant type amplified without using the mutation-introducing primer 902... Binding region of fluorescently labeled probe 903... Position to be mutagenized904... Secondary structure of single-stranded DNA of G13D mutant type amplified using mutation-introducing primer 905... Mutation introduction position 906... Tag sequence

[0077] SEQ ID NOs: 1 to 7: DNA (artificial sequences, synthetic polynucleotides)

Claims

1. amplifying a region of a test nucleic acid containing a target nucleic acid in the presence of a primer pair containing a forward primer and a reverse primer and a probe; measuring the binding of the nucleic acid amplified by the forward primer and the reverse primer to the probe; A method for detecting a target nucleic acid comprising: Either or both of the forward primer and the reverse primer contain a mutation in their sequences for introducing a mutation into a region of the amplified nucleic acid containing the target 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 and the amplified nucleic acid are bound to each other; The method, wherein the mutation is introduced into a region of the amplified nucleic acid that contains the target nucleic acid by the amplifying step.

2. The method of claim 1 , wherein the reverse primer comprises the mutation.

3. The method of claim 1 , wherein the probe comprises a fluorescent dye or comprises a fluorescent dye and a quenching dye, and the fluorescent dye is used to measure binding of the amplified nucleic acid to the probe.

4. The method of claim 1 , wherein the 3′ and 5′ terminal sequences of the probe have complementary sequences.

5. The method of claim 1 , wherein the amplifying step is carried out by an asymmetric nucleic acid amplification reaction.

6. 2. The method of claim 1, wherein either the forward primer or the reverse primer, or both, further comprise a tag sequence, such that the amplified nucleic acid comprises the tag sequence.

7. The method of claim 1 , wherein the percentage of single-stranded bases in the probe binding region in the amplified nucleic acid is 50% or more.

8. The method of claim 1, wherein the binding between the amplified nucleic acid and the probe is measured with a change in temperature, and the melting temperature of the double strand of the amplified nucleic acid and the probe is calculated from the change in the binding with the change in temperature.

9. The method of claim 1, further comprising measuring the binding between the amplified nucleic acid and the probe for each cycle of temperature change, and detecting the target nucleic acid from the relationship between the number of cycles and the change in the binding.

10. The method of claim 1 , comprising detecting a plurality of target nucleic acids using a plurality of primers containing the mutation.

11. The method according to claim 10, further comprising measuring the binding between the amplified nucleic acid and the probe for each cycle of temperature change, and calculating a ratio of the initial concentrations of the multiple target nucleic acids in the test nucleic acid from the relationship between the number of cycles and the change in binding.

12. The method of claim 1, further comprising measuring the binding between the amplified nucleic acid and 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 known concentration of target nucleic acid, 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 detecting the target nucleic acid comprises determining the genotype of the target nucleic acid.

14. A primer for introducing a mutation into a region of a test nucleic acid containing a target nucleic acid and amplifying the nucleic acid, the primer comprising a mutation in its sequence for introducing a mutation into the region of the test nucleic acid containing the target 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 is bound to a probe.

15. The primer of claim 14 which is a reverse primer.

16. The primer of claim 14, further comprising a tag sequence.

17. A kit for detecting a target nucleic acid, comprising: a primer pair including a forward primer and a reverse primer for amplifying a region including the target nucleic acid; a probe that binds to a nucleic acid that is amplified using a forward primer and a reverse primer; Including, The kit, wherein either the forward primer or the reverse primer, or both, contain a mutation in their sequence for introducing a mutation into a region of the amplified nucleic acid containing the target 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 and the amplified nucleic acid bind to each other.

18. 18. The kit of claim 17, wherein either the forward primer or the reverse primer, or both, further comprise a tag sequence.

19. The kit of claim 17, wherein the forward primer and the reverse primer are at different concentrations.

20. 20. The kit of claim 17, wherein the probe comprises a fluorescent dye or comprises a fluorescent dye and a quenching dye.

21. The kit according to claim 17, wherein the 3' and 5' terminal sequences of the probe have complementary sequences.

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

23. and / or The kit according to claim 17 , comprising a plurality of types of the probe for each of a plurality of target nucleic acids.

24. 20. The kit of claim 17, further comprising a DNA polymerase.