Inspection method and inspection device for genetic mutation

Abstract

The purpose of the present invention is to provide a gene mutation inspection method capable of quantitatively analyzing the abundance ratio of a gene mutation to be inspected by using a nucleic acid probe.　This gene mutation inspection method is characterized by comprising: a step for performing a nucleic acid amplification reaction for amplifying a region including a gene mutation to be inspected; a step for confirming that the nucleic acid amplification reaction is in an exponential amplification period by detecting an amplification product in a reaction liquid of the nucleic acid amplification reaction; a step for detecting a target nucleic acid and a non-target nucleic acid in the gene mutation that are contained in the reaction liquid which has been confirmed to be in an exponential amplification period, the target nucleic acid including a to-be-detected base and the non-target nucleic acid including a not-to-be-detected base that corresponds to the to-be-detected base, the detection carried out by using a nucleic acid probe for target nucleic acid detection, the probe having a sequence complimentary to the region containing the to-be-detected base in the target nucleic acid, and a nucleic acid probe for non-target nucleic acid detection, the probe having a sequence complementary to a region including a not-to-be-detected base in the non-target nucleic acid, or a common probe having a sequence complementary to a common region that is common to the target nucleic acid and the non-target nucleic acid and does not overlap with the region including the to-be-detected base or the not-to-be-detected base; and a step for quantitatively analyzing the target nucleic acid contained in the reaction liquid.

Description

Gene mutation testing method and testing device

[0001] The present invention relates to a method and an apparatus for testing gene mutations using nucleic acid probes used in DNA chips and the like.

[0002] Causative and marker gene mutations have been identified for various diseases, including cancer. Gene mutations related to cancer prognosis prediction and drug side effects have also been identified. For example, single nucleotide variants (SNVs) and single nucleotide polymorphisms (SNPs) are typical examples of such gene mutations.

[0003] One known system for testing such genetic mutations is a system that uses a DNA chip (also called a DNA microarray). The DNA chip has a configuration in which nucleic acid probes corresponding to the genetic mutation to be tested (a wild-type probe corresponding to the wild-type and a mutant probe corresponding to the mutant) are immobilized on a substrate. Using genomic DNA collected from a subject as a template, a nucleic acid amplification reaction is carried out using a pair of primer sets that sandwich a region containing the genetic mutation to be tested, thereby obtaining labeled nucleic acid fragments. A solution containing the obtained nucleic acid fragments is brought into contact with the DNA chip under specified reaction conditions. If the solution contains a nucleic acid fragment containing a genetic mutation, a signal from the mutant probe can be detected.

[0004] Based on the basic principle described above, the presence or absence of genetic mutations in the genomic DNA of a subject can be examined. Genetic mutations are not limited to SNVs and SNPs. DNA chips having nucleic acid probes corresponding to sequences are also used for microsatellite, DNA methylation, and chimeric gene testing.

[0005] As described above, in a system using a DNA chip, nucleic acid fragments obtained at the end of nucleic acid amplification (endpoint) can be used to test whether they contain the target gene mutation. In other words, a system using a DNA chip enables qualitative evaluation of the target gene mutation, rather than quantitative measurement.

[0006] On the other hand, as a system for quantifying nucleic acids using a DNA chip, as disclosed in Patent Document 1, a system is known in which the signal intensity based on hybridization between a target nucleic acid and a probe is measured and compared with the signal intensity based on hybridization between a known amount of oligonucleotide and the probe, thereby correcting and quantifying the amount of target nucleic acid. Furthermore, Patent Document 2 discloses a method in which quantitative analysis or qualitative analysis by real-time PCR and qualitative analysis using a DNA chip are simultaneously performed in a single reaction system. However, these prior art techniques are techniques for quantifying the amount (copy number) of target nucleic acid present.

[0007] JP 2007-097574 A Japanese Patent No. 5689446

[0008] As described above, genetic mutation testing using nucleic acid probes used in DNA chips, etc., has the problem that it only measures the presence or absence of genetic mutations in the test subject or quantifies the abundance (copy number), but is unable to quantitatively analyze the abundance ratio of genetic mutations relative to wild-type genes. Therefore, an object of the present invention is to provide a genetic mutation testing method and testing device that can quantitatively analyze the abundance ratio of genetic mutations in the test subject using nucleic acid probes.

[0009] In order to achieve the above-mentioned object, the inventors conducted extensive research and found that, when a nucleic acid fragment containing a genetic mutation to be tested is amplified by a nucleic acid amplification reaction, the nucleic acid amplification reaction can be confirmed to be in the exponential amplification phase by detecting the amplified product in the reaction solution in the nucleic acid amplification reaction, and then the nucleic acid fragment containing the genetic mutation contained in the reaction solution in the exponential amplification phase can be detected using a nucleic acid probe, thereby making it possible to quantitatively analyze the genetic mutation, thereby completing the present invention.

[0010] That is, the gist of the present invention is as follows: (1) A method for testing for a genetic mutation, comprising the steps of: carrying out a nucleic acid amplification reaction to amplify a region containing a genetic mutation to be tested; confirming that the nucleic acid amplification reaction is in the exponential amplification phase by detecting an amplification product in a reaction solution of the nucleic acid amplification reaction; detecting a target nucleic acid containing a target base to be detected in the genetic mutation and a non-target nucleic acid containing a non-target base corresponding to the target base, which are contained in the reaction solution confirmed to be in the exponential amplification phase, using a nucleic acid probe for detecting a target nucleic acid having a sequence complementary to a region containing the target base in the target nucleic acid, a nucleic acid probe for detecting a non-target nucleic acid having a sequence complementary to a region containing the non-target base in the non-target nucleic acid, or a common probe having a sequence complementary to a common region common to the target nucleic acid and the non-target nucleic acid, the common region not overlapping with a region containing the target base or the non-target base; and quantitatively analyzing the target nucleic acid contained in the reaction solution. (2) The method for testing for genetic mutations according to (1), wherein the detecting step uses a DNA chip on which the nucleic acid probe for detecting a target nucleic acid and the nucleic acid probe for detecting a non-target nucleic acid or the common probe are immobilized on a carrier. (3) The method for testing for genetic mutations according to (1), wherein there are multiple regions containing the genetic mutation to be tested, and the step of carrying out the nucleic acid amplification reaction and the step of confirming that the nucleic acid amplification reaction is in the exponential amplification phase are carried out for each of the regions, and the target nucleic acid and non-target nucleic acid contained in the reaction solution for each of the regions confirmed to be in the exponential amplification phase are detected. (4) The method for testing for genetic mutations according to (1), wherein the reaction solution contains an amplification detection probe, and both the step of confirming that the nucleic acid amplification reaction is in the exponential amplification phase and the step of detecting the target nucleic acid and the non-target nucleic acid are carried out by detecting a signal from the amplification detection probe. (5) The method for testing for genetic mutations according to (4), wherein the Tm value of the amplification detection probe is higher than the Tm values ​​of the nucleic acid probe for detecting a target nucleic acid, the nucleic acid probe for detecting a non-target nucleic acid, and the common probe.(6) The method for testing for a genetic mutation according to (4), wherein the amplification detection probe has a sequence complementary to a region in the target nucleic acid and the non-target nucleic acid that does not overlap with a region having a sequence complementary to the nucleic acid probe for detecting a target nucleic acid, the nucleic acid probe for detecting a non-target nucleic acid, and the common probe. (7) The method for testing for a genetic mutation according to (1), wherein the quantitative analysis step calculates the abundance ratio of the target base in the genetic mutation to be tested based on the signal intensity from the nucleic acid probe for detecting a target nucleic acid and the signal intensity from the nucleic acid probe for detecting a non-target nucleic acid or the common probe. (8) The method for testing for a genetic mutation according to (1), wherein the quantitative analysis step quantifies the abundance ratio of the target base in the genetic mutation to be tested based on the signal intensity from the nucleic acid probe for detecting a target nucleic acid and the signal intensity from the nucleic acid probe for detecting a non-target nucleic acid or the common probe using a calibration curve created using multiple samples containing target nucleic acids and non-target nucleic acids in known proportions. (9) The method for testing a genetic mutation according to (1), wherein the quantitative analysis step uses a determination value calculated by the formula: [signal intensity from nucleic acid probe for detecting target nucleic acid] / ([signal intensity from nucleic acid probe for detecting target nucleic acid]+[signal intensity from nucleic acid probe for detecting non-target nucleic acid]), or a determination value calculated by the formula: [signal intensity from nucleic acid probe for detecting target nucleic acid] / [signal intensity from common probe]. (10) The method for testing a genetic mutation according to (1), wherein the detection step is performed in the presence of a blocking nucleic acid comprising a base sequence complementary to the non-target nucleic acid comprising the non-detection target base.(11) A genetic mutation testing device comprising: a nucleic acid amplification reaction detection unit that performs a nucleic acid amplification reaction to amplify a region containing a genetic mutation to be tested and confirms that the nucleic acid amplification reaction is in the exponential amplification phase by detecting an amplified product in a reaction solution in the nucleic acid amplification reaction; a detection unit that receives the reaction solution from the nucleic acid amplification reaction detection unit and detects at least the target nucleic acid using a nucleic acid probe for detecting a target nucleic acid having a sequence complementary to a region containing a target base in a target nucleic acid containing a target base to be detected in the genetic mutation, a nucleic acid probe for detecting a non-target nucleic acid having a sequence complementary to a region containing a non-target base in a non-target nucleic acid containing a non-target base corresponding to the target base to be detected, or a common probe having a sequence complementary to a common region common to the target nucleic acid and the non-target nucleic acid, the common region not overlapping with the region containing the target base or the non-target base to be detected; and a liquid delivery device that supplies the reaction solution from the nucleic acid amplification reaction detection unit to the detection unit. (12) The genetic mutation testing device according to (11), wherein the detection unit comprises a DNA chip mounting unit mounting a DNA chip having the nucleic acid probe for detecting the target nucleic acid and the nucleic acid probe for detecting the non-target nucleic acid or the common probe immobilized on a carrier. (13) The genetic mutation testing device according to (11), wherein the nucleic acid amplification reaction detection unit comprises a plurality of reaction chambers for performing nucleic acid amplification reactions that amplify regions containing genetic mutations of different test subjects, and the liquid delivery device comprises a switching device that switches a flow path that supplies reaction liquid to the detection unit between the plurality of reaction chambers. (14) The genetic mutation testing device according to (11), wherein the detection unit detects the target nucleic acid and the non-target nucleic acid using the common probe. (15) The genetic mutation testing device according to (11), wherein the liquid delivery device comprises a flow path connecting the nucleic acid amplification reaction detection unit and the detection unit, a valve disposed on the flow path, and a pump device connected to the flow path.(16) The genetic mutation testing device according to (11), further comprising a hybridization buffer tank that supplies a hybridization buffer to the detection unit, wherein the liquid delivery device supplies a reaction solution of a nucleic acid amplification reaction in the exponential amplification phase from the nucleic acid amplification reaction detection unit to the detection unit, and supplies a hybridization buffer from the hybridization buffer tank to the detection unit. (17) The genetic mutation testing device according to (16), wherein the hybridization buffer contains a blocking nucleic acid that contains a base sequence complementary to a non-target nucleic acid that contains the non-target base. This specification incorporates the disclosure of Japanese Patent Application No. 2024-112187, from which the present application claims priority.

[0011] According to the genetic mutation testing method and testing device of the present invention, the genetic mutation can be quantitatively analyzed using a nucleic acid probe corresponding to the genetic mutation. By applying the genetic mutation testing method and testing device of the present invention, it is possible to quantitatively analyze the genetic mutation, for example, the ratio of mutant to wild type.

[0012] 1 is a characteristic diagram showing the number of thermal cycles on the horizontal axis and the amount of DNA amplified on the vertical axis when a nucleic acid amplification reaction is performed using template DNA containing 80% wild-type DNA and 20% mutant DNA. FIG. 1 is a configuration diagram schematically showing an example of an apparatus for testing for gene mutations according to the present invention. FIG. 2 is a flowchart showing the steps of testing for gene mutations using the apparatus for testing for gene mutations according to the present invention. FIG. 3 is a configuration diagram schematically showing another example of an apparatus for testing for gene mutations according to the present invention. FIG. 4 is a configuration diagram schematically showing yet another example of an apparatus for testing for gene mutations according to the present invention. FIG. 5 is a characteristic diagram showing a calibration curve obtained using a PCR reaction solution for the V617F mutation of the JAK2 gene in Example 1. FIG. 6 is a characteristic diagram showing a logarithmically transformed calibration curve obtained using a PCR reaction solution for the V617F mutation of the JAK2 gene in Example 1. FIG. 7 is a characteristic diagram showing the relationship between the mutation rate quantified in Example 1 and the actually measured mutation rate. FIG. 8 is a characteristic diagram showing a calibration curve obtained using a PCR reaction solution for the V617F mutation of the JAK2 gene in Example 2. 1 is a characteristic diagram showing a logarithmically transformed calibration curve obtained using a PCR reaction solution for the V617F mutation of the JAK2 gene in Example 2. FIG. 2 is a characteristic diagram showing the relationship between the mutation rate quantified in Example 2 and the actually measured mutation rate.

[0013] The genetic mutation testing method and testing device according to the present invention quantitatively detect a target nucleic acid containing a genetic mutation to be tested using a nucleic acid probe.

[0014] Examples of genetic mutations to be tested include single nucleotide variants (SNVs) and single nucleotide polymorphisms (SNPs), which refer to differences in bases at specific positions in genomic DNA. In other words, a specific genetic mutation involves multiple bases. In the following description, one of these bases is referred to as a target base, and bases other than the target base are referred to as non-target bases.

[0015] The base to be detected refers to, for example, a specific nucleic acid residue at a specific position in genomic DNA, and is not particularly limited, but refers to a specific type of base in a base sequence such as a single nucleotide polymorphism (SNP). For example, if a specific single nucleotide polymorphism can take A (adenine) or C (cytosine), either one of the bases, i.e., A (adenine) in the single nucleotide polymorphism, can be used as the base to be detected. Here, the base to be detected may be either a major allele or a minor allele in a genetic polymorphism, and may or may not be a risk allele.

[0016] Here, the term "target nucleic acid" refers to a nucleic acid molecule containing a base to be detected, i.e., a nucleic acid fragment. The target nucleic acid may be a nucleic acid molecule made of DNA, a nucleic acid molecule made of RNA, a nucleic acid molecule containing DNA and RNA (DNA-RNA complex), or a fragmented molecule such as cell-free DNA. The term "nucleic acid" also refers to adenine, cytosine, guanine, thymine, and uracil, as well as artificial nucleic acids such as peptide nucleic acid (PNA) and locked nucleic acid (LNA).

[0017] A target nucleic acid containing a target base can be prepared by amplifying a predetermined region of genomic DNA containing a gene mutation using a nucleic acid amplification method. Alternatively, the target nucleic acid may be cDNA obtained by reverse transcription from a transcription product collected from an individual organism, tissue, or cell. The base length of the target nucleic acid is not particularly limited, but can be, for example, 60 to 1,000 bases, preferably 60 to 500 bases, and more preferably 60 to 200 bases.

[0018] In addition, for a target nucleic acid containing a target base, a nucleic acid molecule (nucleic acid fragment) containing a non-target base corresponding to the target base is called a non-target nucleic acid.For example, when one base among a plurality of bases that can be taken at a predetermined position in genomic DNA is taken as a target base, the bases other than the target base are taken as non-target bases.More specifically, when a single nucleotide polymorphism at a predetermined position can take A (adenine) or C (cytosine), if A (adenine) in the single nucleotide polymorphism is taken as a target base, C (cytosine) in the single nucleotide polymorphism is taken as a non-target base.

[0019] Furthermore, for example, when a mutant type (the forward or reverse strand of a minor allele) in a specific polymorphism or variant is used as a base to be detected, a nucleic acid fragment containing the mutant type can be used as a target nucleic acid, and a nucleic acid fragment containing a wild type (the forward or reverse strand of a major allele), which is a base not to be detected, can be used as a non-target nucleic acid.

[0020] When the non-target base is present in genomic DNA for a specific gene mutation, the non-target nucleic acid containing the non-target base is simultaneously obtained when the target nucleic acid containing the target base is obtained as described above. For example, when the target nucleic acid is obtained by a nucleic acid amplification reaction such as a polymerase chain reaction, if one allele is a non-target base, the non-target nucleic acid will be amplified together with the target nucleic acid.

[0021] In the genetic mutation testing method and testing device of the present invention, a target nucleic acid containing a target base is detected by using a nucleic acid probe having a base sequence complementary to at least a region of the target nucleic acid containing the target base. The nucleic acid probe is not particularly limited, but may be, for example, 10 to 30 bases long, preferably 15 to 25 bases long. Furthermore, when the bases constituting the nucleic acid probe are viewed as a character string, the base complementary to the target base is preferably located at the center of the character string. Note that, for nucleic acid probes consisting of an even number of bases, the center of the character string includes cases where the base is shifted by one base toward the 5' end or the 3' end.

[0022] In particular, the genetic mutation testing method and testing device according to the present invention can use a common probe having a sequence complementary to a common region common to the target nucleic acid and the non-target nucleic acid. The common region is a region of base sequence common to the target nucleic acid and the non-target nucleic acid, and does not contain target bases or non-target bases. For example, the common region can be a region that does not overlap with the region to which the nucleic acid probe for detecting the target nucleic acid hybridizes and the region to which the nucleic acid probe for detecting the non-target nucleic acid hybridizes.

[0023] The genetic mutation testing method and testing device of the present invention can be applied to any system that includes hybridization, which refers to the complementary binding between nucleic acid molecules in a target nucleic acid and a nucleic acid probe. That is, the genetic mutation testing method and testing device of the present invention can be based on Southern hybridization, Northern hybridization, or in situ hybridization. In particular, the genetic mutation testing method and testing device of the present invention are preferably used in a system in which a nucleic acid probe is immobilized on a carrier (including a substrate, hollow fiber, or microparticle) and the immobilized nucleic acid probe is used to detect (including qualitative and quantitative) the target nucleic acid. More specifically, the genetic mutation testing method and testing device of the present invention are preferably used in a system that uses a DNA chip (DNA microarray) in which a nucleic acid probe is immobilized on a substrate.

[0024] The following describes an exemplary system for detecting a target nucleic acid using a DNA chip (DNA microarray) to detect a gene mutation using the method and device for detecting a gene mutation according to the present invention. The nucleic acid probe for detecting the target nucleic acid (nucleic acid probe for detecting a target nucleic acid), the nucleic acid probe for detecting a non-target nucleic acid (nucleic acid probe for detecting a non-target nucleic acid), and the common probe are preferably single-stranded DNA, and can be obtained by chemical synthesis using a nucleic acid synthesizer, for example. The nucleic acid synthesizer may be a device known as a DNA synthesizer, a fully automated nucleic acid synthesizer, an automatic nucleic acid synthesizer, or the like.

[0025] In this example, the nucleic acid probe for detecting a target nucleic acid, the nucleic acid probe for detecting a non-target nucleic acid, and the common probe are preferably used in the form of a microarray by immobilizing their 5'-end or 3'-end on a carrier. Materials for the carrier can be those known in the art and are not particularly limited. Examples include precious metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten, and their compounds, and conductive materials such as carbon, represented by graphite and carbon fiber; silicon materials, represented by single crystal silicon, amorphous silicon, silicon carbide, silicon oxide, and silicon nitride; composite materials of these silicon materials, represented by SOI (silicon-on-insulator); inorganic materials, such as glass, quartz glass, alumina, sapphire, ceramics, forsterite, and photosensitive glass; polyethylene, polypropylene, and cyclic polyisoprene. Examples of the carrier include organic materials such as olefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene-acrylonitrile copolymer, acrylonitrile-butadiene-styrene copolymer, polyphenylene oxide, and polysulfone. The shape of the carrier is not particularly limited, but is preferably flat.

[0026] The carrier preferably has a carbon layer such as diamond-like carbon (DLC) on its surface and chemically modified groups such as amino groups, carboxyl groups, epoxy groups, formyl groups, hydroxyl groups, and active ester groups. Carriers having a carbon layer and chemically modified groups on their surface include those having a carbon layer and chemically modified groups on the surface of a substrate, and those having chemically modified groups on the surface of a substrate made of a carbon layer. Materials known in the art can be used for the substrate, and are not particularly limited, and the same materials as those listed above as carrier materials can be used.

[0027] The genetic mutation testing method and testing device of the present invention can quantitatively analyze a target nucleic acid in a subject using the DNA chip prepared in this manner. Specifically, the method includes the steps of first extracting DNA from a sample derived from the subject, amplifying a region containing the genetic mutation to be tested by a nucleic acid amplification reaction using the extracted DNA as a template, and detecting at least the target nucleic acid using the DNA chip during the exponential amplification phase of the nucleic acid amplification reaction.

[0028] The subject is typically a human and is the subject of genetic testing, including patients suffering from a disease associated with a specific genetic mutation. The subject-derived sample is not particularly limited. Examples include blood-related samples (blood, serum, plasma, etc.), lymph, feces, cancer cells, tissue or organ fragments, and extracts.

[0029] First, DNA is extracted from a sample collected from a subject. The extraction method is not particularly limited. For example, cells may be disrupted using a disrupter called a blender, mixer, or homogenizer, and then purified using an organic solvent (phenol / chloroform). Alternatively, cells may be disrupted using a surfactant, adsorbed onto a silica column, and then centrifuged. Alternatively, DNA may be adsorbed onto silica-coated magnetic beads and then attracted with a magnet to separate the DNA.

[0030] Next, an amplification reaction is performed using the obtained DNA as a template to amplify the nucleic acid region containing the gene mutation to be tested, preferably the DNA. Examples of applicable amplification reactions include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN). In the amplification reaction, it is desirable to add a label so that the amplified region can be identified. The method for labeling the amplified nucleic acid is not particularly limited, but for example, a method in which the primers used in the amplification reaction are pre-labeled may be used, or a method in which labeled nucleotides are used as substrates in the amplification reaction may be used. The labeling substance is not particularly limited, but radioisotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin may be used.

[0031] This reaction system also contains a buffer necessary for nucleic acid amplification and labeling, a heat-resistant DNA polymerase, a pair of primers specific to the region to be amplified, a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate labeled with a fluorescent label or the like), a nucleotide triphosphate, magnesium chloride, and the like.

[0032] Nucleic acid amplification reactions consist of three steps: thermal denaturation, annealing, and extension. These three steps constitute one cycle, and by repeating multiple cycles, nucleic acid fragments containing the target gene mutation can be synthesized. The procedure for repeating these three steps is called a thermal cycle, and the number of times the three steps are repeated is called the number of thermal cycles.

[0033] Alternatively, the nucleic acid amplification reaction may be performed at the same temperature, which is called two-step PCR, and consists of two steps: a thermal denaturation step and an annealing and extension step. In this case, these two steps are repeated as one cycle to synthesize a nucleic acid fragment containing the gene mutation to be tested. In this case, the procedure for repeating these two steps is called a thermal cycle, and the number of times the two steps are repeated is called the number of thermal cycles.

[0034] The thermal denaturation step is a step in which a reaction solution containing template DNA is heated to typically 90°C or higher to form single-stranded DNA. The first thermal denaturation step in the thermal cycle is preferably carried out for a relatively long time (1 to 5 minutes) when genomic DNA is used as the template. The second and subsequent thermal denaturation steps are preferably carried out for a relatively short time (1 to 90 seconds) because the purpose of the steps is to convert the double-stranded amplification product obtained into single strands.

[0035] The annealing step is a step in which a reaction solution containing a single-stranded template DNA and a pair of primer sets is heated to a temperature typically in the range of 40 to 75°C, and these primers are specifically bound (annealed) to the template DNA. The temperature of the annealing step is set appropriately depending on the sequence and length of the primers. The temperature of the annealing step is usually set within a range of ±5°C of the Tm value of the primers used.

[0036] The extension step involves synthesizing a complementary strand from the end of the primer using a thermostable polymerase (e.g., Taq DNA polymerase) at a temperature of approximately 72°C while the primer is annealed to the template DNA. In this reaction, a complementary strand of the template DNA is synthesized from a labeled primer, or a complementary strand of the template DNA is synthesized while incorporating labeled free nucleotides. Thus, for the genetic mutation being tested, both the target nucleic acid containing the target base and the non-target nucleic acid containing the non-target base are labeled. The duration of the extension step is appropriately determined depending on the length of the nucleic acid fragment to be amplified and the reactivity of the enzyme used.

[0037] The exponential amplification phase in thermal cycling is the period during which the amplification efficiency in thermal cycling is 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, and most preferably 98% or higher. Here, amplification efficiency refers to the percentage increase in the amount of nucleic acid fragment per cycle. In other words, an amplification efficiency of 100% means that the amplified fragment doubles per cycle. Therefore, when defining the exponential amplification phase, the upper limit of the amplification efficiency is not particularly limited, and can be, for example, a value of 100 to 110%.

[0038] In the genetic mutation testing method and testing device of the present invention, the exponential amplification phase of a nucleic acid amplification reaction is confirmed by detecting an amplified product in a reaction solution during the nucleic acid amplification reaction. More specifically, the nucleic acid amplification reaction is carried out using a reaction solution containing a fluorescent dye (an amplification detection probe), and the amplification of nucleic acids by polymerase chain reaction is monitored in real time. The exponential amplification phase of the nucleic acid amplification reaction can be confirmed based on the obtained fluorescence intensity value.

[0039] The amplification detection probe can be any fluorescent probe whose fluorescence intensity changes with nucleic acid amplification by PCR. Examples of amplification detection fluorescent probes include TaqMan® probe, Eprobe®, QProbe®, and dyes (SYBR green I) that specifically intercalate into double-stranded nucleic acids and emit fluorescence. The TaqMan probe method uses a sequence-specific oligonucleotide labeled with a reporter and a quencher. As the PCR reaction progresses, the TaqMan probe is hydrolyzed, increasing the distance between the reporter and quencher, thereby emitting a fluorescent signal. Eprobe is typically quenched by the two fluorescent dye moieties bound to each other, but emits fluorescence upon hybridization with template DNA. Furthermore, QProbe binds to PCR amplification products and reduces its fluorescence upon annealing. Therefore, the fluorescence during annealing in PCR can be measured and the process of nucleic acid amplification can be monitored by monitoring the increase in the fluorescence quenching rate. The amplification detection probe is preferably designed so as not to affect the hybridization reaction between the amplification product and the nucleic acid probe for detecting the target nucleic acid, the nucleic acid probe for detecting the non-target nucleic acid, and the common probe.

[0040] The genetic mutation testing method and testing device of the present invention detect target nucleic acids and non-target nucleic acids contained in a nucleic acid amplification reaction solution confirmed to be in the exponential amplification phase as described above, and quantitatively analyze the target nucleic acid based on this. To quantitatively analyze the target nucleic acid, a hybridization reaction between a nucleic acid probe for detecting the target nucleic acid and the target nucleic acid is carried out, and the amount of nucleic acid hybridized to the nucleic acid probe for detecting the target nucleic acid can be measured, for example, by detecting a label. For example, when a fluorescent label is used, the signal from the label can be detected using a fluorescent scanner, and the signal intensity can be quantified by analyzing the fluorescent signal using image analysis software.

[0041] The hybridization reaction is preferably carried out under stringent conditions. Stringent conditions refer to conditions under which specific hybrids are formed and nonspecific hybrids are not formed, such as conditions in which the hybridization reaction is carried out at 50°C for 16 hours, followed by washing at 2×SSC / 0.2% SDS at 25°C for 10 minutes and then at 2×SSC at 25°C for 5 minutes. That is, the hybridization buffer composition of the present invention may contain salts necessary for the hybridization reaction, such as SSC, or known blocking agents, such as SDS.

[0042] The ability of the genetic mutation testing method and testing device according to the present invention to quantitatively analyze a target nucleic acid will be described with reference to FIG. 1 . In FIG. 1 , a nucleic acid amplification reaction was performed using template DNA containing 80% wild-type DNA and 20% mutant DNA, with the horizontal axis representing the number of thermal cycles and the vertical axis representing the amount of DNA amplified. As shown in FIG. 1 , the 80:20 ratio of wild-type DNA to mutant DNA contained in the template DNA is not maintained near the end of the thermal cycle. Therefore, it can be seen that quantitative analysis of the target nucleic acid is not possible using the reaction solution after the thermal cycle. On the other hand, within the range indicated by the dashed line in FIG. 1 , i.e., during the exponential amplification phase of the thermal cycle, the 80:20 ratio of wild-type DNA to mutant DNA contained in the template DNA is roughly maintained. Therefore, quantitative analysis of the target nucleic acid is possible by detecting the target nucleic acid during the exponential amplification phase of the thermal cycle.

[0043] More specifically, by detecting the target nucleic acid and non-target nucleic acid contained in the nucleic acid amplification reaction solution during the exponential amplification phase, the abundance ratio of the target nucleic acid to the non-target nucleic acid (i.e., the mutation ratio) can be calculated.

[0044] Specifically, when quantitatively analyzing a target nucleic acid based on signals from a nucleic acid probe for detecting a target nucleic acid and a nucleic acid probe for detecting a non-target nucleic acid, the signal intensities of the nucleic acid probe for detecting a target nucleic acid and the nucleic acid probe for detecting a non-target nucleic acid are measured, and a judgment value for evaluating the signal intensity from the nucleic acid probe for detecting a target nucleic acid is calculated. An example of calculating the judgment value is a method using the formula: [signal intensity from the nucleic acid probe for detecting a target nucleic acid] / ([signal intensity from the nucleic acid probe for detecting a target nucleic acid]+[signal intensity from the nucleic acid probe for detecting a non-target nucleic acid]).

[0045] The genetic mutation testing method and testing device according to the present invention are not limited to the above-described configuration. To prevent non-target nucleic acids in a nucleic acid amplification reaction solution from nonspecifically hybridizing with a nucleic acid probe for detecting a target nucleic acid, a so-called blocking nucleic acid can also be used. The blocking nucleic acid can be mixed with a reaction solution containing a target nucleic acid and a non-target nucleic acid, and the resulting mixture can be brought into contact with a DNA chip to allow a hybridization reaction between the target nucleic acid and the nucleic acid probe for detecting a target nucleic acid to proceed. Alternatively, the reaction solution containing the target nucleic acid and the non-target nucleic acid can be mixed with a solution containing a blocking nucleic acid on a DNA chip to allow specific hybridization between the target nucleic acid and the nucleic acid probe for detecting a target nucleic acid to proceed simultaneously. In either case, hybridization of the blocking nucleic acid with the non-target nucleic acid prevents the non-target nucleic acid from hybridizing with the nucleic acid probe for detecting a target nucleic acid.

[0046] The blocking nucleic acid has a base sequence complementary to a region containing a non-target base in the non-target nucleic acid, and can hybridize with the non-target nucleic acid containing the non-target base under conditions that allow hybridization of the target nucleic acid containing the non-target base with the nucleic acid probe.

[0047] In the blocking nucleic acid, the position of the base corresponding to the non-detection target base is not particularly limited. Furthermore, the blocking nucleic acid is not particularly limited, but preferably has a length of 60% or more of the base length of the nucleic acid probe. Furthermore, the blocking nucleic acid preferably has a length of 140% or less of the base length of the nucleic acid probe. For example, if the nucleic acid probe is 25 bases long, the blocking nucleic acid preferably has a base length of 15 to 35 bases long.

[0048] Furthermore, the blocking nucleic acid may contain mismatched bases (non-complementary bases) at positions corresponding to bases other than the non-target bases to be detected contained in the non-target nucleic acid. When the blocking nucleic acid is 15 bases long, the number of mismatched bases may be 1 to 3, preferably 1 to 2. When the blocking nucleic acid is 25 bases long, the number of mismatched bases may be 1 to 5, preferably 1 to 4.

[0049] Furthermore, the concentration of the blocking nucleic acid is not particularly limited and can be appropriately set depending on, for example, the concentrations of the non-target nucleic acids and / or the target nucleic acids, or the primer concentrations. Specifically, the concentration of the blocking nucleic acid in the composition can be 0.01 to 2 μM, preferably 0.02 to 1.5 μM, and more preferably 0.05 to 1.5 μM.

[0050] Note that when a blocking nucleic acid is used to prevent nonspecific hybridization between non-target nucleic acids and nucleic acid probes for detecting target nucleic acids, specific hybridization between non-target nucleic acids and nucleic acid probes for detecting non-target nucleic acids is also inhibited. In this case, the signal intensity from the nucleic acid probes for detecting non-target nucleic acids is significantly reduced. In this case, the value determined by the above formula: [signal intensity from nucleic acid probe for detecting target nucleic acids] / ([signal intensity from nucleic acid probe for detecting target nucleic acids]+[signal intensity from nucleic acid probes for detecting non-target nucleic acids]) may be insufficient for quantitative analysis of target nucleic acids. In this case, it is preferable to measure the total amount of target nucleic acids and non-target nucleic acids using a common probe.

[0051] As described above, the common probe has a sequence complementary to a common region common to the target nucleic acid and the non-target nucleic acid, and therefore can hybridize to the same non-target nucleic acid even if the blocking nucleic acid has hybridized to the same non-target nucleic acid.

[0052] When a common probe is used, the formula for calculating the aforementioned judgment value can be [signal intensity from the nucleic acid probe for detecting target nucleic acid] / [signal intensity from the common probe]. This formula allows the ratio of the target nucleic acid to the total combined target and non-target nucleic acids to be calculated, eliminating the influence of the blocking nucleic acid. When a nucleic acid fragment containing a mutant form is used as the target nucleic acid, the "signal intensity from the nucleic acid probe for detecting target nucleic acid" may be referred to as "MPFI" (Mutant Probe Fluorescent Intensity), and the "signal intensity from the common probe" may be referred to as "CPFI" (Common Probe Fluorescent Intensity).

[0053] Furthermore, absolute quantification of the target nucleic acid can be achieved by preparing a calibration curve in advance. The calibration curve can be prepared by detecting the target nucleic acid and non-target nucleic acid contained in the nucleic acid amplification reaction solution during the exponential amplification phase using various samples with known mutation ratios (samples containing target nucleic acid and non-target nucleic acid in known ratios), and then calculating the determination value using the above formula. In other words, the calibration curve represents the relationship between the determination value calculated as described above and the ratio of target nucleic acid and non-target nucleic acid contained in the nucleic acid amplification reaction solution, or the amount of target nucleic acid.

[0054] The method for creating a calibration curve is not particularly limited, but may include a method in which a preset fluorescence intensity in a nucleic acid amplification reaction is set as the exponential amplification phase, target nucleic acids and non-target nucleic acids are detected at that fluorescence intensity, and a calibration curve is created from the determination value calculated using the above formula. Alternatively, the method for creating a calibration curve may include a method in which the exponential amplification phase (a region with high linearity in the amplification curve) is examined based on an amplification curve obtained in a nucleic acid amplification reaction using a sample with a known mutation rate, the number of thermal cycles in the exponential amplification phase is determined, target nucleic acids and non-target nucleic acids are detected at that number of thermal cycles, and a calibration curve is created from the determination value calculated using the above formula.

[0055] Ideally, the calibration curve should be a linear line of y = x, but due to various errors (operator error, reagent error, measuring equipment error, etc.), it often becomes y = ax + b. Therefore, by normalizing the slope a and intercept b of the calibration curve, it can be made closer to y = x.

[0056] Specifically, a nucleic acid amplification reaction is carried out using a sample with a standard mutation rate (e.g., a sample with a 100% mutation rate), the fluorescence of the reaction solution is detected at predetermined intervals (e.g., every cycle), the reaction solution that has reached a preset fluorescence intensity is removed, and the target nucleic acid and non-target nucleic acids contained in the reaction solution are detected. Here, the fluorescence of the reaction solution is detected by a fluorescent probe for amplification detection, the target nucleic acid is detected by a nucleic acid probe for target nucleic acid detection, and the target nucleic acid and non-target nucleic acids are detected by a common probe.

[0057] First, as a method for determining the exponential amplification phase, there is a method for determining the exponential amplification phase from the amplification curve of a fluorescent probe for detecting amplification. Specifically, a method for calculating the exponential amplification phase from the fluorescence intensity that reaches a predetermined threshold is known. However, this method may not be suitable when the dynamic range of detection changes. Therefore, for example, the exponential amplification phase can be calculated using the following method.

[0058] To determine the fluorescence intensity threshold for the exponential amplification phase, first, several tentative fluorescence intensity thresholds (TTh.PFIs) are set based on the amplification curve of a nucleic acid fragment obtained from the amplification detection fluorescent probe, with the reached fluorescence intensity value being the upper limit of the dynamic range of the amplification detection fluorescent probe. Then, at the set Th.PFIs, the reaction solution is sampled, and a judgment value is calculated using the above formula based on the signal intensities from the nucleic acid probe for detecting the target nucleic acid, the nucleic acid probe for detecting the non-target nucleic acid, and the common probe. The true fluorescence intensity threshold (Th.PFI) is determined from the linearity of the resulting calibration curve. Alternatively, the exponential amplification phase may be determined by performing regression analysis or the like on the amplification curve of a nucleic acid fragment detected with the amplification detection fluorescent probe.

[0059] When calculating the judgement value in TTh.PFI, the reciprocal of the judgement value obtained from a sample with a 100% mutation rate can be used as a correction coefficient. This correction coefficient can be multiplied by the judgement value obtained from known mutation rate samples (e.g., a sample with a 1% mutation rate, a 10% mutation rate, a 50% mutation rate, a 100% mutation rate, etc.) including the 100% mutation rate sample to correct the slope a of the calibration curve. More specifically, if the judgement value for a sample with a 100% mutation rate is calculated by dividing the signal intensity from the nucleic acid probe for detecting the target nucleic acid by the signal intensity from the common probe, the correction coefficient is calculated as [signal intensity from the common probe] / [signal intensity from the nucleic acid probe for detecting the target nucleic acid]. Then, by multiplying the judgement values ​​calculated for various known mutation rate samples by this correction coefficient, a corrected judgement value can be calculated from the judgement values ​​calculated for various known mutation rate samples. The corrected judgement value for a sample with a 100% mutation rate is 1, and the judgement values ​​for the other mutation rate samples fall within a range of 0 to 1.

[0060] Next, based on the known mutation rate samples with normalized slope a, a scatter plot is created with the corrected judgment value (judgment value × correction coefficient) on the horizontal axis and the known mutation rate on the vertical axis, and a linear approximation line can be obtained using the least squares method. In this case, the ratio of the rate of change on the horizontal axis to the rate of change on the vertical axis is constant, so the intercept b can be accurately corrected by logarithmicizing both axes.

[0061] By this procedure, the slope a and intercept b can be corrected, and a calibration curve in which various errors have been normalized can be obtained.

[0062] One method for determining the MPFI and CPFI for calculating the decision value is to create an approximation curve for the relationship between fluorescence intensity and temperature cycle value. This method allows the MPFI and CPFI corresponding to a predetermined TTh.PFI to be determined from the created approximation curve. The approximation curve is not particularly limited as long as it is obtained by regression analysis, and examples include a linear approximation curve, an exponential approximation curve, a logarithmic approximation curve, a polynomial approximation curve, a power approximation curve, a moving average curve, and a sigmoid curve.

[0063] In this way, a calibration curve for a preset TTh.PFI can be created. The genetic mutation testing method and testing device according to the present invention may use the calibration curve created as described above, but it is preferable to set multiple TTh.PFIs to create calibration curves in the same way, and then select and use from the multiple calibration curves the calibration curve (Th.PFI) that has a more linear relationship between the corrected judgment value and the mutation rate.

[0064] The multiple TTh.PFIs can be appropriately set so that their values ​​are equally spaced. For example, when photographing with a 16-bit camera, the upper limit of the fluorescence intensity is 65535, and the multiple TTh.PFIs can be set so that the interval width of the TTh.PFIs is in the range of 1 to 65535, preferably in the range of 5 to 50,000, and more preferably in the range of 10 to 10,000. Calibration curves are created for multiple TTh.PFIs set within such ranges, and the calibration curve with the highest linearity is selected. Specifically, the coefficient of determination R for the multiple calibration curves obtained is calculated. 2Calculate the coefficient of determination R 2 A calibration curve with a value closer to 1 can be selected as a more linear calibration curve. However, it is also possible to extrapolate the fluorescence intensity by drawing an approximate line using multiple exposure images. In such cases, the equal interval width of TTh.PFI is also extended, so the equal interval width of TTh.PFI can also be set using the extended maximum value as the upper limit.

[0065] Furthermore, the genetic mutation testing method and testing device of the present invention are not limited to the use of the calibration curve showing the relationship between the corrected judgment value and the mutation rate described above, and it is preferable to also use a "logarithmically transformed calibration curve" obtained by logarithmically transforming both sides of the equation showing the calibration curve.

[0066] That is, it is preferable to prepare calibration curves for a plurality of TTh.PFIs as described above, and further prepare logarithmically transformed calibration curves corresponding to the prepared calibration curves. After preparing a set of calibration curves and logarithmically transformed calibration curves showing the relationship between the corrected judgment value and the mutation rate for a plurality of TTh.PFIs, a set with high linearity is selected for both calibration curves. Specifically, the coefficient of determination of the calibration curve showing the relationship between the corrected judgment value and the mutation rate is set to a predetermined value (for example, R 2 ≥ 0.99), a set of calibration curves with the highest coefficient of determination in the corresponding logarithmically transformed calibration curve is selected. In the genetic mutation testing method and testing device of the present invention, the calibration curves included in the selected set can be used.

[0067] In addition to the above-mentioned methods, another method for determining the fluorescence intensity threshold during the exponential amplification phase is to use a pseudo target with a copy number equivalent to that of the amplification product. By adjusting the copy number of the pseudo target to be equivalent to that of the amplification product during the exponential amplification phase and reacting them, the dynamic ranges of the nucleic acid probe for detecting target nucleic acids, the nucleic acid probe for detecting non-target nucleic acids, and the common probe can be calculated. Multiple pseudo targets can be used to calculate Th.PFI using the same procedure as above, or the Tm can be calculated based on the results obtained from a single pseudo target. Examples of pseudo targets include synthetic DNA such as oligonucleotide DNA and plasmid DNA, and DNA spiked with target nucleic acids. These pseudo targets are pre-labeled with fluorescent substances, allowing the abundance of the nucleic acid probe for detecting target nucleic acids, the nucleic acid probe for detecting non-target nucleic acids, and the common probe to be measured using a detection system such as a cooled CCD camera, thereby determining their dynamic ranges.

[0068] The dynamic ranges of the nucleic acid probes for detecting target nucleic acids, the nucleic acid probes for detecting non-target nucleic acids, and the common probe can also be measured using an analytical device or the like. In this case, surface analysis methods such as SEM, TEM, XRD, XPS, AES, GDS, ellipsometry, and absorbance measurement can be used. The dynamic range can be determined by measuring the amounts of the nucleic acid probes for detecting target nucleic acids, the nucleic acid probes for detecting non-target nucleic acids, and the common probe present on the surface of the DNA chip using these spectroscopic devices.

[0069] The calibration curve prepared as described above can be used to analyze the proportion of target nucleic acids (mutation proportion) in a subject. Specifically, a portion or all of the reaction solution from a nucleic acid amplification reaction confirmed to be in the exponential amplification phase is removed, and the target nucleic acids and non-target nucleic acids contained in the removed reaction solution are detected. Specifically, for example, a judgment value is calculated from the fluorescence intensity (MPFI) observed with the nucleic acid probe for detecting the target nucleic acid and the fluorescence intensity (CPFI) observed with the common probe, and this is multiplied by a correction coefficient to calculate a corrected judgment value, and the mutation proportion can be analyzed based on the calibration curve.

[0070] Furthermore, the genetic mutation testing method and testing device of the present invention can quantitatively analyze each genetic mutation when there are multiple regions containing genetic mutations to be tested. For example, myeloproliferative neoplasms (MPN) are thought to be associated with mutations in multiple genes, including three genes: JAK2, CALR, and MPL. Quantitative analysis can be performed on these multiple genetic mutations. Specifically, for each region containing a genetic mutation, a nucleic acid amplification reaction is performed and a step of confirming that the nucleic acid amplification reaction is in the exponential amplification phase is performed. Then, the reaction solutions in the exponential amplification phase are combined, and the target nucleic acid and non-target nucleic acids corresponding to each genetic mutation contained in the resulting reaction solution are detected using the corresponding nucleic acid probes for detecting the target nucleic acid and the nucleic acid probes for detecting the non-target nucleic acid or a common probe. The next detection step is performed using only the reaction solutions for which the nucleic acid amplification reaction for each genetic mutation has been confirmed to be in the exponential amplification phase (i.e., the reaction solutions optimally timed for quantification). This allows for more accurate quantitative analysis of each target nucleic acid.

[0071] In the detection step, multiple nucleic acid probes with different target bases can be arranged so that multiple genetic mutations can be tested using a single DNA chip. That is, detection can be performed using a DNA chip on which nucleic acid probes for detecting target nucleic acids corresponding to each of multiple genetic mutations and nucleic acid probes for detecting non-target nucleic acids or a common probe are immobilized on a carrier. Then, in each reaction solution, the above-mentioned nucleic acid amplification reaction is performed using a pair of primer sets specific to regions containing different genetic mutations, and part or all of the reaction solution is sampled from each reaction solution at the optimal timing during the exponential amplification phase for that reaction solution, and the target nucleic acid for multiple genetic mutations can be quantitatively analyzed using a single DNA chip.

[0072] In the above-described embodiment, the fluorescence of the reaction solution is detected using a fluorescent probe for amplification detection to confirm that the nucleic acid amplification reaction is in the exponential amplification phase. Meanwhile, when quantitatively analyzing the target nucleic acid, pre-labeled primers or labeled nucleotides are used as amplification reaction substrates, and the fluorescence of these labels is measured to detect the target nucleic acid hybridized to the nucleic acid probe for detecting the target nucleic acid. However, this is not limited to this example. That is, both the step of confirming that the nucleic acid amplification reaction is in the exponential amplification phase and the step of detecting the target nucleic acid and non-target nucleic acid may be performed by detecting the fluorescence of the fluorescent probe for amplification detection. In this case, labels added to the primers or nucleotide substrates can be omitted. When a fluorescent probe for amplification detection is used in the detection step, it is necessary to select a fluorescent probe for amplification detection that emits light even when the target nucleic acid is hybridized to the nucleic acid probe for detecting the target nucleic acid on the DNA chip. An example of an applicable fluorescent probe for amplification detection is Eprobe (registered trademark). Furthermore, when an amplification detection probe such as Eprobe is also used in the step of detecting target nucleic acids and non-target nucleic acids, it is preferable to design the amplification detection probe so that its Tm value is higher than the Tm values ​​of the nucleic acid probe for detecting target nucleic acids, the nucleic acid probe for detecting non-target nucleic acids, and the common probe. This prevents the amplification product from dissociating when the amplification product hybridizes to each probe, allowing the amplification products (target nucleic acids and non-target nucleic acids) hybridized to each probe to be properly detected. Furthermore, when an amplification detection probe is used in the detection step, it is preferable to design the amplification detection probe so that it has a sequence complementary to a region of the target nucleic acid and non-target nucleic acid that does not overlap with the region having a sequence complementary to the nucleic acid probe for detecting target nucleic acids, the nucleic acid probe for detecting non-target nucleic acids, and the common probe. This prevents the hybridization of the target nucleic acid and non-target nucleic acid to each probe, allowing for smooth detection of the target nucleic acid and non-target nucleic acid.

[0073] A genetic mutation testing device according to the present invention will be described below with reference to the drawings. As shown schematically in Figure 2, the testing device 1 includes a nucleic acid amplification reaction detection unit 2 that performs a nucleic acid amplification reaction to amplify a region containing a genetic mutation to be tested and confirms that the nucleic acid amplification reaction is in the exponential amplification phase by detecting an amplified product in the reaction solution of the nucleic acid amplification reaction, and a detection unit 3 that receives the reaction solution from the nucleic acid amplification reaction detection unit 2 and detects the target nucleic acid contained in the reaction solution. The nucleic acid amplification reaction detection unit 2 includes a reaction vessel 4 that stores reagents and substances necessary for the nucleic acid amplification reaction and a first temperature control unit 5 that controls the reaction solution in the reaction vessel 4 to a predetermined temperature. Furthermore, the reaction vessel 4 has at least a portion of its surface that is transparent, and an imaging unit 9A is provided facing the transparent portion to measure fluorescence from the reaction solution. The detection unit 3 includes a DNA chip mounting unit 7 that mounts a DNA chip 6 having nucleic acid probes for detecting target nucleic acids, nucleic acid probes for detecting non-target nucleic acids, and a common probe, a second temperature control unit 8 that controls the hybridization reaction solution supplied to the DNA chip 6 mounted on the DNA chip mounting unit 7 to a predetermined temperature, and an imaging unit 9B that is disposed in a position facing the surface of the DNA chip 6 on which the various probes are immobilized. In the testing device 1, the nucleic acid amplification reaction detection unit 2 and the detection unit 3 are connected via a flow path 10 that allows the solution to pass through. The first temperature control unit 5 and the second temperature control unit 8 each include a metal block with high thermal conductivity and a temperature control device that heats / cools the metal block.

[0074] The testing device 1 also includes a hybridization buffer tank 12 connected to the DNA chip mounting section 7 via a flow path 11, and a cleaning solution tank 14 connected to the DNA chip mounting section 7 via a flow path 13. The testing device 1 also includes a waste liquid tank 16 connected to the DNA chip mounting section 7 via a flow path 15. The testing device 1 also includes a pump device 18 connected to the waste liquid tank 16 via a flow path 17.

[0075] In the testing device 1, liquid supply shutoff valves 19A, 19B, 19C, and 19D are provided in the flow paths 10, 11, 13, and 15. In the testing device 1, intake valves 20A, 20B, 20C, and 20D are attached to the reaction chamber 4, the hybridization buffer tank 12, the washing solution tank 14, and the waste tank 16, respectively. These liquid supply shutoff valves 19A, 19B, 19C, and 19D can open and close the flow paths, directing the pressure of the pump device 18 to the flow path to which liquid is to be sent. That is, the testing device 1 is equipped with a liquid supply device including the pump device 18, the liquid supply shutoff valves 19A, 19B, 19C, and 19D, and the intake valves 20A, 20B, 20C, and 20D. This liquid supply device can supply various solutions to the detection unit 3 via the flow paths 10, 11, and 13, and can supply waste liquid to the waste tank 16 via the flow path 15.

[0076] Furthermore, when the reagent in the reaction chamber 4 or the DNA chip mounting portion 7 temporarily expands or contracts due to temperature fluctuations, these liquid supply shutoff valves 19A, 19B, 19C, and 19D can be moved to another portion connected by a flow path. The liquid supply shutoff valve is not particularly limited, and any valve that has been subjected to a hydrophobic or hydrophilic treatment, or a material that changes shape due to heat or the like can be used without any restrictions, but a shape that can be physically opened and closed by external stress is preferred, and as an example, it is preferable to use a diaphragm valve that is pressed down by an actuator, or a rotary valve that is opened and closed by rotation.

[0077] The testing device 1 configured as described above can detect target nucleic acids and non-target nucleic acids contained in a nucleic acid amplification reaction solution confirmed to be in the exponential amplification phase, as described above, and quantitatively analyze the target nucleic acid based on this. A flowchart for quantitatively analyzing a target nucleic acid is shown in FIG. 3 . To quantitatively analyze a target nucleic acid, a nucleic acid amplification reaction is first performed in a reaction chamber 4 for a region containing a genetic mutation to be tested. At this time, the reaction chamber 4 is filled with a reaction solution containing reagents necessary for the nucleic acid amplification reaction. The reaction solution may be filled into the reaction chamber 4 by an operator, or it may be filled into the reaction chamber 4 from a separate flow path (not shown). Furthermore, by setting a thermal cycle for the nucleic acid amplification reaction in advance, the first temperature control unit 5 can control the temperature and processing time of the reaction solution in the reaction chamber 4 according to the set thermal cycle.

[0078] In the testing device 1, the fluorescence generated by the amplification detection fluorescent probe contained in the reaction solution in the reaction vessel 4 is measured by the imaging unit 9A, and the nucleic acid amplification reaction is monitored based on the fluorescence intensity. Then, when a predetermined fluorescence intensity corresponding to the exponential amplification phase is observed, a part or all of the reaction solution is removed and sent to the DNA chip mounting unit 7 on which the DNA chip 6 is mounted. Specifically, the reaction solution in the reaction vessel 4 can be sent to the DNA chip mounting unit 7 by operating the pump device 18 while the intake valve 20A and the liquid supply shutoff valves 19A and 19D are open and the other intake valves 20B, 20C, and 20D and the liquid supply shutoff valves 19B and 19C are closed.

[0079] At this time, the inspection device 1 can be set to temporarily suspend the thermal cycle performed by the first temperature control unit 5 and wait until the liquid transfer is completed in one temperature range of the thermal cycle (for example, 95°C).

[0080] In addition to the reaction solution, a hybridization buffer solution is supplied from the hybridization buffer solution tank 12 to the DNA chip mounting section 7. To supply (feed) the hybridization buffer solution to the DNA chip mounting section 7, the intake valve 20B and the feed shutoff valves 19B and 19D are opened, and the other intake valves 20A, 20C, and 20D and the feed shutoff valves 19A and 19C are closed, and the pump device 18 is operated. This allows the hybridization buffer solution in the hybridization buffer solution tank 12 to be fed to the DNA chip mounting section 7. The hybridization buffer solution may have a composition containing the above-mentioned blocking nucleic acid.

[0081] As a result, hybridization of the target nucleic acid and non-target nucleic acid contained in the reaction solution with the various probes on the DNA chip 6 proceeds in the DNA chip mounting section 7. At this time, the second temperature control section 8 adjusts the temperature of the solution supplied to the DNA chip mounting section 7 to a preset hybridization temperature. The second temperature control section 8 can also preheat the solution before the reaction, if necessary. In this example, the reaction solution and the hybridization buffer solution are sent separately through different flow paths, but it is also possible to mix the reaction solution and the hybridization buffer solution and then send the mixed solution to the DNA chip mounting section 7.

[0082] Next, in the testing device 1, the detection unit 3 detects the target nucleic acid and non-target nucleic acid hybridized to the probe. Specifically, first, a cleaning solution is sent to the DNA chip mounting unit 7 to clean the DNA chip 6. The cleaning solution can be sent from the cleaning solution tank 14. To supply (send) the cleaning solution to the DNA chip mounting unit 7, the intake valve 20C and the liquid supply shutoff valves 19C and 19D are opened, and the other intake valves 20A, 20B, and 20D and the liquid supply shutoff valves 19A and 19B are closed, and the pump device 18 is operated. During this cleaning, the second temperature control unit 8 can also lower the cleaning temperature to a temperature (e.g., 25°C) lower than the hybridization reaction temperature.

[0083] When washing the DNA chip 6, the mixture of the reaction solution and hybridization buffer solution may be discharged into the waste tank 16, and then the washing solution may be sent as described above, or the mixture of the reaction solution and hybridization buffer solution may be discharged into the waste tank 16 by sending the washing solution as described above.

[0084] The surface of the DNA chip 6 on which the nucleic acid probes for detecting target nucleic acids, the nucleic acid probes for detecting non-target nucleic acids, and the common probe are immobilized is imaged by the imaging unit 9B, and the signal intensity of each probe is measured by image analysis software in a computer (not shown). Based on the measured signal intensities, the target nucleic acid can be quantitatively analyzed by the same computer.

[0085] As described above, by using the testing device 1 according to the present invention, it is possible to deliver a nucleic acid amplification reaction solution in which it has been confirmed that the nucleic acid amplification reaction is in the exponential amplification phase, and detect target nucleic acids and non-target nucleic acids based on hybridization reactions with each probe using a DNA chip 6. In the example described above, the reaction vessel 4 and the DNA chip mounting section 7 are connected via a flow path 10, and the nucleic acid amplification reaction solution is delivered using a configuration including a pump device 18, delivery shutoff valves 19A, 19B, 19C, and 19D, and intake valves 20A, 20B, 20C, and 20D. However, the mechanism for delivering the nucleic acid amplification reaction solution (delivery device) is not limited to this configuration, and some or all of the nucleic acid amplification reaction solution can also be delivered using a pipette device disposed on a drive arm.

[0086] 2, the above-described liquid delivery operation is performed by suction using the pump device 18. However, the test device 1 can also perform the above-described liquid delivery operation by using a pressure pump connected upstream of the nucleic acid amplification reaction detection unit 2, the hybridization buffer tank 12, and the washing liquid tank 14.

[0087] In addition, in the testing device 1 shown in Figure 2, the hybridization buffer solution is sent directly to the DNA chip mounting section 7 from the hybridization buffer solution tank 12 connected to the DNA chip mounting section 7 via the flow path 11, but it may also be sent to the DNA chip mounting section 7 after first merging with the flow path 10.

[0088] 2 is provided with a DNA chip mounting section 7 on which a DNA chip 6 can be mounted, and is configured to allow the DNA chip 6 to be attached and detached. However, the testing device 1 is not limited to this configuration, and may be configured such that each probe is directly fixed to the detection section 3.

[0089] 2 may also include, as needed, a nucleic acid extraction unit (not shown) for extracting template DNA from a subject-derived sample upstream of the nucleic acid amplification reaction detection unit 2. The nucleic acid extraction unit may be configured from a disrupter such as a blender, mixer, or homogenizer for disrupting cells, or a nucleic acid extraction device including an organic solvent (phenol / chloroform), a silica membrane, an anion exchange resin column, magnetic beads, etc.

[0090] The DNA extracted in the nucleic acid extraction unit is mixed with reagents necessary for the nucleic acid amplification reaction, and the mixture is sent to the reaction chamber 4 of the nucleic acid amplification reaction detection unit 2 connected via a flow path. The mixing operation of the extracted DNA and the reagents may be performed after the DNA and the reagents are each filled into the reaction chamber 4. That is, the DNA and the reagents can be filled into the reaction chamber 4 separately, and the filled DNA and reagents can be mixed in the reaction chamber 4. Alternatively, the DNA and the reagents may be mixed in advance, and the resulting mixture may be filled into the reaction chamber 4.

[0091] FIG. 4 is a schematic diagram illustrating another example of a genetic mutation testing device according to the present invention. In the testing device 1 shown in FIG. 2, the nucleic acid amplification reaction solution is delivered from a nucleic acid amplification reaction detection unit 2 having a single reaction chamber 4, but the testing device according to the present invention is not limited to this configuration. That is, as shown schematically in FIG. 4, the genetic mutation testing device 1 according to the present invention may be configured to include a nucleic acid amplification reaction detection unit 2 having multiple reaction chambers 4 (five in the example of FIG. 4). In this case, the temperatures of the multiple reaction chambers 4 may be collectively controlled by a single first temperature control unit 5, or may be individually controlled by multiple first temperature control units 5.

[0092] The inspection device 1 of FIG. 4 also includes a liquid delivery device having flow paths connected to each of the multiple reaction vessels 4 and a switching device disposed on the flow paths for switching between the multiple reaction vessels 4 as the flow paths for supplying reaction liquid to the detection unit 3. In the inspection device 1 of FIG. 4, each of the multiple reaction vessels 4 and the detection unit 3 are connected via a flow path 10, and a liquid delivery shutoff valve 19A is disposed midway along the flow path 10. In addition, an intake valve 20A is attached to each reaction vessel 4. These liquid delivery shutoff valves 19A and intake valves 20A connected to the multiple reaction vessels 4 collectively function as a switching device for switching between the multiple reaction vessels 4 as the flow paths for supplying reaction liquid to the detection unit 3. That is, by opening the liquid delivery shutoff valve 19A and intake valve 20A connected to a specific reaction vessel among the multiple reaction vessels 4 at the appropriate timing, the pressure of the pump device 18 can be directed to the flow path 10 to which liquid is to be delivered, thereby connecting one of the multiple reaction vessels 4 to the detection unit 3. The switching device may include a control device for controlling the timing of opening and closing of each of the liquid supply cutoff valve 19A and the intake valve 20A, as required.

[0093] 4, the multiple reaction chambers 4 of the nucleic acid amplification reaction detection unit 2 can be configured to perform nucleic acid amplification reactions that amplify regions containing different gene mutations to be tested. The DNA chip 6 mounted on the DNA chip mounting unit 7 has nucleic acid probes for detecting target nucleic acids and nucleic acid probes for detecting non-target nucleic acids or a common probe immobilized on a carrier, which correspond to the respective gene mutations, and can detect the target nucleic acids and non-target nucleic acids contained in the reaction solution in the multiple reaction chambers 4.

[0094] The reaction solutions sent from the reaction chambers 4 to the DNA chip mounting section 7 are mixed, and then, as in Figure 2, hybridization reaction and washing steps are carried out in the DNA chip mounting section 7, followed by detection. This allows the step of hybridization reaction between the reaction solution and the probes on the DNA chip 6 to be carried out only once, enabling efficient quantitative analysis.

[0095] 4, the flow paths 10 extending from the reaction chambers 4 to the DNA chip mounting section 7 join just before the DNA chip mounting section 7, but a reservoir may be provided at the joining point to ensure smooth joining and mixing of the reaction solutions from the reaction chambers 4. Furthermore, the pump device 18 is not limited to the configuration shown in the figure, and may also be configured such that a pressure pump is connected upstream of each reaction chamber 4.

[0096] As a method for amplifying multiple regions each containing a different genetic mutation to be tested, in addition to using the testing device 1 of Figure 4, it is also possible to use the testing device 1 of Figure 2 and an amplification detection fluorescent probe of a different fluorescent color for each region.

[0097] Fig. 5 is a schematic diagram showing another example of a genetic mutation testing device according to the present invention. The testing device 1 in Fig. 5 is equipped with a nucleic acid amplification reaction detection unit 2 having a plurality of reaction chambers 4, similar to the example in Fig. 4.

[0098] 4, the flow paths from each reaction chamber 4 are connected to an independent DNA chip mounting section 7, and these multiple DNA chip mounting sections 7 form the detection section 3. In Fig. 5, a connection port 21 is provided upstream of the intake valve 20A of each reaction chamber 4. Furthermore, the hybridization buffer tank and the washing solution tank are not directly connected to the DNA chip mounting section 7 and are not shown.

[0099] In the testing device 1 of Figure 5, each reaction chamber 4 is filled with a reaction solution consisting of reagents necessary for the nucleic acid amplification reaction from the connection port 21, and the nucleic acid amplification reaction is carried out until a predetermined fluorescence intensity corresponding to the exponential amplification phase is observed, which is the same as the testing device 1 shown in Figure 2.

[0100] 5, when it is confirmed that the reaction solution is in the exponential amplification phase, it is drawn back to the connection port 21, a hybridization buffer is injected into the connection port 21 and mixed with the reaction solution at the connection port 21, and the mixed solution is sent to the DNA chip mounting section 7. Thereafter, in the DNA chip mounting section 7, the target nucleic acid and non-target nucleic acid contained in the reaction solution are hybridized with various probes on the DNA chip 6, as in FIG. 2. When the reaction solution is sent from the reaction vessel 4 to the DNA chip mounting section 7, a predetermined intake valve 20A and the corresponding liquid supply shutoff valves 19A and 19D are opened, and the pump device 18 is operated.

[0101] Next, a cleaning solution for washing away excess reagent is sent from the connection port 21 to the DNA chip mounting section 7 via the reaction chamber 4, washing the DNA chip 6 and discharging it into the waste tank 16. Subsequently, a rinse solution is sent from the connection port 21 to the DNA chip mounting section 7 via the reaction chamber 4, rinsing the DNA chip 6 and discharging it into the waste tank 16. When the cleaning solution and rinse solution are sent from the reaction chamber 4 to the DNA chip mounting section 7 and then discharged into the waste tank 16, this is done by opening the predetermined intake valve 20A and the corresponding liquid supply shutoff valves 19A and 19D and operating the pump device 18, as in the case of sending the reaction solution. Thereafter, the DNA chip 6 is imaged by the imaging section 9B, and the signal intensity of each probe is measured and analyzed, as in FIG. 2 .

[0102] The injection of various liquids from the connection port 21 may be performed by an operator, or may be performed by connecting tanks (not shown) for the various liquids to the connection port 21 via flow paths.

[0103] 5, the flow paths are joined immediately before the waste liquid tank 16, which reduces loss of reaction liquid and simplifies and miniaturizes the flow path design. In addition, in the apparatus of Fig. 5, the pump device 18 is disposed downstream of the waste liquid tank 16, but this configuration is not limiting, and a pressure pump may be connected upstream of the connection port 21 and the liquid may be sent by operating the pressure pump.

[0104] The device in Figure 4 and the device in Figure 5 are not alternatives, and when amplifying multiple regions in each reaction chamber 4, the reaction solutions in some of the reaction chambers 4 may be merged and detected in one DNA chip mounting section 7, and the reaction solutions in the other reaction chambers 4 may be sent to individual DNA chip mounting sections 7 for detection.

[0105] Other configurations of the inspection device 1 in FIGS. 4 and 5 are similar to those of the inspection device 1 shown in FIG.

[0106] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

[0107] Example 1 [Mutation Rate Quantification Test Using IC5] 1. Preparation of Standard Curve Samples In this example, the V617F mutation in the JAK2 gene was the mutation to be tested. Regarding the gene mutation, a mutant-type plasmid containing the mutant form and a wild-type plasmid containing the wild-type form were prepared using FASMAC's artificial gene synthesis service. The wild-type plasmid for the JAK2 gene was a plasmid containing the base sequence shown in SEQ ID NO: 1. The mutant-type plasmid was a plasmid containing the same region inserted except for the above-mentioned mutation (the V617F mutation in the JAK2 gene).

[0108] The copy numbers of the prepared mutant and wild-type JAK2 gene plasmids were quantified in advance using Bio-Rad's ddPCR, and the JAK2 V617F mutation was mixed so that the mutation rate was 1%, 5%, 20%, 50%, or 100%. The total copy number of the mutant and wild-type JAK2 gene plasmids mixed was 5 × 10 3 The concentration was adjusted with TE buffer to 1 / μL and used as a calibration curve sample.

[0109] 2. Real-time PCR: Using five prepared standard curve samples (mutation rates: 1%, 5%, 10%, 50%, or 100%), a specific region of the JAK2 gene was amplified with a primer set. The primer set shown in Table 1 was designed for this PCR and prepared through Thermo Fisher Scientific's oligo synthesis service. Of the primer sets shown in Table 1, the forward primer marked "F" was fluorescently labeled (IC5).

[0110]

[0111] Next, the amplification detection fluorescent probe (Eprobe (registered trademark)) shown in Table 2, which is necessary for fluorescence detection during PCR, was designed and produced through DNAFORM's oligo synthesis service. E The fluorescent probe for amplification detection hybridizes with the amplified product, but is designed to have a sequence complementary to a region in the amplified product that does not overlap with the regions having sequences complementary to the nucleic acid probe for detecting target nucleic acids, the nucleic acid probe for detecting non-target nucleic acids, and the common probe.

[0112]

[0113] The primer set designed as above and the fluorescent probe for amplification detection were mixed to the composition shown in Table 3 to prepare a primer-probe mix.

[0114]

[0115] Then, a PCR reaction solution having the composition shown in Table 4 was prepared using the calibration curve samples and the primer-probe mix.

[0116]

[0117] Using the PCR reaction solution prepared as described above, PCR was thermal cycled at 95°C for 5 minutes, followed by 22, 25, 28, 31, 34, 37, and 40 cycles, each consisting of 95°C for 30 seconds, 59°C for 30 seconds, and 72°C for 45 seconds, with a final temperature of 4°C. PCR was performed using a Thermo Fisher Scientific StepOne real-time PCR system, and fluorescence detection was performed during annealing (59°C for 30 seconds) using the FAM setting.

[0118] 3. Preparation of amplification curves The obtained fluorescence intensity values ​​were used to prepare a sixth-order polynomial approximation curve, after previously subtracting the average fluorescence intensity value from cycles 1 to 5, where no fluorescence was detected, as the background value.

[0119] 4. Setting of TTh.PFI and calculation of MPFI and CPFI From the obtained amplification curve, the upper limit of the dynamic range was set to 20,000, and TTh.PFI = 2,000, 4,000, 6,000, 8,000, 10,000, 15,000, and 20,000.

[0120] 5. DNA Chip In this example, a mutant probe (nucleic acid probe for detecting target nucleic acid) corresponding to the V617F mutation in the JAK2 gene, a corresponding wild-type probe (nucleic acid probe for detecting non-target nucleic acid), and a common probe common to both the wild-type and mutant types were designed. The common probe specifically hybridizes to the amplified nucleic acid regardless of whether or not the gene mutation is present in the amplified nucleic acid. The base sequences of the designed probes are shown in Table 5.

[0121]

[0122] 6. Preparation of Hybridization Reaction Solution In this example, 30 μL of reaction solution containing PCR amplification products extracted from cycles 22, 25, 28, 31, 34, 37, and 40 of PCR thermal cycling and 15 μL of hybridization buffer (2.25×SSC / 0.23% SDS / 0.2 nM JAK2 gene blocker 150 nM (Thermo Fisher Scientific)) were added to a designated PCR tube (4titude 4ti-0750 / TA) and mixed several times by pipetting, taking care not to create bubbles. The JAK2 gene blocker is shown in Table 6.

[0123]

[0124] 7. Hybridization Reaction The DNA chips with the various probes immobilized as described in Section 5 above were placed in a BioShot HT-32 genetic analyzer (manufactured by Toyo Kohan Co., Ltd.) and hybridization reactions were carried out. PCR tubes containing the solution prepared in Section 6 above, DNA chip needles with DNA chips immobilized for insertion into the designated PCR tubes, and a washing solution (0.1x SSC / 0.1% SDS solution, room temperature) for washing away excess reagent after the hybridization reaction of the DNA chip, a rinse solution (1x SSC solution, room temperature), and a detection solution for fluorescence detection (1x SSC solution, room temperature) were placed in the BioShot HT-32 and operation was started.

[0125] The reaction solution in the designated PCR tube was heated to 49.5°C by automatic operation, and the DNA chip needle was inserted and the hybridization reaction was carried out for 30 minutes. After the hybridization reaction, the DNA chip needle was quickly immersed in a washing solution bath and agitated 30 times so that the DNA chip went in and out of the washing solution surface. After washing, the DNA chip needle was immersed in a rinse solution bath and agitated 80 times so that the DNA chip went in and out of the rinse solution surface.

[0126] The rinsed DNA chip needle was slowly immersed in a detection solution tank, and the fluorescence was captured by a CCD camera for 2 seconds by irradiating it with a 640 nm single wavelength laser.

[0127] 8. Preparation of a calibration curve In this example, the fluorescence intensity values ​​obtained were used after subtracting the background value from the area where no probe was spotted. First, a sixth-order polynomial approximation curve was prepared from the seven fluorescence intensity values ​​for each temperature cycle obtained from a calibration curve sample with a 100% mutation rate (hereinafter referred to as M100).

[0128] The MPFI and CPFI were calculated by substituting the multiple TTh.PFIs set in 4. above into the resulting sixth-order polynomial. The judgment value was calculated using the formula: [MPFI] / [CPFI], and the reciprocal of the judgment value at M100 (i.e., [CPFI] / [MPFI]) was used as the correction coefficient A.

[0129] Next, for the standard curve samples with mutation rates of 1%, 5%, 10%, and 50% (hereinafter referred to as M1, M5, M10, and M50), the MPFI and CPFI in TTh.PFI and the judged value were calculated in the same manner as for M100. The judged values ​​obtained for M1, M5, M10, M50, and M100 were each multiplied by the correction coefficient A to calculate the corrected judged value.

[0130] A scatter plot was prepared by plotting the corrected judgement values ​​for M1, M5, M10, M50, and M100 on the X axis and the known mutation rates on the Y axis, and linear approximation line I (Figure 6) was constructed using the least squares method. In addition, a scatter plot was prepared by plotting the logarithmic transformation values ​​(Log%) of the corrected judgement values ​​for M1, M5, M20, M50, and M100 on the X axis and the logarithmic transformation values ​​(Log%) of the known mutation rates on the Y axis, and linear approximation line II (Figure 7) was constructed using the least squares method.

[0131] 9. Results For the V617F mutation in the JAK2 gene, the correction coefficient, corrected judgement value, slope a, intercept b and R of linear approximation line I and linear approximation line II for each TTh.PFI were calculated. 2 The values ​​are shown in Table 7.

[0132]

[0133] In Table 7, "○" indicates that the coefficient of determination of the linear approximation line I is R 2 ≧0.99, which means that there is linearity. Also, "×" indicates that the coefficient of determination is R 2< 0.99, which means there is no linearity. "○○" indicates that the coefficient of determination of the linear approximation line I is R 2 ≧0.99 and indicates the condition where the coefficient of determination of linear approximation line II is the highest. Under the conditions marked with "○○" in Table 7, the mutation rate can be accurately calculated based on the judgment value. Based on these linear approximation lines I and II, a TTh.PFI of 6000 that corresponds to "○○" was defined as the true Th.PFI.

[0134] 10. Preparation of samples for quantification The mutant and wild-type plasmids prepared in 1 above were mixed so that the mutation rates were 0%, 0.25%, 0.5%, 1%, 2.5%, 5%, 10%, or 100%, respectively. The concentrations were quantified using Qubit (Thermo Fisher Scientific). The mixed plasmid DNA was diluted with TE buffer to 0.08 and 0.32 pg / μL, respectively, to prepare samples for quantification.

[0135] 11. Real-time PCR: Using eight quantitative samples (0%, 0.25%, 0.5%, 1%, 2.5%, 5%, 10%, and 100%), a specific region of the JAK2 gene was simultaneously amplified using the primer-probe set used in Section 2 above. The composition of the reaction solution is shown in Table 8.

[0136]

[0137] Using the PCR reaction solution prepared as above, a PCR thermal cycle was performed at 95°C for 5 minutes, followed by one cycle of 95°C for 30 seconds, 59°C for 30 seconds, and 72°C for 45 seconds, and the reaction solution was removed from the device at the temperature cycle where Th.PFI was reached. Next, a DNA chip and hybridization reaction solution similar to those in 5 and 6 above were prepared, and a hybridization reaction was performed under the same conditions as in 7 above.

[0138] 12. Calculation of judgment value The ratio of the obtained MPFI to CPFI was calculated and multiplied by the correction coefficient A obtained in 8. above to calculate the corrected judgment value. That is, the corrected judgment value was calculated as follows: [MPFI] / [CPFI] × correction coefficient A. The obtained corrected judgment value was logarithmically transformed and substituted into the calibration curve (linear approximation curve II) obtained in 8. above to convert it into the mutation ratio. That is, the formula: y = 1.0970x -0.2490 was applied, resulting in a correction coefficient A = 0.784.

[0139] 13. Comparison of mutation rates with ddPCR For the quantitative samples in 10. above, the mutation rates (Log%) measured by ddPCR were plotted on the X axis and the mutation rates calculated using the standard curve on the Y axis, and the results of linear approximation using the least squares method are shown in Figure 8. As shown in Figure 8, the coefficient of determination R 2 The correlation was ≧0.95, and the results showed a very high correlation.

[0140] (Example 2) [Mutation rate quantification test using OY] In Example 1, a calibration curve was created using the fluorescent dye IC5 (Ex 640 nm / Em 660 nm) of the hybridized PCR product, whereas in Example 2, a calibration curve was created using the amplification detection fluorescent probe Oxazole Yellow (Ex 490 nm / Em 510 nm) that binds to the hybridized PCR product. Only the changes from Example 1 are described below. The item numbers for each item correspond to those in Example 1.

[0141] 7. Hybridization Method In this example, the hybridization reaction was carried out in the same manner as in 7. of Example 1, except that the light source and filter shown in Table 9 were installed in a genetic analyzer BIOSHOT HT-32 (manufactured by Toyo Kohan Co., Ltd.), the rinsed DNA chip needle was slowly immersed in a detection solution tank, and the fluorescence was captured by a CCD camera for 2 seconds upon irradiation with a 488 nm single-wavelength laser.

[0142]

[0143] 8. Preparation of a calibration curve In this example, the fluorescence intensity values ​​obtained were used after subtracting the background value from the area where no probe was spotted. First, a sixth-order polynomial approximation curve was prepared from the seven fluorescence intensity values ​​for each temperature cycle obtained from a calibration curve sample with a 100% mutation rate (hereinafter referred to as M100).

[0144] The MPFI and CPFI were calculated by substituting the multiple TTh.PFIs set in 4 of Example 1 into the obtained sixth-order polynomial. The judgment value was calculated using the formula: [MPFI] / [CPFI], and the reciprocal of the judgment value at M100 (i.e., [CPFI] / [MPFI]) was used as the correction coefficient A.

[0145] Next, for the standard curve samples with mutation rates of 1%, 5%, 10%, and 50% (hereinafter referred to as M1, M5, M10, and M50), the MPFI and CPFI in TTh.PFI and the judged value were calculated in the same manner as for M100. The judged values ​​obtained for M1, M5, M10, M50, and M100 were each multiplied by the correction coefficient A to calculate the corrected judged value.

[0146] A scatter plot was prepared by plotting the corrected judgement values ​​for M1, M5, M10, M50, and M100 on the X axis and the known mutation rates on the Y axis, and linear approximation line I (Figure 9) was prepared using the least squares method. In addition, a scatter plot was prepared by plotting the logarithmic transformation values ​​(Log%) of the corrected judgement values ​​for M1, M5, M20, M50, and M100 on the X axis and the logarithmic transformation values ​​(Log%) of the known mutation rates on the Y axis, and linear approximation line II (Figure 10) was prepared using the least squares method.

[0147] 9. Results For the V617F mutation in the JAK2 gene, the correction coefficient, corrected judgement value, slope a, intercept b and R of linear approximation line I and linear approximation line II for each TTh.PFI were calculated. 2 The values ​​are shown in Table 10.

[0148]

[0149] In Table 10, "○" indicates that the coefficient of determination of the linear approximation line I is R 2 ≧0.99, which means that there is linearity. Also, "×" indicates that the coefficient of determination is R 2< 0.99, which means there is no linearity. "○○" indicates that the coefficient of determination of the linear approximation line I is R 2 ≧0.99 and indicates the condition where the coefficient of determination of linear approximation line II is the highest. Under the conditions marked with "○○" in Table 10, the mutation rate can be accurately calculated based on the judgment value. Based on these linear approximation lines I and II, a TTh.PFI of 6000 that corresponds to "○○" was defined as the true Th.PFI.

[0150] 12. Calculation of judgment value The ratio of the obtained MPFI to CPFI was calculated and multiplied by the correction coefficient A obtained in Example 2 to calculate the judgment value. That is, judgment value = [MPFI] / [CPFI] × correction coefficient A. The obtained judgment value was logarithmically transformed and substituted into the calibration curve (linear approximation curve II) obtained in 8. above to convert it into a mutation ratio. That is, the formula: y = 1.0985x -0.2863 was applied, resulting in a correction coefficient A = 0.978.

[0151] 13. Comparison of Mutation Rate with ddPCR For the quantification sample of this example (the same as the quantification sample in 10. of Example 1), the mutation rate (Log%) measured by ddPCR was plotted on the X axis, and the mutation rate calculated using the calibration curve was plotted on the Y axis. The results of linear approximation using the least squares method are shown in Figure 11. As shown in Figure 11, the coefficient of determination R 2 The correlation was ≧0.95, and the results showed a very high correlation.

[0152] 1 Inspection device 2 Nucleic acid amplification reaction detection section 3 Detection section 4 Reaction tank 5 First temperature control section 6 DNA chip 7 DNA chip mounting section 8 Second temperature control section 9A, 9B Imaging section 10, 11, 13, 15, 17 Flow path 12 Hybridization buffer tank 14 Washing solution tank 16 Waste liquid tank 18 Pump device 19A, 19B, 19C, 19D, 19E Liquid supply shut-off valve 20A, 20B, 20C, 20D Intake valve 21 Connection port All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

1. A method for testing for a genetic mutation, comprising: a step of carrying out a nucleic acid amplification reaction to amplify a region containing a genetic mutation to be tested; a step of confirming that the nucleic acid amplification reaction is in the exponential amplification phase by detecting an amplification product in a reaction solution of the nucleic acid amplification reaction; a step of detecting a target nucleic acid containing a target base to be detected in the genetic mutation and a non-target nucleic acid containing a non-target base corresponding to the target base, which are contained in the reaction solution confirmed to be in the exponential amplification phase, using a nucleic acid probe for detecting a target nucleic acid having a sequence complementary to a region containing the target base to be detected in the target nucleic acid, and a nucleic acid probe for detecting a non-target nucleic acid having a sequence complementary to a region containing the non-target base to be detected in the non-target nucleic acid, or a common probe having a sequence complementary to a common region common to the target nucleic acid and the non-target nucleic acid, which does not overlap with a region containing the target base or the non-target base to be detected; and a step of quantitatively analyzing the target nucleic acid contained in the reaction solution.

2. A method for testing gene mutations according to claim 1, wherein the detection step uses a DNA chip on which the nucleic acid probe for detecting the target nucleic acid and the nucleic acid probe for detecting the non-target nucleic acid or the common probe are immobilized on a carrier.

3. A method for testing for a genetic mutation according to claim 1, wherein there are a plurality of regions containing the genetic mutation to be tested, and for each of the regions, a step of carrying out the nucleic acid amplification reaction and a step of confirming that the nucleic acid amplification reaction is in the exponential amplification phase are carried out, and the target nucleic acid and non-target nucleic acid contained in the reaction solution for each of the regions confirmed to be in the exponential amplification phase are detected.

4. A method for testing a gene mutation according to claim 1, wherein the reaction solution contains an amplification detection probe, and both the step of confirming that the nucleic acid amplification reaction is in the exponential amplification phase and the step of detecting the target nucleic acid and the non-target nucleic acid are carried out by detecting a signal from the amplification detection probe.

5. The method for detecting a gene mutation according to claim 4, wherein the Tm value of the amplification detection probe is higher than the Tm values ​​of the nucleic acid probe for detecting a target nucleic acid, the nucleic acid probe for detecting a non-target nucleic acid, and the common probe.

6. A method for testing genetic mutations as described in claim 4, wherein the amplification detection probe has a sequence complementary to a region in the target nucleic acid and the non-target nucleic acid that does not overlap with a region having a sequence complementary to the nucleic acid probe for detecting the target nucleic acid, the nucleic acid probe for detecting the non-target nucleic acid, and the common probe.

7. A method for testing a genetic mutation according to claim 1, wherein the quantitative analysis step calculates the abundance ratio of the target base in the genetic mutation being tested based on the signal intensity from the nucleic acid probe for detecting the target nucleic acid and the signal intensity from the nucleic acid probe for detecting the non-target nucleic acid or the common probe.

8. The method for testing a genetic mutation according to claim 1, wherein the quantitative analysis step uses a calibration curve prepared using a plurality of samples containing target nucleic acids and non-target nucleic acids in known proportions, and quantifies the proportion of the target base in the genetic mutation being tested based on the signal intensity from the nucleic acid probe for detecting the target nucleic acid and the signal intensity from the nucleic acid probe for detecting non-target nucleic acids or the common probe.

9. The method for testing a gene mutation according to claim 1, wherein the quantitative analysis step uses a determination value calculated by the formula: [signal intensity from nucleic acid probe for detecting target nucleic acid] / ([signal intensity from nucleic acid probe for detecting target nucleic acid]+[signal intensity from nucleic acid probe for detecting non-target nucleic acid]), or a determination value calculated by the formula: [signal intensity from nucleic acid probe for detecting target nucleic acid] / [signal intensity from common probe].

10. A method for testing a gene mutation according to claim 1, wherein the detecting step is carried out in the presence of a blocking nucleic acid containing a base sequence complementary to the non-target nucleic acid containing the non-detection target base.

11. A genetic mutation testing device comprising: a nucleic acid amplification reaction detection unit that performs a nucleic acid amplification reaction to amplify a region containing a genetic mutation to be tested, and confirms that the nucleic acid amplification reaction is in the exponential amplification phase by detecting an amplified product in a reaction solution from the nucleic acid amplification reaction; a detection unit that receives the reaction solution from the nucleic acid amplification reaction detection unit and detects at least the target nucleic acid using a nucleic acid probe for detecting a target nucleic acid having a sequence complementary to a region containing a target base in a target nucleic acid that contains a target base to be detected in the genetic mutation, a nucleic acid probe for detecting a non-target nucleic acid having a sequence complementary to a region containing a non-target base in a non-target nucleic acid that contains a non-target base corresponding to the target base to be detected, or a common probe that has a sequence complementary to a common region that is common to the target nucleic acid and the non-target nucleic acid and that does not overlap with the region containing the target base or the non-target base; and a liquid delivery device that supplies the reaction solution from the nucleic acid amplification reaction detection unit to the detection unit.

12. A genetic mutation testing device as described in claim 11, wherein the detection unit includes a DNA chip mounting unit that mounts a DNA chip on which the nucleic acid probe for detecting the target nucleic acid and the nucleic acid probe for detecting the non-target nucleic acid or the common probe are fixed to a carrier.

13. A genetic mutation testing device as described in claim 11, wherein the nucleic acid amplification reaction detection unit comprises a plurality of reaction chambers for carrying out nucleic acid amplification reactions that amplify regions containing different genetic mutations to be tested, and the liquid delivery device includes a switching device that switches the flow path for supplying reaction liquid to the detection unit between the plurality of reaction chambers.

14. The genetic mutation testing device according to claim 11, wherein the detection unit detects target nucleic acids and non-target nucleic acids using the common probe.

15. A genetic mutation testing device as described in claim 11, wherein the liquid delivery device comprises a flow path connecting the nucleic acid amplification reaction detection unit and the detection unit, a valve disposed on the flow path, and a pump device connected to the flow path.

16. A genetic mutation testing device as described in claim 11, which is provided with a hybridization buffer tank that supplies a hybridization buffer to the detection unit, and the liquid delivery device supplies the reaction solution of the nucleic acid amplification reaction in the exponential amplification phase from the nucleic acid amplification reaction detection unit to the detection unit, and also supplies the hybridization buffer from the hybridization buffer tank to the detection unit.

17. The genetic mutation testing device according to claim 16, wherein the hybridization buffer solution contains a blocking nucleic acid that contains a base sequence complementary to the non-target nucleic acid that contains the non-detection target base.

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