DNA detection method and DNA detection system

Abstract

The present application provides a method and system for accurately distinguishing between micro-partitions configured with a target gene and micro-partitions configured with a pseudo gene, and counting the target gene with high precision in digital PCR using melting curve analysis. The method includes: a step of configuring DNA solution in multiple partitions; a step of performing nucleic acid amplification reaction; a step of changing the temperature of each partition and measuring the fluorescence intensity; a step of calculating the melting temperature of the double-stranded DNA for each partition; a step of counting the number of partitions for each type of DNA; a step of outputting the number of counted partitions for each type of DNA; and a step of distinguishing the first gene and the pseudo gene based on the melting temperature and the number of counted partitions.

Description

Technical Field

[0001] This invention relates to DNA detection methods and DNA detection systems, and particularly to digital PCR. Background Technology

[0002] Previous genetic testing methods include PCR (Patent Documents 2-4) and real-time PCR (Non-Patent Document 1). These methods suffer from reduced reproducibility when the amount of the gene being tested (referred to as the "target gene" in this specification) is extremely small.

[0003] As a solution to this problem, digital PCR was developed (Patent Document 1). When digital PCR is used, the extremely diluted sample is used to determine and detect whether DNA is 0 (absent) or 1 (present), thereby enabling the quantification of trace amounts of DNA.

[0004] The following illustrates an example of a digital PCR detection method. First, DNA polymerase, primers, and fluorescently labeled probes are added to a minimally diluted sample to prepare the PCR reaction solution. The PCR reaction solution is then divided into tiny partitions, such as wells or droplets. At this point, each partition is designed to contain either one molecule of the target gene or none at all.

[0005] Next, the target gene within the microregions was amplified by PCR. After PCR, the fluorescence intensity of each microregion was measured, and the number of microregions with fluorescence intensity exceeding a threshold was counted, thereby enabling quantification of the target gene.

[0006] In such digital PCR, because extremely diluted samples are used, the influence of sample components, which are the main cause of hindering the PCR reaction, can be suppressed. Furthermore, since a standard curve is not required, the absolute amount of DNA being tested can be directly measured.

[0007] However, it is known that in previous PCR processes, reaction efficiency has been reduced due to the presence of reaction inhibitors in the reaction solution, the formation of secondary structures of template DNA, and insufficient primer design.

[0008] On the other hand, in digital PCR, since the measurement is performed at the endpoint of the reaction, the PCR reaction efficiency itself does not have a significant impact on the measurement results. However, in reality, even when the measurement is performed at the endpoint, the fluorescence intensity deviation caused by the non-uniformity of PCR reaction efficiency in each tiny region is large, which reduces the reproducibility and accuracy of digital PCR measurements.

[0009] Therefore, in order to improve the reproducibility and accuracy of digital PCR, the inventors have developed a technique (Patent Document 5) that allows the identification of target genes within micro-regions by measuring the melting temperature (Tm) of the PCR amplification products, even when the PCR reaction efficiency of each micro-region is uneven. Specifically, for example, after PCR, by measuring the melting temperature (Tm) at which the target gene amplified within the micro-region dissociates from the fluorescently labeled probe, the genotype of the target gene can be identified based on the difference in melting temperature, even if the PCR reaction efficiency is uneven.

[0010] The presence of pseudogenes poses a problem in the detection of target genes. During biological evolution, new genes are acquired, but functional pseudogenes are also created. A pseudogene is a copy of a gene made during evolution; one gene retains its original function while the other is altered, resulting in a modified gene that loses its function without acquiring the new one. Therefore, for example, the KRAS gene and its pseudogene, the KRASP1 gene, share extremely high sequence homology.

[0011] Therefore, when pseudogenes exist in the target gene, it is sometimes impossible to distinguish between the target gene and its pseudogenes, and the pseudogenes are incorrectly counted as the target gene, making the copy number of the target gene higher than the true value.

[0012] In previous quantitative PCR, since the target gene and pseudogene exist in the same region, if primers are designed with the part of the target gene and pseudogene whose sequences differ by only a few bases, the amplification of the target gene and pseudogene will compete, the amplification of the pseudogene will be suppressed, and the target gene will be amplified preferentially.

[0013] Existing technical documents

[0014] Patent documents

[0015] Patent Document 1: Japanese Patent Publication No. 2013-521764

[0016] Patent Document 2: U.S. Patent No. 4,683,195

[0017] Patent Document 3: U.S. Patent No. 4,683,202

[0018] Patent Document 4: U.S. Patent No. 4800159

[0019] Patent Document 5: Japanese Patent Application Publication No. 2018-108063

[0020] Non-patent literature

[0021] Non-patent literature 1: Genome Res., 10, pp. 986-994, 1996 Summary of the Invention

[0022] The problem that the invention aims to solve

[0023] However, digital PCR presents the problem of difficulty in identifying the aforementioned pseudogenes.

[0024] In digital PCR, unlike quantitative PCR, the reaction solution containing the sample is divided into tiny partitions, allowing a single molecule of the target gene to either enter or not enter a single partition. Therefore, only either the target gene or the pseudogene exists in a single partition.

[0025] Therefore, the following problem exists: even if a few bases exist in the pseudogene in the part that is inconsistent with the primer region designed for the target gene, the pseudogene will amplify in the tiny partition containing only the pseudogene because there is no competition with the target gene, and the pseudogene will become a false positive for the target gene.

[0026] Distinguishing between target genes and pseudogenes is important for reducing false positives and false negatives, and improving assay reproducibility and accuracy.

[0027] Therefore, the purpose of this invention is to provide a new DNA detection method and DNA detection system, which, in digital PCR using melting curve analysis, accurately distinguishes between micro-regions containing target genes and micro-regions containing pseudogenes through a measuring device, and counts target genes with high precision.

[0028] Methods for solving problems

[0029] The inventors discovered that in digital PCR using melting curve analysis, if probes are designed with different melting temperatures for the target gene and pseudogene, the target gene and pseudogene can be identified as different genes based on the melting temperature, and they can be identified as the target gene and its pseudogene based on the correspondence between the counts of the two genes, thus completing the present invention.

[0030] An example of the DNA detection method of the present invention includes the following steps:

[0031] The process of preparing DNA solutions involves preparing DNA solutions in multiple partitions, wherein the DNA solutions can contain multiple DNAs including a first gene and its pseudogenes, and the DNA solutions contain fluorescently labeled probes or DNA intercalators.

[0032] The process of performing nucleic acid amplification reactions in each of the aforementioned partitions;

[0033] The process of changing the temperature of each of the said partitions during or after the nucleic acid amplification reaction, and measuring the fluorescence intensity of each of the said partitions as a function of the temperature change;

[0034] The process of calculating the melting temperature of the double strand of DNA disposed in each partition based on the change in fluorescence intensity with the change in temperature;

[0035] The process of identifying the type of DNA in each of the partitions based on the melting temperature, and counting the number of partitions for each type of DNA;

[0036] The process of outputting the number of partitions for each class of the DNA; and

[0037] The process of distinguishing the first gene and the pseudogene based on the melting temperature and the number of counted partitions.

[0038] An example of the DNA detection system of the present invention includes:

[0039] An imaging device that captures images of a device capable of configuring DNA solutions in multiple partitions, the DNA solutions containing multiple DNAs including a first gene and its pseudogenes, and the DNA solutions containing fluorescently labeled probes or DNA intercalators;

[0040] A temperature adjustment unit that adjusts the temperature of each of the partitions in order to perform nucleic acid amplification reactions in each of the partitions;

[0041] The database stores information representing the reference melting temperature for the first gene and the pseudogene of the first gene, respectively.

[0042] The imaging control unit enables the imaging device to capture images in order to obtain the fluorescence intensity that varies with temperature for each of the aforementioned zones.

[0043] A melting temperature memory, which stores information representing the melting temperature of the double strand of DNA disposed in each of the said partitions, the melting temperature being obtained based on the change in fluorescence intensity as a function of temperature in an image captured by the imaging device; and

[0044] The analysis unit uses the database and the melting temperature memory to count the number of partitions containing the first gene and the number of partitions containing the pseudogene. The analysis unit outputs the number of partitions obtained by counting the various types of the DNA, and distinguishes the first gene and the pseudogene based on the melting temperature and the number of partitions obtained by counting.

[0045] Invention Effects

[0046] According to the present invention, a novel DNA detection method and DNA detection system are provided, which, in digital PCR using melting curve analysis, more accurately distinguishes between micro-regions containing target genes and micro-regions containing pseudogenes through a measuring device, thereby counting target genes with higher precision. Attached Figure Description

[0047] Figure 1 This is an example of a measurement result based on the principle of the DNA detection method in Implementation Method 1.

[0048] Figure 2 This is an example of digital PCR assay results that detect wild-type and variant types of a target gene by measuring the fluorescence intensity of two different fluorescent dyes.

[0049] Figure 3 This is a schematic diagram of the fluorescence measuring unit in Embodiment 1.

[0050] Figure 4 This is a schematic diagram of the digital PCR system of Implementation Method 1.

[0051] Figure 5 This is an example of a database used in the DNA detection method of Implementation Method 1.

[0052] Figure 6 This is a schematic diagram illustrating a method for determining the melting temperature of DNA using a fluorescently labeled probe in the DNA detection method of Embodiment 1.

[0053] Figure 7 This is an example of data related to the fluorescence intensity of the fluorescent dye contained in the pores as the temperature changes, as described in Embodiment 1.

[0054] Figure 8 This is an example of data related to the melting temperature obtained from the changes in fluorescence intensity of each well accompanied by temperature changes, as described in Implementation 1.

[0055] Figure 9 This is an example of the result obtained by counting the genes in each partition in Implementation 1.

[0056] Figure 10 It means to use Figure 3 and Figure 4 A flowchart of an embodiment of a method for measuring melting temperature using an apparatus.

[0057] Figure 11 This is an example of a measurement result displayed on a monitor.

[0058] Figure 12 This is an example of the measurement results of Implementation Method 1. Detailed Implementation

[0059] The objects, features, advantages, and concepts involved in this invention will be apparent to those skilled in the art from the description herein. Based on this description, those skilled in the art can readily reproduce the invention. The embodiments and specific examples described below represent preferred embodiments of the invention and are shown for illustrative purposes only, and are not intended to limit the invention thereto. Various changes and modifications can be made based on the description without departing from the intent and scope of the invention disclosed herein, and this will be apparent to those skilled in the art.

[0060] [Implementation Method 1]

[0061] (1) The principle and effect of DNA detection methods

[0062] Figure 1 This example illustrates a measurement result based on the principle of the DNA detection method of Embodiment 1. This example is conceived in a representative embodiment of the method where, for PCR amplification products, the melting temperature of the DNA double strand is calculated based on changes in fluorescence intensity accompanying temperature variations, thereby detecting the wild-type, variant, and pseudogene of the target gene.

[0063] The relationship between fluorescence intensity and melting temperature was plotted for microregion 101 containing wild-type alleles of the target gene, microregion 102 containing variant alleles of the target gene, and microregion 103 containing pseudogenes of the target gene.

[0064] in addition, Figure 2 The image shows an example of digital PCR assay results for detecting wild-type and variant types of a target gene by measuring the fluorescence intensity of two different fluorescent dyes.

[0065] In digital PCR, sometimes different colored fluorescently labeled probes are used for each variant (genotype) of DNA, allowing multiple variants to be detected in a single assay. Figure 2 The example is a diagram illustrating the results of using a yellow fluorescently labeled probe on the wild-type allele of the target gene and a green fluorescently labeled probe on the variant allele of the target gene. The relationship between the green fluorescence intensity and the yellow fluorescence intensity was plotted for micro-region 201 containing the wild-type allele of the target gene, micro-region 202 containing the variant allele of the target gene, and micro-region 203 containing the pseudogene of the target gene.

[0066] Here, the fluorescently labeled probe has a sequence complementary to that of the primer pair used for PCR, and is configured such that the probe is decomposed and the fluorescent label emits fluorescence upon primer elongation. Specifically, the TaqMan (registered trademark) probe can be cited as an example.

[0067] During DNA amplification in PCR, in micro-region 201 containing the wild-type allele of the target gene, the yellow fluorescently labeled probe corresponding to the wild-type allele of the target gene is decomposed, emitting yellow fluorescence. Additionally, in micro-region 202 containing the variant allele of the target gene, the green fluorescently labeled probe corresponding to the variant A allele of the target gene is decomposed, emitting green fluorescence. Empty droplets not containing the target gene do not show either green or yellow fluorescence (not illustrated).

[0068] However, in reality... Figure 2 As shown, pseudogenes with very similar sequences sometimes exist within the target gene. Even if the portion of the pseudogene is inconsistent with the primer region designed for the target gene by a few bases, it amplifies in the micro-region 203 containing the pseudogene because there is no competition with the target gene. Consequently, the fluorescently labeled probe of the target gene is decomposed, emitting yellow fluorescence, sometimes overlapping with the distribution of the micro-region 201 containing the wild-type allele of the target gene.

[0069] In digital PCR using melting curve analysis, the melting temperature of fluorescently labeled probes and DNA varies depending on the genotype, enabling genotype discrimination. Figure 1 The example is a diagram illustrating the results of measuring the melting temperature of DNA in each microregion using fluorescently labeled probes corresponding to wild-type, variant, and pseudogenes for the target gene.

[0070] Here, a known molecular beacon can be used as a fluorescently labeled probe. The following describes the DNA detection method in detail using a molecular beacon as an example. A molecular beacon is constructed as an oligonucleotide having a sequence complementary to the sequence between primer pairs used in PCR to amplify the target gene. Furthermore, the molecular beacon has complementary sequences at both ends, with a fluorescent dye at one end and an extinction dye (quenching agent) at the other end.

[0071] When a molecular beacon hybridizes with a target gene, the fluorescent pigments at both ends separate from the extinct pigments and emit fluorescence. However, as the temperature rises and the beacon dissociates from the target gene, the complementary sequences at both ends hybridize to form a stem-loop structure, and the fluorescent pigments and extinct pigments become close together, causing the fluorescent pigments to become extinct.

[0072] In a small partition 101 containing the wild-type allele of the target gene, a fluorescently labeled probe corresponding to the wild-type allele of the target gene hybridizes with DNA amplified by PCR and emits fluorescence. The melting temperature corresponding to the fluorescently labeled probe of the wild-type allele is observed.

[0073] In addition, in the micro-region 102 containing the variant allele of the target gene, the fluorescently labeled probe corresponding to the variant allele of the target gene hybridizes with the DNA amplified by PCR and emits fluorescence, and the melting temperature corresponding to the fluorescently labeled probe of the variant allele is observed.

[0074] Furthermore, in the micro-region 103 containing the pseudogene of the target gene, the fluorescently labeled probe corresponding to the wild-type allele of the target gene hybridizes with the DNA amplified by PCR in the form of mismatched bases and emits fluorescence, and the melting temperature corresponding to the mismatch hybridization is observed.

[0075] In this way, based on fluorescence intensity, fluorescence type (e.g., color) and melting temperature, it is possible to determine whether there is a target gene with wild-type alleles, a target gene with variant alleles, or a target gene with pseudogenes.

[0076] The melting temperature of DNA is unaffected by PCR reaction efficiency or in-plane measurement bias during fluorescence assays, thus enabling high-precision identification of DNA genotypes within micro-regions. For example, by determining the sequence of fluorescently labeled probes based on their different melting temperatures (Tm) relative to the target gene, and analyzing the changes in fluorescence intensity accompanying temperature variations within micro-regions, melting curve analysis can be performed to compare melting temperatures, thereby enabling genotype identification of DNA within micro-regions.

[0077] Furthermore, the relationship (e.g., ratio) between the copy number of the target gene and the copy number of its pseudogene is determined by the location of each gene on the chromosome. For example, if both are located on autosomes, their copy numbers are equal. Therefore, by counting the genotypes of DNA in each micro-region and confirming whether the sum of the wild-type and variant copy numbers of the target gene and the copy number of the pseudogene matches the expected relationship (e.g., 1:1), higher precision gene detection can be achieved.

[0078] (2) Example of the structure of a DNA detection device

[0079] The DNA detection system of Embodiment 1 includes a DNA detection device for detecting a target gene in a DNA solution and performs the DNA detection method described in this specification. In this embodiment, the DNA detection device constitutes the DNA detection system on its own, but it may also be designed so that multiple devices cooperate to constitute the DNA detection system.

[0080] The DNA detection device includes: a temperature adjustment unit for heating a DNA solution; a fluorescence measurement unit for measuring the intensity of fluorescence emitted from the DNA solution; a computer for calculating the melting temperature of the DNA double strand based on a melting curve representing the change in fluorescence intensity accompanying a change in the temperature of the DNA solution; and a monitor for displaying information transmitted from the computer.

[0081] DNA solutions can be provided in any carrier, for example, as microdroplets in oil, or configured within the wells of a plate, etc. As an example of a DNA detection device, [the following is an example]... Figure 3 and Figure 4 This refers to a DNA detection device equipped with a fluorescence measurement section.

[0082] Figure 3 This is a schematic diagram of the fluorescence measuring unit in Embodiment 1. Figure 4 This is a schematic diagram of the digital PCR system of Implementation Method 1. Figure 3 The fluorescence assay unit measures the color and fluorescence intensity of fluorescent dyes contained in microdroplets or wells that have been extremely diluted. Figure 4 Digital PCR systems have Figure 3 The illustrated fluorescence measurement unit, the computer for analyzing the measurement data, and the monitor for displaying the results utilize melting curve analysis. In these examples, the fluorescence measurement unit measures the color and fluorescence intensity of the fluorescent dye contained in the DNA solution in the droplets or wells, but the structure of the DNA detection device of the present invention is not limited thereto.

[0083] exist Figure 3 In the example of the fluorescence measurement unit shown in (A), a microfluidic path is used to measure the fluorescence intensity of the microdroplet. The microdroplet 301 flows in the direction of the arrow within the microfluidic path 303. If the microdroplet flows to the measurement position (in... Figure 3 (A) indicates the position of droplet 302. The droplet is heated by a temperature adjustment unit (not shown) while being irradiated with excitation light by a light source 304. The fluorescent substance contained in the droplet is excited by the light source 304, and the emitted fluorescence is detected by a photomultimeter 306 through a fluorescence filter 305. The photomultimeter 306 is an example of an imaging device that can capture images of a device capable of preparing DNA solutions in multiple compartments.

[0084] The fluorescence measurement unit includes a light source 304, a fluorescence filter 305, and a photoelectric multimeter 306. The fluorescence measurement unit can be set up separately for each color of the fluorescent dye, or it can be configured as follows: Figure 3 As shown in (A), the configuration involves using the excitation light from one light source to excite two fluorescent dyes, and using two fluorescent filters to simultaneously detect the fluorescence of each dye.

[0085] Alternatively, it can be like Figure 3 (B) and Figure 3 As in (C), droplets are arranged on a plane, and the color and fluorescence intensity of the fluorescent pigment in each droplet are measured.

[0086] To explain in more detail, for example, multiple droplets 311 are arranged in an array in a droplet detection cartridge 310 and placed on a temperature control stage 312, which serves as a temperature adjustment unit. The temperature control stage 312 changes the temperature of each zone to allow for nucleic acid amplification reactions in each zone. By changing the temperature of the droplet detection cartridge using the temperature control stage 312, the change in fluorescence intensity of the droplets accompanying the temperature change is measured. Different changes in fluorescence intensity are observed in droplets 301 containing the target gene and droplets 302 not containing the target gene.

[0087] The measurement sequence is as follows. First, excitation light is emitted from light source 304 through lens 308, fluorescence filter 305, and dichroic mirror 309 to each microdroplet 311. The excitation light excites the fluorescent material contained in each microdroplet 311, and the emitted fluorescence is detected by CCD camera 307 through dichroic mirror 309, fluorescence filter 305, and lens 308. CCD camera 307 is an example of an imaging device.

[0088] exist Figure 3 In (A), droplets need to be processed one by one, but in terms of being able to process multiple droplets at once, it is preferable to... Figure 3 (B) and Figure 3 The device of (C). Additionally, in Figure 3 (B) and Figure 3 In the apparatus of (C), the temperature control stage 312 can also be used for DNA amplification reactions, which is also superior to... Figure 3 (A) is more preferred.

[0089] Furthermore, it can also be like Figure 3 Instead of droplets, wells arranged in an array are used (D). Specimens are added in one or zero doses of the target gene into each well, and PCR is performed inside the wells to measure the color and fluorescence intensity of the fluorescent dye.

[0090] To explain in more detail, for example, a reaction solution containing the sample is added to the wells of the well-type detection kit 313. Then, PCR is performed in the wells on a temperature control unit 312. The temperature of the kit 313 is changed by the temperature control unit 312, and the changes in fluorescence intensity in the wells corresponding to the temperature change are measured. Different changes in fluorescence intensity are observed in wells 314 containing the target gene and wells 315 not containing the target gene.

[0091] The measurement sequence is as follows: First, excitation light is irradiated from the light source 304 through the lens 308, fluorescence filter 305, and dichroic mirror 309 into each aperture. The excitation light excites the fluorescent substance contained in the reaction solution within the aperture, and the emitted fluorescence is detected by the CCD camera 307 through the dichroic mirror 309, fluorescence filter 305, and lens 308. (The text abruptly ends here.) Figure 3 In the case of using wells as in (D), without the step of arranging droplets in the droplet detection kit, analysis from PCR to melting curve can be performed within the kit 313.

[0092] When observing changes in fluorescence intensity of the wells with temperature variations, a tilt adjustment section (not shown) can be provided below the temperature control stage 312. The tilt adjustment section removes air bubbles generated in the chamber 313 due to the heating of the temperature control stage 312. This prevents the inability to obtain fluorescence images due to air bubbles when measuring the fluorescence intensity of each well while the sample temperature is subsequently lowered by the temperature control stage 312.

[0093] like Figure 4 As shown, the fluorescence data detected by the fluorescence measurement unit 401 is sent to the computer 402. In the computer 402, the analysis unit 403 calculates the melting temperature of the amplified product and stores it in the memory 405. Thus, the memory 405 functions as a melting temperature memory, storing information representing the melting temperature of the double strand of DNA located in each partition. Furthermore, as described later, the melting temperature is obtained based on the change in fluorescence intensity accompanying temperature changes in an image captured by an imaging device (e.g., a photomultimeter 306).

[0094] In addition, the relationships between gene types, melting temperatures, and quantities are pre-prepared in database 404. Specifically, database 404 stores information representing predetermined melting temperatures (reference melting temperatures) as benchmarks for both the target gene (first gene) and its pseudogenes. Referring to database 404, the genotype of the target gene is determined based on the measured melting temperature values ​​in memory 405, and counting is performed according to genotype.

[0095] The counting results are displayed on monitor 406. Monitor 406 is an example of an output device, and the display processing in monitor 406 can also be replaced by output processing to other output devices (printing devices, non-volatile storage devices, etc.).

[0096] The DNA detection device of Embodiment 1 may include a sample splitting unit. The sample splitting unit divides the DNA solution containing the target gene into tiny partitions. These tiny partitions may be configured as wells arranged in an array within the cartridge, or as droplets dispersed in oil. This partitioning allows for appropriate extreme dilution.

[0097] Alternatively, the DNA detection device of Embodiment 1 may also include an amplification unit for amplifying DNA in micro-regions.

[0098] (3) Examples of methods for analyzing melting curves

[0099] Figure 5 This represents an example of a database prepared prior to digital PCR measurements that stores information representing the baseline melting temperature of genes. Figure 5 The data shown can be measured in advance through pre-experiments and stored in database 404.

[0100] exist Figure 5 In this example, for each gene, a baseline melting temperature is stored for both the gene itself and its pseudogenes. Alternatively, in this example, the baseline melting temperature is stored for each genotype. Multiple variants can also be defined for a single gene.

[0101] The information representing the reference melting temperature is defined in this example as a single temperature value, but it can also be defined as information representing a range of temperatures.

[0102] In addition, in this example, besides the baseline melting temperature, information representing the color of the fluorescent dye is also stored for each genotype of each gene.

[0103] Additionally, in this example, for each gene, information is stored indicating the relationship between the number of target genes (regardless of genotype) and the number of their pseudogenes. This relationship is expressed, for example, as a ratio. Figure 5 In the example, regarding gene A, it is shown that when there are x copies of the target gene, there are x copies of the pseudogene, that is, it shows that the ratio of the number of pseudogenes to the number of target genes is 1.

[0104] The copy number relationship between a target gene and its pseudogene is determined by their respective positions on the chromosome; therefore, the copy number relationship can also be defined according to each position on the chromosome. A specific example is shown below.

[0105] First, let's address the case where the target gene is located on an autosome. In this case, if the pseudogene is located on an autosome, the number of pseudogenes is equal to the number of the target gene. Figure 5 Gene A has the potential to correspond to this situation. When the pseudogene is located on the X chromosome, in a female sample, the number of pseudogenes is equal to the number of target genes; on the other hand, in a male sample, the number of pseudogenes is half the number of target genes. Figure 5 Gene B is equivalent to this case. If the pseudogene is located on the Y chromosome, the number of pseudogenes is 0 in female samples and half the number of target genes in male samples.

[0106] Next, we will explain the case where the target gene is located on the X chromosome. In this case, when the pseudogene is located on an autosome, the number of pseudogenes is equal to the number of the target gene in a female sample; conversely, the number of pseudogenes is twice that of the target gene in a male sample. When the pseudogene is located on the X chromosome, the number of pseudogenes is equal to the number of the target gene. Figure 5 Gene A may also correspond to this situation. When the pseudogene is located on the Y chromosome, the number of pseudogenes is 0 in female samples and equals the number of target genes in male samples.

[0107] Furthermore, the case where the target gene is located on the Y chromosome is explained. In this case, if the sample is from a female, the number of target genes is 0. If the sample is from a male and the pseudogene is located on an autosome, the number of pseudogenes is twice the number of target genes. If the sample is from a male and the pseudogene is located on the X chromosome, the number of pseudogenes is equal to the number of target genes. If the sample is from a male and the pseudogene is located on the Y chromosome, the number of pseudogenes is equal to the number of target genes.

[0108] Figure 6 This is a schematic diagram illustrating the principle of measuring the melting temperature of target genes and pseudogenes using fluorescently labeled probes. As the fluorescently labeled probe, a molecular beacon designed with a structure capable of hybridizing with the target gene is preferred.

[0109] like Figure 6 As shown in (A), when the fluorescently labeled probe 602 exists alone in its free state, a stem-loop forms, and the fluorescent dye 603 and quencher 604 are close to each other, thus not emitting fluorescence. If the fluorescently labeled probe 602 is added to the sample solution after the PCR reaction, at a temperature of approximately room temperature, as... Figure 6 As shown in (B), the loop portion of the fluorescently labeled probe 602 anneals the DNA 601 amplified in the sample solution. This causes the fluorescent dye 603 to separate from the quenching agent 604, resulting in the fluorescently labeled probe 602 emitting strong fluorescence.

[0110] Then, when heating the sample solution, such as Figure 6 As shown in (C), DNA601 and fluorescently labeled probe 602 dissociate, as shown again... Figure 6 As shown in (A), a stem-loop is formed within the fluorescently labeled probe 602, thus reducing the fluorescence intensity from the fluorescently labeled probe 602.

[0111] Figure 6(D) represents an example of plotting the melting curve (a curve representing the change in fluorescence intensity relative to the change in temperature) at this point. Furthermore, this fluorescently labeled probe can be used in conjunction with fluorescently labeled probes for PCR, or a probe different from the one used for PCR can be created.

[0112] In addition, the determination of the melting curve can be performed in parallel with the nucleic acid amplification reaction, or it can be performed by heating the sample solution independently of the nucleic acid amplification reaction (e.g., after the nucleic acid amplification reaction is completed).

[0113] Figure 6 (E) indicates in Figure 6 The melting curve of (D) is a differential curve showing the result of differentiating the fluorescence intensity with respect to temperature (or dividing the change in fluorescence intensity by the change in temperature). Additionally, in Figure 6 The sign is reversed in (E). The temperature corresponding to the inflection point of fluorescence intensity is calculated as the melting temperature of the DNA double strand, 605. Figure 6 The temperature at the inflection point of the fluorescence intensity of (D) corresponds to Figure 6 The temperature at the maximum value of the differential curve of (E).

[0114] In this way, the melting temperature can be used as the inflection point of fluorescence intensity for calculation. Based on this calculation method, the melting temperature can be calculated with high accuracy.

[0115] Furthermore, the melting temperature of fluorescently labeled probes used to detect target genes can be controlled using known techniques. For example, this can be achieved by altering the probe's sequence or chain length. Alternatively, it can be controlled using artificial DNA such as peptide nucleic acid (PNA) or locked nucleic acid (LNA).

[0116] In the fluorescently labeled probe 602 used here, the combination of fluorescent dye 603 and quenching agent 604 is not particularly limited as long as it is a combination commonly used in real-time PCR. For example, examples of fluorescent dye 603 include FAM, VIC, ROX, Cy3, Cy5, etc., and examples of quenching agent 604 include TAMRA, BHQ1, BHQ2, BHQ3, etc.

[0117] Regarding the sequence of the fluorescently labeled probe 602, separate sequences specific to the wild-type, variant, and pseudogene of the target gene can be prepared and detected separately. In this case, such as Figure 6As shown in (F), the fluorescently labeled probe 602 may include a first fluorescently labeled probe having a sequence corresponding to the target gene and a second fluorescently labeled probe having a sequence corresponding to the pseudogene. In this configuration, it is easier to distinguish between the target gene and the pseudogene.

[0118] Alternatively, specific sequences can be prepared for both the wild-type and variant forms of the target gene. For pseudogenes, fluorescently labeled probes of these specific sequences can be hybridized with the wild-type form of the target gene for detection. In this case, such as... Figure 6 As shown in (G), the fluorescently labeled probe 602 may contain a first fluorescently labeled probe having a sequence corresponding to the target gene. This structure reduces the number of fluorescently labeled probes that need to be prepared.

[0119] In a variation of Embodiment 1, a DNA intercalating agent can be used as an alternative to the fluorescently labeled probe. Specifically, the DNA intercalating agent is first added to a PCR reaction solution to prepare a sample solution, and then a nucleic acid amplification reaction, such as PCR, is performed. At approximately room temperature, the DNA intercalating agent binds to the two strands of DNA amplified in the sample solution, emitting strong fluorescence.

[0120] Subsequently, as the temperature of the sample solution rises, the two strands of DNA in the sample solution dissociate into one strand of DNA, and the DNA intercalating agent no longer binds, thus reducing the fluorescence intensity. Alternatively, the measurement of fluorescence intensity changes relative to temperature changes can also be performed independently of the nucleic acid amplification reaction (e.g., after the nucleic acid amplification reaction is complete) by heating the sample solution.

[0121] Furthermore, the melting temperature of the target gene can be controlled by altering the primer design, depending on the sequence of the PCR amplification product and the chain length of the sequence.

[0122] As DNA intercalating agents, intercalating agents that increase fluorescence intensity by binding to two strands of DNA and can be used for the detection of two strands of DNA are used. Specifically, SYBR Green I, SYBR Gold, PicoGreen, SYTO Blue, SYTO Green, SYTO Orange, SYTO Red, POPO-1, BOBO-1, YOYO-1, TOTO-1, JOJO-1, POPO-3, LOLO-1, BOBO-3, YOYO-3, TOTO-3, PO-Pro-1, YO-Pro-1, TO-Pro-1, JO-Pro-1, PO-Pro-3, YO-Pro-3, TO-Pro-3, TO-Pro-5, ethidium bromide, etc., can be used. If the DNA intercalating agent is heat-resistant, it can be pre-added to the well or droplets before performing the PCR reaction.

[0123] The following shows the use Figure 6 The principle of this method is to analyze the melting temperature of target genes and pseudogenes.

[0124] The PCR reaction solution, containing the sample, fluorescently labeled probe, and enzymes, was divided into tiny partitions. After PCR processing, the fluorescence intensity of each partition was calculated based on fluorescence images obtained while the temperature was being varied. Figure 7 The data is stored in memory 405. Figure 7 This illustrates an example of data stored in the memory in Embodiment 1. This data, for example, is obtained by analyzing the fluorescence intensity of the fluorescent dye as it changes with temperature for each well using a resolution unit.

[0125] exist Figure 7 In the example, fluorescence intensity was measured for multiple colors. This allows for more precise discrimination. However, fluorescence intensity can also be measured for a single color.

[0126] Next, based on the changes in fluorescence intensity relative to temperature variations in each region, melting curves for each region were constructed. Then, based on the melting curves of each region, the melting temperature of each region was calculated. Figure 8 The melting temperature shown is stored in memory 405. Hereinafter, the melting temperature calculated based on the measurement of fluorescence intensity will be referred to as the "measured melting temperature," distinguishing it from the predefined reference melting temperature.

[0127] Figure 9 This is an example of the result obtained by counting genes in each region. For example... Figure 9As shown, it is determined whether the gene configured in each partition is the wild type of the target gene in database 404, a variant of the target gene, a pseudogene of the target gene, or none of the above. Next, the number of wild types of the target gene (the number of partitions configured with wild types of the target gene), the number of variants of the target gene (the number of partitions configured with variants of the target gene), and the number of pseudogenes of the target gene (the number of partitions configured with pseudogenes of the target gene) are counted.

[0128] Next, referring to database 404, the relationship between the number of target genes (the total number of wild-type and variant genes) and the number of pseudogenes is determined. This determination is made, for example, by whether the ratio of the measured number of pseudogenes to the number of target genes matches the ratio recorded in database 404.

[0129] As a specific example, in the detection Figure 5 In the case of gene A shown, if the ratio of the number of detected pseudogenes to the number of target genes is 1, it is considered a match; otherwise, it is considered a mismatch. Furthermore, matching can be determined using a range, for example, in the detection... Figure 5 In the case of gene A shown, if the measured ratio is within the range of 0.8 to 1.2, it is considered a match; otherwise, it is considered a mismatch.

[0130] In addition, Figure 5 In the case of gene A, the ratio is 1 or about 1, but the ratio varies depending on the gene and can be any of 1 / 2, 1, 2, or 0.

[0131] If the measured ratio matches the ratio recorded in database 404, information indicating a match is displayed on monitor 406. If they do not match, information indicating a mismatch (e.g., a measurement error) is displayed on monitor 406. This information can be reflected in the accuracy management of digital PCR. Additionally, if measurement errors occur at a frequency exceeding a preset baseline, an alarm can be displayed on monitor 406. This alarm may include, for example, information instructing the user to perform adjustments to the digital PCR system.

[0132] (4) Method for determining melting temperature

[0133] Reference Figure 10 The flowchart illustrates an example of a method for determining melting temperature. This example uses... Figure 3 and Figure 4 This is one embodiment of a method for measuring melting temperature using the apparatus and container shown. In this example, using... Figure 3 The device of (B), equipped with Figure 3 (D) pore boxes, DNA intercalators, or molecular beacons.

[0134] First, prepare a database 404 (S1001) associated with the target gene to be determined by digital PCR. Next, prepare a sample solution (DNA solution) from a biological sample containing DNA. The sample solution may contain various DNA molecules including the target gene (first gene) and its pseudogenes. Add this sample solution to the PCR reaction solution. The PCR reaction solution contains DNA polymerase, primers, DNA intercalating agents or molecular beacons, deoxyribonucleotides, and buffer. Thus, the sample solution contains fluorescently labeled probes or DNA intercalating agents.

[0135] The PCR reaction solution is divided into wells arranged in an array within the cartridge 313 (S1002). This step involves preparing the sample solution in multiple compartments separately. Here, in this embodiment, digital PCR processing is performed. That is, the sample solution prepared in each compartment is extremely diluted, thereby enabling digital PCR to be performed.

[0136] Next, box 313 is placed in a thermal cycler, and PCR (S1003) is performed under temperature control of the thermal cycler. This is the procedure for nucleic acid amplification reactions in each partition. DNA is amplified by repeatedly performing cycles that include denaturation, extension, and annealing processes.

[0137] When a DNA intercalating agent is used, it intercalates into the amplified DNA; when a molecular beacon is used, it hybridizes with the amplified DNA. This results in increased fluorescence intensity. Reaction conditions, including temperature, time, and cycle number for each step, can be easily set by those skilled in the art. After PCR, when the temperature is lowered to room temperature, the DNA forms two strands.

[0138] After PCR, a fluorescence image is obtained (S1004). Detailed steps are described below. The cartridge 313 is placed on the temperature control stage 312 of the DNA detection device. While the temperature of the cartridge 313 is changed by the temperature control stage 312, the fluorescence measurement unit 401 measures the fluorescence intensity of the DNA intercalating agent or molecular beacon from each well. Thus, a fluorescence image is obtained.

[0139] The S1004 process involves changing the temperature of each zone and measuring the fluorescence intensity of each zone as a function of the temperature change. The S1004 process can be performed during the nucleic acid amplification reaction or after the nucleic acid amplification reaction.

[0140] In process S1004, the computer 402 or other components may also function as an imaging control unit. The imaging control unit enables the imaging device (e.g., photomultimeter 306) to capture images in order to obtain the fluorescence intensity that varies with temperature for each zone.

[0141] Next, the obtained fluorescence image is sent to computer 402, and the analysis unit 403 calculates the fluorescence intensity of each micro-region and stores it in memory 405 (S1005).

[0142] The analysis unit 403 generates a melting curve based on the fluorescence intensity data (S1006), calculates the melting temperature using the melting curve, and stores it in the memory 405 (S1007). This process includes the following steps: based on the change in fluorescence intensity with temperature, calculating the melting temperature of the double strand of DNA disposed in each partition. The measured melting temperature is thus obtained.

[0143] In addition, the analysis unit 403 refers to the data related to the reference melting temperature in the database 404 and counts the number of wild-type, variant, and pseudogenes of the target gene based on the measured melting temperature in the memory 405 (S1008). The process of S1008 includes the process of identifying the type of DNA in each partition based on the reference melting temperature and the measured melting temperature, and counting the number of partitions for each type of DNA.

[0144] Thus, in S1008, the analysis unit 403 uses the database 404 and the memory 405 to count the number of partitions containing the target gene (first gene) and the number of partitions containing pseudogenes. Additionally, in S1008, the analysis unit 403 can also determine whether the sample solution contains the target gene and its pseudogenes.

[0145] The distinction between wild-type, variant, and pseudogenotype can be based on the baseline melting temperature and the measured melting temperature. For example, in Figure 5 In the case of gene A, if the melting temperature is measured to be 63℃, it can be determined to be the wild type of the target gene.

[0146] This determination can also be based on a range. For example, if the measured melting temperature of a well is within a predetermined range (e.g., within ±1°C of the reference melting temperature recorded in database 404 for a particular genotype), it can be determined that the well contains the gene for that genotype. By using such a wide range, a robust determination that appropriately takes into account the permissible range can be made.

[0147] Next, the analysis unit 403 determines the relationship between the number of target genes (the total number of wild-type and variant types) and the number of pseudogenes. That is, it distinguishes between target genes and pseudogenes based on the melting temperature and the number of partitions obtained from the count. For example, it determines whether the ratio is 1, 1 / 2, 2, 0, or none of these. The result is output (S1009). Here, as described above, this determination can also be based on a range. For example, in step S1009, the analysis unit 403 determines whether the ratio of the number of partitions containing pseudogenes to the number of partitions containing the target gene (first gene) is a value within the range corresponding to 1, 1 / 2, 2, 0, or not within these ranges. In this way, by automatically determining the correspondence, the user of the device can more easily understand the contents of the sample solution.

[0148] Finally, monitor 406 outputs the number of target genes and the total number of genes in the box to monitor (S1010). For example, it outputs the number of partitions for counting various classes of DNA.

[0149] In S1008, the fluorescence intensity information can also be used to determine whether the DNA in each well is positive (i.e., the gene is configured in the well) or negative (i.e., the gene is not configured in the well).

[0150] Information about fluorescence intensity can be obtained, for example, by using the fluorescence intensity value itself. If the fluorescence intensity of a well is within a predetermined range, the well is considered positive; otherwise, it is considered negative.

[0151] Alternatively, the ratio or difference of fluorescence intensity at different temperatures can be used as information about fluorescence intensity. For example, fluorescence intensity can be standardized by using the ratio or difference between fluorescence intensity at temperatures below the reference melting temperature and fluorescence intensity at temperatures above the reference melting temperature. For example, for a given well, if the ratio or difference is within a predetermined range, the well is determined to be positive; otherwise, the well is determined to be negative.

[0152] For example, by subtracting the fluorescence intensity at 85°C from the fluorescence intensity at 50°C, the influence of the fluorescence of the fluorescently labeled probe itself, i.e., the influence of the background, can be removed.

[0153] Furthermore, the methods for determining the range of fluorescence intensity, the range of reference melting temperature, and the range of the correspondence (e.g., ratio) between the copy numbers of the target gene and the pseudogene can be arbitrarily chosen. For example, preliminary experiments can be conducted beforehand, and the operator can statistically determine these ranges based on the results, or the DNA detection system can determine them automatically. Alternatively, the threshold for fluorescence intensity and the predetermined range of reference melting temperature can be statistically determined using the measurement data from each well in the kit during each digital PCR assay.

[0154] Data used to statistically determine DNA within wells may include any or all of the following items, or may include items other than these.

[0155] - Fluorescence intensity at temperatures below the reference melting temperature

[0156] - Fluorescence intensity at temperatures above the reference melting temperature

[0157] - The ratio of fluorescence intensity at temperatures below the reference melting temperature to fluorescence intensity at temperatures above the reference melting temperature.

[0158] - The difference between fluorescence intensity at temperatures below the reference melting temperature and fluorescence intensity at temperatures above the reference melting temperature.

[0159] - Characteristic quantities representing the reference melting temperature

[0160] - Characteristic quantities representing the shape of the melting curve

[0161] There are no particular limitations on the sample solution used, as long as it contains the DNA of the test subject. Examples include biological samples (body fluids, tissues, cells, excrement, etc. of animals and plants) or soil samples (containing fungi, bacteria, etc.).

[0162] Examples of bodily fluids include blood, saliva, and bone marrow fluid. Blood contains cell-free DNA (cf DNA) and circulating tumor DNA (ct DNA). Examples of tissues include diseased areas obtained through surgery or biopsy (e.g., cancerous tissue in the breast or liver). Tissues can be fixed tissues, such as formalin-fixed paraffin-embedded tissue sections (FFPE). Examples of cells include cells collected through biopsy (cells in or near the affected area) and circulating tumor cells in the blood.

[0163] There are no particular limitations on the pretreatment of these specimens. Substances obtained by collecting samples from organisms or the environment and adding them to a suspension for homogenization or by dissolving them in a dissolving solution can be used directly. However, it is preferred to use substances obtained by extracting or purifying the nucleic acids contained in them.

[0164] It is preferable to add oil to the upper surface of the PCR reaction solution to prevent evaporation of the well-divided PCR reaction solution during PCR and melting curve analysis. The oil is preferably a chemically inert substance that is insoluble or poorly soluble in the PCR reaction solution, and more preferably a substance that is stable against temperature changes at high temperatures, similar to PCR. Fluorinated oils, silicone oils, hydrocarbon oils, etc., can be used.

[0165] Examples of fluorinated oils include perfluorocarbons and hydrofluoroethers. Fluorinated oils are preferred because they have long carbon chains and low volatility. Examples of silicone oils include polyphenylmethylsiloxanes and trimethylsiloxysilicates. Examples of hydrocarbon oils include mineral oils, liquid paraffin, and hexadecane.

[0166] Oils can be used by adding surfactants. There are no particular restrictions on the type of surfactant used; Tween20, Tween80, Span80, Triton X-100, etc., can be used.

[0167] (5) Display results

[0168] Figure 11 This is an example of the measurement results displayed on a monitor. This example shows the results of testing two cancer-related genes, A and B, as target genes. In this example, equal amounts of cancer-related genes A and B were detected.

[0169] It is possible Figure 11 As shown in (A), the number of DNA counts in the sample solution is displayed according to the type and type of mutation of cancer-related genes. Alternatively, it can be shown as... Figure 11 As shown in (B), the proportion of counted DNA in the sample solution is displayed according to the type and type of mutation of cancer-related genes. Figure 11 In example (B), the proportion of variant genes in the target gene is shown.

[0170] like Figure 11 As shown in (A), the total number of cancer-related genes can be displayed (in this example, the same number of cancer-related genes A and B were detected). The total number of genes can be a value calculated by combining the wild-type and variant types of the target gene, a value corrected for the results of pseudogene assays of the target gene, or a value calculated based on the number of target genes or pseudogenes. Such a display allows the user of the device to easily understand the contents of the sample solution.

[0171] The results displayed on the monitor can be Figure 11 The quantity or proportion of DNA in such a sample solution can also be... Figure 1 Such a graph, which plots the measured values ​​of the sample solution on two axes—fluorescence intensity of the fluorescently labeled probe and melting temperature—may also include both axes. Additionally, it may include a histogram showing the amount of DNA in the sample solution relative to either the fluorescence intensity of the fluorescently labeled probe or the melting temperature.

[0172] The range of fluorescence intensity of the fluorescently labeled probe used for counting DNA can be arbitrarily changed by the user. Similarly, the range of the reference melting temperature used for counting DNA can also be arbitrarily changed by the user. The DNA detection system can also accept operations to change these ranges. In this way, by observing the graphs and histograms of the measurement results and changing the range of fluorescence intensity and / or the reference melting temperature, the user can recount the amount of DNA in the sample solution within the new range.

[0173] In addition to displaying the number or proportion of target genes, the count of pseudogenes can also be displayed. Furthermore, the DNA detection device can determine whether the correspondence between the counts of target genes and the counts of pseudogenes matches, and display the result on monitor 406. For example, it can also determine whether the ratio of the count of pseudogenes to the count of target genes matches a value (or range) stored in database 404. If they match, information indicating that these counts match (e.g., a message indicating that the measurement was performed correctly) is displayed on monitor 406; if they do not match, information indicating that these counts do not match (e.g., a message indicating that a measurement error occurred) is displayed on monitor 406.

[0174] In addition, as mentioned above, the sample solution is treated as a solution in wells or droplets, so the number of wells or droplets can be expressed instead of the amount of DNA in the sample solution.

[0175] (6) Procedure

[0176] Another embodiment of the present invention is a procedure for enabling a DNA detection device to perform a DNA detection method. Here, the DNA detection device may be, for example, the DNA detection device of Embodiment 1, using the device detailed in section (2) above, as the DNA detection method, and performing the method detailed in section (1) above.

[0177] Another embodiment of the present invention is a recording medium for storing the above-described program.

[0178] [Example]

[0179] In this embodiment, the results of using fluorescently labeled probes to determine the melting temperature of DNA in the well and to identify the BRAF gene and its pseudogenes are shown.

[0180] The BRAF gene is known to reside on chromosome 7, an autosome, while its pseudogene, BRAFP1, resides on the X chromosome. The sample used in this example is genomic DNA from HCT116, a human colorectal cancer cell line. Since this is male genomic DNA, the copy number of the BRAF pseudogene should be half the combined copy number of the wild-type and variant types.

[0181] First, for the BRAF gene, prepare wild-type and V600E variant genomic DNA (final concentration 133 molecules / μL). Add the necessary PCR primers (final concentration 0.25 μM), reverse primers (final concentration 2.0 μM), the corresponding fluorescently labeled probe for wild-type (final concentration 0.5 μM), and 1x master mixture (including DNA polymerase and dNTPs) to prepare the PCR reaction solution. At this point, primers are added asymmetrically to achieve overamplification of the complementary DNA strand of the fluorescently labeled probe.

[0182] The sequences of the primers and probes are as follows. Furthermore, the fluorescently labeled probes all have complementary sequences near both ends, and they are designed to form double strands within the molecule. Additionally, CAL Fluorescein 610, a fluorescent dye, is bound at the 5' end, and BHQ-2, a quenching agent, is bound at the 3' end.

[0183] Forward primer: 5'-CATGAAGACCTCACAGTAAAAATAGGTGAT-3' (serial number 1)

[0184] Reverse primer: 5'-TGGGACCCACTCCATCGA-3' (serial number 2)

[0185] Fluorescently labeled probe corresponding to the wild-type BRAF gene: 5'-GGTCTAGCTACAGTGAAATC-3' (Sequence No. 3)

[0186] Then, for each well, in order to allow wild-type DNA of the BRAF gene to enter one well, or V600E variant DNA to enter one well, or to allow none of them to enter, 15 μL of PCR reaction solution was added, and the DNA was amplified by PCR.

[0187] The PCR reaction was treated at 96℃ for 10 minutes, followed by 59 cycles (60℃ for 2 minutes, then 98℃ for 30 seconds), and finally treated at 60℃ for 2 minutes. After the reaction, the well-shaped chip was cooled from 85℃ to 50℃ on a temperature-controlled platform while the fluorescence intensity changes in each well were observed, and the melting curves were measured and analyzed.

[0188] The results are shown in Figure 12 Furthermore, when using two or more fluorescent dyes, it is possible to obtain [specific results] for each dye. Figure 12 That would be the outcome.

[0189] Figure 12 (A) is a graph plotted using the ratio of fluorescence intensity at 50°C to fluorescence intensity at 85°C as the horizontal axis and the measured melting temperature as the vertical axis, showing the results of testing samples containing the BRAF gene in wild-type and pseudogene samples. Based on the measured melting temperature, there are two distributions: group 1201 (e.g., within the range of 64°C to 66°C) distributed around 65°C represents wells containing the BRAF gene in wild-type samples, while group 1203 (e.g., within the range of 60°C to 62°C) distributed around 61°C represents wells containing the BRAF gene in pseudogene samples.

[0190] Figure 12 (B) shows the results of testing specimens containing the wild-type, V600E variant, and pseudogene of the BRAF gene. Based on the different melting temperatures, three distributions were identified: group 1201 (e.g., within the range of 64℃ to 66℃), distributed around 65℃, represents wells containing the wild-type BRAF gene; group 1203 (e.g., within the range of 60℃ to 62℃), distributed around 61℃, represents wells containing the pseudogene of the BRAF gene; and group 1202 (e.g., within the range of 57℃ to 59℃), distributed around 58℃, represents wells containing the V600E variant of the BRAF gene.

[0191] Compare Figure 12 The number of counts for each group, in Figure 12 In (A), the number of pseudogenes of the BRAF gene is half the number of wild-type BRAF genes. Figure 12 In (B), the number of pseudogenes of the BRAF gene is half the total number of wild-type and V600E BRAF genes.

[0192] In this way, the melting temperature is measured to distinguish the target gene and pseudogene in the genotype discrimination of digital PCR, and the copies are counted to confirm that they are consistent with the correspondence with the copy number calculated in advance based on the position on the chromosome, thereby ensuring the accuracy of the measurement.

[0193] Symbol Explanation

[0194] 301, 302, 311… microdroplets (partitions)

[0195] 303…Microflow

[0196] 304…light source

[0197] 305…fluorescent filter

[0198] 306… Photoelectric multimeter (photography device)

[0199] 307…CCD camera (image capturing device)

[0200] 308…lens

[0201] 309… Dichroic mirror

[0202] 310… Microdroplet Detection Kit

[0203] 312… Temperature Control Panel (Temperature Adjustment Section)

[0204] 313…box

[0205] Holes 314, 315… (divided)

[0206] 401…Fluorescence Measurement Section

[0207] 402... Computer

[0208] 403…Analysis Department

[0209] 404... Database

[0210] 405… Memory (Melting Temperature Memory)

[0211] 406…monitor

[0212] 601…DNA

[0213] 602…fluorescently labeled probe

[0214] 603…Fluorescent Pigment

[0215] 604…Quenching agent

[0216] 605…melting temperature

[0217] 1201…contains wild-type pores

[0218] 1202… Contains variant pores

[0219] 1203…a pore containing pseudogenes.

Claims

1. A DNA detection method, characterized in that, The process includes the following steps: The process of preparing a DNA solution involves preparing a DNA solution in multiple partitions such that each partition is either a molecule containing or not containing the first gene. The DNA solution can contain multiple DNAs including the first gene and its pseudogene. The DNA solution contains a fluorescently labeled probe or a DNA intercalator. The fluorescently labeled probe is a fluorescently labeled probe for the wild-type and pseudogenes of the first gene that is bound to a single-color fluorescent dye, or it is the same probe for the wild-type and pseudogenes of the first gene. The process of performing nucleic acid amplification reactions in each of the aforementioned partitions; The process of changing the temperature of each of the said partitions during or after the nucleic acid amplification reaction, and measuring the fluorescence intensity of each of the said partitions as a function of the temperature change; The process of calculating the melting temperature of the double strand of DNA disposed in each partition based on the change in fluorescence intensity with the change in temperature; The process of identifying the type of DNA in each of the partitions based on the melting temperature, and counting the number of partitions for each type of DNA; The process of outputting the number of partitions for each class of the DNA; The process of distinguishing the first gene and the pseudogene based on the melting temperature and the number of counted partitions; The process of determining whether the ratio of the number of partitions configured with the pseudogene to the number of partitions configured with the first gene is a value within a range corresponding to 1, a value within a range corresponding to 1 / 2, a value within a range corresponding to 2, a value within a range corresponding to 0, or a value outside these ranges; and The process of determining whether the ratio matches a predetermined value or range, displaying information indicating a match if the ratio matches, and displaying information indicating a mismatch if the ratio does not match.

2. The DNA detection method according to claim 1, characterized in that, The fluorescently labeled probe comprises a first fluorescently labeled probe having a sequence corresponding to the first gene.

3. The DNA detection method according to claim 2, characterized in that, The fluorescently labeled probe includes a second fluorescently labeled probe having a sequence corresponding to the pseudogene.

4. The DNA detection method according to claim 1, characterized in that, The method includes the following steps: displaying the number of the first gene, or displaying the proportion of variant genes in the first gene, or displaying the total number of genes calculated based on the number of the first gene or the number of pseudogenes.

5. The DNA detection method according to claim 1, characterized in that, The DNA solution configured in each of the partitions was extremely diluted.

6. The DNA detection method according to claim 1, characterized in that, The process of identifying the type of DNA in each of the partitions based on the melting temperature and counting the number of partitions for each type of DNA is also performed based on information indicating a predetermined reference melting temperature.

7. The DNA detection method according to claim 1, characterized in that, The partitions are formed as microdroplets dispersed in pores or oil arranged in an array.

8. The DNA detection method according to claim 1, wherein, The melting temperature was calculated as the inflection point of the fluorescence intensity.

9. A DNA detection system, characterized in that, have: The imaging device captures images of a device capable of configuring a DNA solution in multiple partitions such that each partition represents either a molecule of a first gene that has entered or not entered. The DNA solution may contain multiple DNAs including the first gene and its pseudogene. The DNA solution contains a fluorescently labeled probe or a DNA intercalator. The fluorescently labeled probe is either a fluorescently labeled probe for the wild-type and pseudogenes of the first gene that is bound to a single-color fluorescent dye, or the same probe for the wild-type and pseudogenes of the first gene. A temperature adjustment unit that adjusts the temperature of each of the partitions in order to perform nucleic acid amplification reactions in each of the partitions; A database that stores information representing the reference melting temperature for the first gene and the pseudogene, respectively; The imaging control unit enables the imaging device to capture images in order to obtain the fluorescence intensity that varies with temperature for each of the aforementioned zones. A melting temperature memory, which stores information for each of the partitions representing the melting temperature of the double strand of the DNA disposed in that partition, the melting temperature being obtained based on the change in fluorescence intensity as a function of temperature in an image captured by the imaging device; as well as The analysis unit uses the database and the melting temperature memory to count the number of partitions containing the first gene and the number of partitions containing the pseudogene. The analysis unit outputs the number of partitions obtained by counting various types of the DNA, and distinguishes between the first gene and the pseudogene based on the melting temperature and the number of partitions obtained by counting. The analysis unit determines whether the ratio of the number of partitions configured with the pseudogene to the number of partitions configured with the first gene is a value within the range corresponding to 1, or a value within the range corresponding to 1 / 2, or a value within the range corresponding to 2, or a value within the range corresponding to 0, or a value outside these ranges. Determine whether the ratio matches a predetermined value or range. If it matches, display information indicating a match; otherwise, display information indicating a mismatch.

10. The DNA detection system according to claim 9, characterized in that, The information representing the reference melting temperature is information representing the threshold of the temperature range.

Images