DNA detection method and DNA detection system

Abstract

To provide a DNA detection method and a DNA detection system.SOLUTION: A DNA detection method includes: a first step of dividing a sample solution containing a fluorescently labeled probe or a DNA intercalator and multiple types of DNAs to be detected into multiple microfractions; a second step of performing a nucleic acid amplification reaction in microcompartments containing the microfractions; a third step of measuring the fluorescence intensity from the fluorescently labeled probe or the DNA intercalator in each of the microcompartments in response to a temperature change; a fourth step of calculating a melting temperature of the DNA to be detected from each of the measured fluorescence intensities; a fifth step of identifying the microcompartments; and a sixth step of discriminating the types of the multiple types of DNAs to be detected based on the melting temperature in each of the microcompartments calculated in the fourth step.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a DNA detection method and a DNA detection system, in particular to digital PCR. [Background technology]

[0002] Conventional genetic testing includes methods such as PCR (Patent Documents 2-4) and real-time PCR (Non-Patent Document 1). In these methods, measurement reproducibility decreases when the gene to be detected (referred to as the "target gene" in this specification) is in a trace amount.

[0003] Digital PCR (Patent Document 1) has been developed as a method to solve this problem. Digital PCR uses limiting dilution samples to determine whether DNA is 0 (absent) or 1 (present), and allows for the quantification of minute amounts of DNA.

[0004] An example of a method for quantifying DNA in a sample using digital PCR is shown below. First, when preparing a PCR reaction solution from a sample, the sample is diluted so that when the PCR reaction solution is divided into wells on a plate or as droplets in oil, each aliquot either contains one molecule of the target gene or does not, and the DNA polymerase, primers, and fluorescently labeled probe required for PCR are added to prepare the PCR reaction solution. The resulting PCR reaction solution is then divided into microfractions as described above.

[0005] Next, the target gene in each microfraction is amplified by PCR. After PCR is completed, the fluorescence intensity of each microfraction is measured, and the number of microcompartments with fluorescence intensity exceeding a threshold is counted. From the obtained value, the amount of the target gene contained in the sample can be calculated.

[0006] In this type of digital PCR, limiting dilution of the sample is used, which can suppress the influence of sample-derived components that inhibit the PCR reaction. In addition, since a standard curve is not required, the absolute amount of the target DNA can be measured directly.

[0007] In conventional PCR, the reaction efficiency is reduced due to the presence of reaction inhibitors in the reaction solution, the formation of secondary structures in the template DNA, and improper primer design.

[0008] On the other hand, in digital PCR, DNA is detected as 0 or 1 at the end point of the reaction, so it has been said that the PCR reaction efficiency itself does not have a significant effect on the measurement results. However, in reality, even when measured at the end point, the fluorescence intensity varies greatly due to the non-uniform reaction efficiency between each microfraction. This has been the cause of reduced measurement reproducibility and measurement accuracy in digital PCR.

[0009] Therefore, in order to improve the measurement reproducibility and measurement accuracy of digital PCR, the inventors have developed a technology that can determine the presence or absence of a target gene in each microfraction even if the PCR reaction efficiency between the microfractions is uneven by measuring the melting temperature (Tm) of the PCR amplified product through melting curve analysis (Patent Document 5). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Publication of JP2013-521764 [Patent Document 2] U.S. Patent No. 4,683,195 [Patent Document 3] U.S. Patent No. 4,683,202 [Patent Document 4] U.S. Patent No. 4,800,159 [Patent Document 5] JP 2018-108063 A [Non-patent literature]

[0011] [Non-Patent Document 1] Genome Res., 10, pp986-994, 1996 Summary of the Invention [Problem to be solved by the invention]

[0012] However, in digital PCR using melting curve analysis, if air bubbles are generated near the microcompartment containing the microfraction of the sample when acquiring a fluorescent image for calculating Tm, the Tm value is calculated as an incorrect value. In digital PCR using melting curve analysis, the sample is divided into microcompartments, and the target gene in the microcompartment is amplified in the microcompartment arranged on a flat surface. Then, the fluorescent image of the microcompartment is acquired while changing the temperature, and the melting temperature of the gene in the microcompartment is calculated from the change in fluorescent intensity for each microcompartment. In this case, air bubbles are likely to be generated in the place where the temperature becomes high during the fluorescent image acquisition. In particular, when through-holes provided in the substrate are used as the microcompartments and the PCR reaction solution added to the through-holes is blocked with oil or the like to cause a reaction, or when droplets in the oil are arranged on a flat surface in the flow channel to cause a reaction, air bubbles may be generated in the oil. When the generated air bubbles move during the fluorescent image acquisition, the fluorescent intensity of each microcompartment fluctuates, causing noise in the melting curve. As a result, a value different from the melting temperature of the gene in the microcompartment is calculated, and the target gene may not be quantified correctly.

[0013] Therefore, an object of the present invention is to provide a novel DNA detection method and DNA detection system that detects microcompartments located near generated air bubbles using a measuring device in digital PCR using melting curve analysis, and accurately counts target genes. [Means for solving the problem]

[0014] The present inventors analyzed micro-compartments in which melting temperatures different from the genotype contained in the added sample were calculated in digital PCR using melting curve analysis, and as a result, it became clear that the locations of such micro-compartments are unevenly distributed, and that the cause is the presence of air bubbles. Therefore, the inventors found that erroneous determination of genotypes can be reduced by removing the micro-compartments located near air bubbles from the analysis data, and thus completed the present invention.

[0015] One embodiment of the present invention is a DNA detection method including a first step of dividing a sample solution containing a fluorescently labeled probe or a DNA intercalator and a plurality of types of DNA to be detected into a plurality of micro fractions, a second step of performing a nucleic acid amplification reaction in a micro compartment containing the micro fractions, a third step of measuring the fluorescence intensity from the fluorescently labeled probe or the DNA intercalator in each of the micro compartments in accordance with a temperature change, a fourth step of calculating the melting temperature of the DNA to be detected from each of the measured fluorescence intensities, a fifth step of identifying the micro compartments affected by air bubbles, and a sixth step of removing data of the micro compartments affected by the air bubbles from the total data obtained from the plurality of micro compartments. In the fifth step, the micro compartments affected by the air bubbles may be identified as the micro compartments having a number of peaks of a differential curve of a melting curve created from each of the fluorescence intensities measured in the third step that is equal to or greater than a predetermined value. In addition, in the fifth step, when two or more adjacent micro-compartments are identified as the micro-compartments affected by the air bubbles, in the sixth step, the data of the two or more adjacent micro-compartments may be removed, and in the fifth step, when a single micro-compartment is identified as the micro-compartment affected by the air bubbles, the data of the single micro-compartment may not be removed. In the fifth step, the micro-compartment affected by the air bubbles may be identified by analyzing an image of the micro-compartment. In the fifth step, a micro-compartment having a number of peaks of a differential curve of a melting curve created from each fluorescence intensity measured in the third step or more may be selected, and the micro-compartment affected by the air bubbles may be identified by confirming whether or not it is affected by the air bubbles by analyzing an image of the selected micro-compartment. In addition, in the fifth step, in two or more adjacent micro-compartments, micro-compartments in which the number of peaks of the differential curve is equal to or greater than a predetermined value may be selected, and in the fifth step, in a single micro-compartment, a micro-compartment in which the number of peaks of the differential curve is equal to or greater than a predetermined value may not be selected. Furthermore, in the first step, the DNA solution may be subjected to limit dilution.In a fourth step, the types of the DNAs to be detected in each of the microcompartments may be determined based on the melting temperatures, and in a fifth step, the microcompartments may be specified for each type of DNA whose type has been determined based on information representing a predetermined reference melting temperature. The microcompartments may be formed of wells arranged in an array or droplets dispersed in oil. In the fourth step, the melting temperature may be calculated as an inflection point of a melting curve created from the fluorescence intensities measured in the third step. Another embodiment of the present invention is a DNA detection system comprising a first device having a plurality of microcompartments for containing a DNA solution containing a fluorescently labeled probe or a DNA intercalator, a second device for capturing an image of the first device, a third device for adjusting the temperature of the microcompartments in order to perform a nucleic acid amplification reaction in the microcompartments, a fourth device for controlling an imaging device to capture an image in order to obtain a fluorescence intensity that changes with a temperature change in the microcompartments, and a fifth device for detecting the generation of air bubbles from the obtained fluorescence intensity of the microcompartments.

[0016] A further embodiment of the present invention includes a first step of dividing a sample solution containing a fluorescently labeled probe or a DNA intercalator and multiple types of DNA to be detected into multiple microfractions, a second step of performing a nucleic acid amplification reaction in microcompartments containing the microfractions, a third step of measuring the fluorescence intensity from the fluorescently labeled probe or the DNA intercalator in accordance with a temperature change in each of the microcompartments, a fourth step of calculating the melting temperature of the DNA to be detected from each of the measured fluorescence intensities, and a fourth step of calculating a melting temperature of the DNA to be detected from each of the measured fluorescence intensities in the third step as the microcompartments. The method includes a fifth step of identifying a microcompartment in which the number of peaks of the differential curve of the melting curve is equal to or greater than a predetermined value and the number of peaks of two or more adjacent microcompartments is equal to or greater than the predetermined value; a sixth step of determining the types of DNA to be detected based on the melting temperatures in each of the microcompartments calculated in the fourth step; and a seventh step of outputting a first analysis result including the microcompartment identified in the fifth step and a second analysis result not including the microcompartment identified in the fifth step for the types of DNA determined in the sixth step. The positions of the microcompartments identified in the fifth step among the entirety of the multiple microcompartments may be displayed. In the seventh step, the first and second analysis results may be displayed on a graph of fluorescence intensity and melting temperature. In the seventh step, the number of the microcompartments or the ratio to the total number may be output as the first and second analysis results for each type of DNA. The types of DNA may be output for different genes and their wild type and mutant type. In the first step, the DNA solution may be subjected to limiting dilution. In the fourth step, the melting temperature may be calculated as an inflection point of a melting curve created from the fluorescence intensities measured in the third step. A further embodiment of the present invention is a DNA detection system including a fluorescence measurement unit, a computer, and a display unit, the computer including an analysis unit and a memory, the memory having information on the relationship between the type of DNA and the melting temperature, the fluorescence measurement unit dividing a sample solution containing a fluorescently labeled probe or a DNA intercalator and a plurality of types of DNA to be detected into a plurality of micro fractions, and performing a nucleic acid amplification reaction in a micro compartment including the micro fractions, in each of the micro compartments, measuring the fluorescence intensity from the fluorescently labeled probe or the DNA intercalator in accordance with a temperature change, and the computer displaying the fluorescence intensity measured in the fluorescence measurement unit. The DNA detection system obtains information about the fluorescence intensity from the fluorescence intensity information stored in the memory, calculates the melting temperature of the amplification product and stores it in the memory, creates a melting curve from the information about the fluorescence intensity stored in the memory and calculates the number of peaks of the differential curve of the melting curve, identifies a micro-compartment in which the number of peaks of the differential curve of the melting curve is equal to or greater than a predetermined value set in advance and the number of peaks of two or more adjacent micro-compartments is equal to or greater than the predetermined value, and discriminates the type of DNA from the melting temperature of the amplification product, and the display unit outputs a first analysis result including the identified micro-compartment and a second analysis result not including the identified micro-compartment for the type of DNA. The display unit may display the position of the identified micro-compartment among the entire plurality of micro-compartments. The display unit may display the first and second analysis results on a graph of the fluorescence intensity and the melting temperature. The display unit may output the number of the micro-compartments or the ratio to the total number for each type of DNA as the first and second analysis results. The types of DNA may be output for different genes and their wild-type and mutant types. Effect of the Invention

[0017] The present invention provides a novel DNA detection method and DNA detection system that can detect microcompartments located near generated air bubbles in digital PCR using melting curve analysis, and quantify target genes with greater accuracy. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram showing a method for measuring the melting temperature of DNA using a fluorescently labeled probe in a DNA detection method according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic diagram showing the relationship between droplets and air bubbles when depressions for capturing droplets of a PCR reaction solution are used as microcompartments in a DNA detection method according to one embodiment of the present invention. [Diagram 3] FIG. 2 is a schematic diagram showing the relationship between wells and air bubbles when wells are used as microcompartments in a DNA detection method according to one embodiment of the present invention. [Figure 4] FIG. 1 shows data relating to the fluorescence intensity of a fluorescent dye contained in a well with temperature change, and data relating to the melting temperature obtained from the change in fluorescence intensity of each well with temperature change, in one embodiment of the present invention. [Diagram 5] FIG. 1 is a diagram showing the measurement results obtained by a DNA detection method according to one embodiment of the present invention. [Figure 6] FIG. 2 is a schematic diagram of a fluorescence measuring unit in one embodiment of the present invention. [Figure 7] FIG. 1 is a schematic diagram of a digital PCR system in one embodiment of the present invention. [Figure 8] FIG. 1 shows a database used in a DNA detection method according to one embodiment of the present invention. [Figure 9] 8 is a flow chart illustrating one embodiment of a method for making melting temperature measurements using the apparatus of FIGS. 6 and 7. [Figure 10] FIG. 11 is a diagram showing measurement results displayed on a monitor in one embodiment of the present invention. [Figure 11] FIG. 11 is a diagram showing measurement results displayed on a monitor in one embodiment of the present invention. [Figure 12] FIG. 1 is a diagram showing measurement results obtained in an example of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The objectives, features, advantages, and ideas related thereto of the present invention will be apparent to those skilled in the art from the description of this specification. From the description of this specification, those skilled in the art can easily reproduce the present invention. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustrative or explanatory purposes, and do not limit the present invention thereto. It is apparent to those skilled in the art that various modifications and alterations can be made based on the description of this specification without departing from the intent and scope of the present invention disclosed in this specification.

[0020] (1) DNA detection method One embodiment disclosed in the present specification is a DNA detection method including a first step of dividing a DNA solution containing a fluorescently labeled probe or a DNA intercalator and multiple types of DNA to be detected into multiple micro fractions, a second step of performing a nucleic acid amplification reaction in a micro compartment containing the micro fractions, a third step of measuring the fluorescence intensity from the fluorescently labeled probe or the DNA intercalator in each of the micro compartments in accordance with the temperature change in the nucleic acid amplification reaction, a fourth step of calculating the melting temperature of the DNA to be detected from each measured fluorescence intensity, a fifth step of identifying the micro compartments affected by air bubbles, and a sixth step of removing the data of the micro compartments affected by air bubbles from the total data obtained in multiple micro compartments. Hereinafter, this DNA detection method will be specifically described with reference to the schematic diagrams of Figures 1 to 7.

[0021] (First step) In this step, a sample solution containing a fluorescently labeled probe or a DNA intercalator and multiple types of DNA to be detected is divided into multiple small fractions.

[0022] First, a specimen solution containing DNA derived from a biological sample is prepared. The specimen solution may contain multiple types of target genes. This specimen solution is added to a PCR reaction solution. The PCR reaction solution contains DNA polymerase, a primer, a DNA intercalator or a fluorescently labeled probe, deoxyribonucleotides, and a buffer solution. As a result, the specimen solution contains the fluorescently labeled probe or the DNA intercalator. As the fluorescently labeled probe, it is preferable to use a molecular beacon designed to have a structure capable of hybridizing to the target gene.

[0023] The sample solution is micro-fractionated into micro-compartments. When micro-fractionating, the sample solution is preferably subjected to limiting dilution so that each micro-compartment contains 0 or 1 DNA molecule.

[0024] The biological sample used is not particularly limited, and may be any sample containing the DNA to be detected, such as an individual sample (body fluids, tissues, cells, excrement, etc. of animals and plants) or a soil sample (including fungi, bacteria, etc.). Examples of body fluids include blood, saliva, and cerebrospinal fluid. Blood contains cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA). Examples of tissues include diseased areas (e.g., cancer tissues of the breast, liver, etc.) obtained by surgery or biopsy. The tissues may be tissues that have already been fixed, such as formalin-fixed paraffin-embedded tissue sections (FFPE). Examples of cells include cells collected by biopsy (from the diseased area or its vicinity) and circulating tumor cells circulating in the blood.

[0025] The pretreatment of these specimens is not particularly limited, and after collection from a living body or the environment, they may be added to a suspension and homogenized, or dissolved in a dissolving solution, and then used as is. However, it is preferable to use specimens after extracting or purifying the nucleic acids contained therein.

[0026] (Second step) In this step, a nucleic acid amplification reaction is carried out in each microcompartment containing the microfraction. The nucleic acid amplification reaction is preferably so-called PCR, particularly digital PCR.

[0027] (Third step) In this step, the fluorescence intensity from the fluorescently labeled probe or DNA intercalator is measured in each of the microcompartments in accordance with the temperature change. This temperature change may be achieved by utilizing the temperature change during the nucleic acid amplification reaction, or by raising the temperature of the sample solution independently of the nucleic acid amplification reaction (for example, after the nucleic acid amplification reaction is completed). The fluorescently labeled probe or DNA intercalator used in this step may be the same as the fluorescently labeled probe or DNA intercalator for PCR, or may be different from the fluorescently labeled probe or DNA intercalator for PCR.

[0028] FIG. 1 is a schematic diagram showing the behavior of a fluorescently labeled probe when the temperature of a reaction solution is changed. Examples of the fluorescently labeled probe include a molecular beacon and a Taqman probe, but FIG. 1 will be explained using a molecular beacon as an example. A molecular beacon is configured as an oligonucleotide and has a sequence complementary to a sequence between a primer pair used in a nucleic acid amplification reaction that amplifies a target gene. A molecular beacon also has complementary sequences at both ends, with a fluorescent dye 103 at one end and a quenching dye (quencher) 104 at the other end. In a nucleic acid amplification reaction, in the initial state, as shown in FIG. 1B, a molecular beacon 102 exists independently and in a free state. At that time, the molecular beacon 102 forms a stem loop, and the fluorescent dye 103 and the quencher 104 are in close proximity to each other, so that no fluorescence is emitted. In the first denaturation step, when the sample solution is heated, it takes on a highly flexible structure as shown in Figure 1C, but the fluorescence remains quenched because the fluorescent dye and quenching dye do not always separate. In the annealing step, when the temperature is lowered to about room temperature, the loop portion of the molecular beacon 102 anneals to the DNA 101 amplified in the sample solution as shown in Figure 1A. As a result, the fluorescent dye 103 and the quencher 104 are always separated, so the fluorescently labeled probe 102 emits strong fluorescence. In the next extension step, the molecular beacon 102 is released, and the structure becomes like Figure 1B again, and the fluorescence is quenched. In the next denaturation step, the structure becomes like Figure 1C again, and the fluorescence remains quenched. In the nucleic acid amplification reaction, this process is repeated, so the fluorescence intensity can be measured at some stage during heating or cooling. The fluorescence intensity can also be measured after the completion of the nucleic acid amplification reaction in the same manner. When a DNA intercalator is used, when the sample DNA is double-stranded, the DNA intercalator intercalates between the double strands and emits fluorescence, but when the sample DNA is single-stranded, the DNA intercalator is released and the fluorescence is quenched. Therefore, in the nucleic acid amplification reaction, the fluorescence intensity may be measured at some stage during heating or cooling, similar to the molecular beacon 102. After the nucleic acid amplification reaction is completed, the fluorescence intensity may be measured by heating or cooling only for the purpose of measuring the fluorescence intensity.

[0029] In the molecular beacon 102 used here, the combination of the fluorescent dye 103 and the quencher 104 is not particularly limited as long as it is a combination generally used in real-time PCR. For example, examples of the fluorescent dye 103 include FAM, VIC, ROX, Cy3, Cy5, etc., and examples of the quencher 104 include TAMRA, BHQ1, BHQ2, BHQ3, etc.

[0030] When two types of target genes with different sequences are used, the sequences of the molecular beacons 102 are prepared to be specific to each of the target genes, and by binding different fluorescent dyes to them, it is possible to detect the two types of target genes in a single reaction system.

[0031] The DNA intercalator is not particularly limited as long as it increases the fluorescence intensity by binding to double-stranded DNA and can be used to detect double-stranded DNA. Specifically, SYBR (registered trademark) Green I, SYBR Gold, PicoGreen (registered trademark), SYTO (registered trademark) Blue, SYTO Green, SYTO Orange, SYTO Red, POPO (registered trademark)-1, BOBO (registered trademark)-1, YOYO (registered trademark)-1, TOTO (registered trademark)-1, JOJO (registered trademark)-1, POPO-3, LOLO (registered trademark)-1, BOBO-3, YOYO-3, TOTO-3, PO-Pro (registered trademark)-1, YO-Pro (registered trademark)-1, TO-Pro (registered trademark)-1, JO-Pro (registered trademark)-1, PO-Pro-3, YO-Pro-3, TO-Pro-3, TO-Pro-5, ethidium bromide, etc. can be applied. When the DNA intercalator is heat-resistant, it can be added to the reaction solution before performing the nucleic acid amplification reaction.

[0032] (Fourth step) In this step, the melting temperature of the DNA to be detected is calculated from each measured fluorescence intensity. The method of calculating the melting temperature is not particularly limited, and the melting temperature may be determined by obtaining a function of fluorescence intensity and temperature, calculating the derivative of the function, and calculating the temperature at which the function has a maximum value. Alternatively, the function of fluorescence intensity and temperature may be plotted on a graph to create a melting curve (i.e., a curve showing the change in fluorescence intensity with respect to the change in temperature), and further differentiating the fluorescence intensity with respect to temperature to create a differential curve, and the temperature corresponding to the peak of the differential curve may be determined as the melting temperature. An example of a melting curve is shown in FIG. 4A, and a differential curve obtained from this melting curve is shown in FIG. 4B. The temperature corresponding to the inflection point of the melting curve corresponds to the peak of the differential curve, and is calculated as the melting temperature 401 of the DNA double strand. The melting temperature of the fluorescently labeled probe for detecting the target gene can be adjusted based on known techniques when the fluorescently labeled probe is designed. For example, it can be adjusted by changing the sequence or chain length of the probe. Alternatively, it can be regulated by using artificial DNA such as Peptide Nucleic Acid (PNA) or Locked Nucleic Acid (LNA).

[0033] (5th step) In this step, the micro-compartments affected by the bubbles are identified based on the melting temperature calculated in the fourth step.

[0034] As an example, Figures 2 and 3 show the behavior of air bubbles generated near microcompartments in digital PCR using melting curve analysis.

[0035] The cartridge 204 illustrated in FIG. 2 has a recess for capturing droplets of a PCR reaction solution as a microcompartment. As shown in FIG. 2A, the inside of the cartridge 204 is filled with oil 203, and the droplet 201 is captured in a recess 202 for capturing droplets provided inside the cartridge 204. In digital PCR using melting curve analysis, a fluorescent image of the droplet is acquired while changing the temperature, and a melting curve analysis is performed for each droplet. At that time, air bubbles 105 may be generated in the cartridge 104, and air bubbles are generated frequently especially at high temperatures. If air bubbles 205 exist under the droplet 201, they will cause noise in the fluorescence intensity measurement, so it is necessary to tilt the cartridge 204 as shown in FIG. 2B to allow the air bubbles to escape from under the droplet 201. However, if the droplets 201 are not evenly captured in the droplet capture wells 202 as shown in FIG. 2C, even if the cartridge 204 is tilted, air bubbles 205 may be captured in empty droplet capture wells 202, making it impossible to accurately measure the fluorescence intensity of the droplets 201 near the air bubbles.

[0036] The cartridge 304 illustrated in FIG. 3 has wells for containing PCR reaction liquid as minute compartments. As shown in FIG. 3A, the inside of the cartridge 304 is filled with oil 303, and PCR reaction liquid 301 is added to the wells of the through-hole chip 302 provided inside the cartridge 304. When the wells are used, air bubbles 305 may be generated in the cartridge 304, as in the case of droplets. Air bubbles 305 are particularly likely to be generated at high temperatures. If air bubbles 305 exist under the through-hole chip 302, they will cause noise in the measurement of fluorescence intensity. Therefore, it is necessary to tilt the cartridge 304 as shown in FIG. 3B to allow the air bubbles to escape from under the through-hole chip 302. However, if the PCR reaction liquid is not evenly added to the through-hole chip 302 as shown in FIG. 3C, even if the cartridge 304 is tilted, air bubbles 305 may be captured under the wells with a small amount of PCR reaction liquid, and the fluorescence intensity of the wells near the air bubbles may not be accurately measured.

[0037] FIG. 4 shows examples of melting curves of microcompartments when bubbles are generated and when they are not generated. When bubbles are not generated, measuring the change in fluorescence intensity of the microcompartment with temperature change results in a smooth melting curve as shown in FIG. 4A, and when its differential curve is calculated, it has a single peak as shown in FIG. 4B, and the temperature of the peak is the melting temperature (Tm) 401. However, when bubbles are generated, measuring the change in fluorescence intensity of the microcompartment with temperature change results in the fluorescence intensity fluctuating with the movement of the bubbles as shown in FIG. 4C. Therefore, when a differential curve is calculated, it has multiple peaks as shown in FIG. 4D, and the melting temperature of the target gene in the microcompartment may not be identified or an incorrect melting temperature may be obtained.

[0038] (Sixth step) In this step, data from microcompartments that are affected by air bubbles is removed from the total data obtained from the multiple microcompartments.

[0039] FIG. 5 shows examples of melting curve analysis results of microcompartments when bubbles are not generated and when bubbles are generated. As shown in FIG. 5A, in a microcompartment containing a target gene having a wild-type allele, a melting temperature 501 corresponding to a fluorescently labeled probe of the wild-type allele is detected, and in a microcompartment containing a target gene having a mutant allele, a melting temperature 502 corresponding to a fluorescently labeled probe of the mutant allele is detected. However, when bubbles are generated, as shown in FIG. 5B, a microcompartment containing a target gene having a wild-type allele may erroneously show melting temperatures 503 and 504 having values ​​different from the melting temperature of the wild-type allele. As a result, the distribution of melting temperatures 503 having values ​​different from the melting temperature of the wild-type allele and the distribution of melting temperatures 502 of the mutant allele may overlap, and the microcompartment containing a target gene having a wild-type allele may be identified as a microcompartment containing a target gene having a mutant allele. Conversely, a micro-compartment containing a target gene having a mutant allele may erroneously show a melting temperature value different from the melting temperature of the mutant allele, and the micro-compartment containing the target gene having the mutant allele may be identified as a micro-compartment containing a target gene having a wild-type allele. Here, if the generation of an air bubble is detected and the position of the micro-compartment near the air bubble is identified, the micro-compartment showing a melting temperature 503 value different from the melting temperature of the wild-type allele can be removed from the analysis data, reducing erroneous determination of genes in the micro-compartment and enabling more accurate gene detection.

[0040] (2)DNA detection system The DNA detection system disclosed in this specification is for detecting a target gene in a DNA solution, and includes a first device having a plurality of microcompartments for receiving a DNA solution containing a fluorescently labeled probe or a DNA intercalator, a second device for capturing an image of the first device, a third device for adjusting the temperature of the microcompartments to perform a nucleic acid amplification reaction in the microcompartments, a fourth device for controlling the image capture device to capture an image in order to obtain the fluorescence intensity that changes with the temperature change in the microcompartments, and a fifth device for detecting the occurrence of bubbles from the obtained fluorescence intensity of the microcompartments. This DNA detection system executes the above-mentioned DNA detection method.

[0041] In addition to the above-mentioned devices, the DNA detection system may include a sixth device 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 that represents the change in the intensity of fluorescence associated with the temperature change of the DNA solution, and a monitor for displaying information transmitted from the computer. These devices may be physically located within a single device, or each may exist as a separate device, or some devices may be combined into a single device while other devices may exist independently.

[0042] The DNA solution may be held by any carrier, and may be present as droplets in depressions provided in a plate, cartridge, or the like as shown in Fig. 2, or may be contained in wells in a plate, cartridge, or the like as shown in Fig. 3. As an example of a DNA detection system, a DNA detection system having a fluorescence measurement unit is shown in Figs. 6 and 7.

[0043] Fig. 6 is a schematic diagram of a fluorescence measurement unit for measuring the intensity of fluorescence emitted from a DNA solution, and Fig. 7 is a schematic diagram of a digital PCR system. The fluorescence measurement unit in Fig. 6 measures the color and fluorescence intensity of a fluorescent dye contained in a limiting diluted droplet or well. The digital PCR system in Fig. 7 includes the fluorescence measurement unit exemplified in Fig. 6, a computer that calculates the melting temperature of the DNA double strand and analyzes the measurement data, and a monitor that displays the results.

[0044] 6A includes a light source 604, a fluorescence filter 605, and a detection unit such as a photomultiplier meter or a camera 607. The fluorescence measurement unit may be provided with multiple light sources and / or multiple detection units for each color of fluorescent dye, or multiple fluorescent dyes with different excitation wavelengths may be excited from a single light source via multiple fluorescence filters 605, and one detection unit may simultaneously detect multiple fluorescences with different wavelengths via multiple fluorescence filters 605.

[0045] For example, as shown in FIG. 6B, a plurality of droplets 611 are arranged in an array in a droplet detection cartridge 610 as shown in FIG. 2, and set on a temperature-adjusted stage 612, which is a temperature adjustment unit. The temperature-adjusted stage 612 changes the temperature of each compartment to carry out a nucleic acid amplification reaction in each compartment. The temperature of the droplet detection cartridge is changed by the temperature-adjusted stage 612, and the change in fluorescence intensity of the droplets associated with the temperature change is measured. The fluorescence intensity changes differently between droplet 601 containing the target gene and droplet 602 not containing the target gene.

[0046] The measurement procedure is, for example, as follows: First, excitation light is irradiated onto each droplet 611 from a light source 604 through a lens 608, a fluorescent filter 605, and a dichroic mirror 609. A fluorescent substance contained in each droplet 611 is excited by the excitation light, and the emitted fluorescence is detected by a CCD camera 607 through the dichroic mirror 609, the fluorescent filter 605, and the lens 608. The CCD camera 607 is an example of an imaging device.

[0047] Alternatively, as shown in Fig. 6C, a well-type detection cartridge 613 as shown in Fig. 3 may be used. A reaction solution containing a sample is added to a well provided in the well-type detection cartridge 613. Then, PCR is performed in the well, and the cartridge is set on a temperature-adjusting stage 612, which is a temperature adjustment unit. The temperature of the cartridge 613 is changed by the temperature-adjusting stage 612, and the change in fluorescence intensity of the well associated with the temperature change is measured. The fluorescence intensity changes differently between well 614 containing the target gene and well 615 not containing the target gene.

[0048] The measurement procedure is, for example, as follows: First, excitation light is irradiated onto each well from a light source 604 through a lens 608, a fluorescent filter 605, and a dichroic mirror 609. The excitation light excites a fluorescent substance contained in the reaction solution in the well, and the emitted fluorescence is detected by a CCD camera 607 through a dichroic mirror 609, a fluorescent filter 605, and a lens 608. When a well is used as shown in Fig. 6D, the process from PCR to melting curve analysis can be carried out within a cartridge 613 without the step of arranging droplets in a droplet detection cartridge.

[0049] When observing the change in fluorescence intensity of the microcompartments accompanying a temperature change, a tilt adjustment unit (not shown) may be provided below the temperature-controlled stage 612. The tilt adjustment unit removes air bubbles generated in the cartridge by heating with the temperature-controlled stage 612. This prevents air bubbles from preventing the acquisition of a fluorescence image when the temperature of the sample is subsequently lowered by the temperature-controlled stage 612 while the fluorescence intensity of each well is measured.

[0050] As shown in Fig. 7, the fluorescence data detected by the fluorescence measurement unit 701 is sent to a computer 702. In the computer 702, the melting temperature of the amplified product is calculated by an analysis unit 703 and stored in a memory 705. In this manner, the memory 705 stores the melting temperature of the double strand of DNA in each microcompartment. As described below, the melting temperature is obtained based on the change in fluorescence intensity accompanying the temperature change in an image captured by an imaging device.

[0051] Furthermore, the relationship between the type of gene and the melting temperature is prepared in advance in database 704. In particular, database 704 stores information indicating a predetermined melting temperature serving as a reference (hereinafter referred to as a reference melting temperature) for each of the wild type and mutant type of the target gene. With reference to database 704, the genotype of the target gene is identified based on the measured melting temperature value in memory 705. Thereafter, the number of identified microcompartments is measured for each genotype.

[0052] The measurement results are displayed on the monitor 706. The monitor 706 is an example of an output device, and the display process on the monitor 706 may be replaced with output process to another output device (printer, non-volatile storage device, etc.).

[0053] The DNA detection system disclosed herein may include a sample dividing device, which performs limit dilution of a DNA solution containing a gene of interest and divides it into minute fractions.

[0054] The DNA detection system may also include an amplification device for amplifying DNA for the microcompartments.

[0055] (3) DNA detection method using a DNA detection system An embodiment of a method for detecting DNA using the above-mentioned DNA detection system will be described below with reference to Figures 8 and 9. In this embodiment, the change in fluorescence intensity in a nucleic acid amplification reaction is used to determine the melting temperature of DNA. The melting temperature is determined using a melting curve and a differential curve.

[0056] FIG. 8 shows an example of a database including information representing the reference melting temperature of a gene, which is prepared prior to digital PCR measurement. The data shown in FIG. 8 can be measured in advance by a pilot experiment or the like, and stored as a database 704. In the example of FIG. 8, data on the reference melting temperature for each gene, for the wild type of the gene and the mutant type of the gene, is stored in memory as a database. In this example, the reference melting temperature corresponds to each genotype. A plurality of mutant types may be defined for one gene. In this example, the reference melting temperature is specified as a value representing a single temperature, but may be specified as information representing a temperature range. In this example, in addition to the reference melting temperature, data indicating the color of the fluorescent dye for each genotype of each gene is also stored.

[0057] First, a DNA solution containing a fluorescently labeled probe or a DNA intercalator and multiple types of DNA to be detected is divided into microcompartments in the cartridge 613 (S901). In this embodiment, wells are used as the microcompartments.

[0058] It is desirable to add oil to the top of the DNA solution so that the DNA solution divided into the wells does not evaporate during the measurement of the nucleic acid amplification reaction and the melting curve analysis. The oil is preferably a substance that is insoluble or poorly soluble in the PCR reaction solution and is chemically inactive, and is also preferably a substance that is stable against temperature changes at high temperatures such as PCR. Fluorine-based oils, silicone-based oils, hydrocarbon-based oils, etc. can be used. Examples of fluorine-based oils include perfluorocarbon and hydrofluoroether. Fluorine-based oils are preferred because the longer the carbon chain, the lower the volatility. Examples of silicone-based oils include polyphenylmethylsiloxane and trimethylsiloxysilicate. Examples of hydrocarbon-based oils include mineral oil, liquid paraffin, and hexadecane. The oil may be used with the addition of a surfactant. The type of surfactant is not particularly limited here, but Tween 20, Tween 80, Span80, Triton X-100, etc. can be applied.

[0059] Next, the cartridge 613 is set in a thermal cycler (S902).

[0060] First, in each well, a nucleic acid amplification reaction is carried out by controlling the temperature of a thermal cycler (S903). DNA is amplified by repeating a cycle including a denaturation step, an extension step, and an annealing step. When a DNA intercalator is used, it intercalates into the amplified DNA, and when a molecular beacon is used, it hybridizes to the amplified DNA. This causes the DNA to amplify and the fluorescence intensity to increase. The reaction conditions including the temperature, time, number of cycles, etc. of each step can be easily set by those skilled in the art. After the nucleic acid amplification reaction, the temperature is lowered to room temperature, and the amplified DNA forms a double strand.

[0061] At this time, while changing the temperature by the temperature control device, the fluorescence intensity that changes with the temperature change is measured for each well (S903). The specific procedure is as follows. The cartridge 613 is placed on the temperature control stage 612 of the DNA detection system. While changing the temperature of the cartridge 613 by the temperature control stage 612, the fluorescence measurement unit 701 measures the fluorescence intensity from the fluorescently labeled probe or DNA intercalator in each well. Here, the fluorescence intensity may be measured directly by the photomultiple meter 606, but may also be obtained by acquiring a fluorescent image and analyzing the image. Specifically, for example, the computer 702 or other components function as an imaging control unit and cause the imaging device to capture an image. The obtained fluorescent image is sent to the computer 702, and the analysis unit 703 calculates the fluorescent intensity of each micro-compartment from the fluorescent image. The obtained fluorescent intensity data of each micro-compartment is stored in the memory 705.

[0062] From the obtained fluorescence intensity data for each microcompartment, positive wells that contain the target gene and negative wells that do not are distinguished (S904).

[0063] On the other hand, the analysis unit 703 creates a melting curve based on the fluorescence intensity data (S905) and calculates the number of peaks in the differential curve (S906). Among the positive wells, the positions of wells in which the number of peaks in the differential curve of the melting curve is equal to or greater than a set number are identified (S907). If the positions of wells in which the number of peaks in the differential curve of the melting curve is equal to or greater than a set number are close to each other, it is determined that an air bubble exists at that position (S908). Here, as information used for detecting air bubbles, round air bubbles may be detected by image recognition using the entire fluorescent image of the micro-section. The melting temperature is calculated from the melting curve of each well and stored in the memory 705 (S909). The melting temperature calculated based on the measurement of the fluorescence intensity in this way is called the "measured melting temperature" and is distinguished from the reference melting temperature defined in advance. The database 704 in the memory 705 is referred to, and the type of DNA in the well is determined by referring to the reference melting temperature from the color of the fluorescence and the measured melting temperature (S910).

[0064] The analysis results, such as a graph of the relationship between fluorescence intensity and melting temperature, including or excluding wells contained in the area determined to contain bubbles, are displayed (S911), and the user selects whether to include or exclude the wells in the bubble area from the analysis results (S912).

[0065] Finally, the number of target genes in the cartridge is displayed on the monitor (S913). A specific display example is shown below.

[0066] As shown in FIG. 10A, a mark 1002 of the location where the bubble is estimated to exist may be superimposed on an illustration or fluorescent image of a chip 1001 in a cartridge with a micro-well. If the positions of the micro-compartments near the bubble and the number of the compartments are also displayed on the monitor in this way, this information can be used for the quality control of digital PCR. Furthermore, the melting temperature range 1003 of the wild type and the melting temperature range 1004 of the mutant type, and the plot 1005 of each micro-compartment calculated by melting curve analysis may be superimposed on a graph of the fluorescence intensity and melting temperature, and the analysis results including the wells included in the area determined to have the bubble (FIG. 10B) and the analysis results excluding the wells (FIG. 10C) may be displayed. This allows the user to use both results.

[0067] FIG. 11 is an example of the measurement results displayed on the monitor. This example shows the results of measuring wild-type and mutant cancer-related genes A and B as target genes. After selecting either the analysis result including the wells included in the area determined to have bubbles or the analysis result excluding them, the number of counted DNAs contained in the sample solution may be displayed for each type of cancer-related gene and each type of mutation as shown in FIG. 11A. In this case, the total number of genes may be calculated from the sum of the wild-type and mutant types of the target genes. This display allows the user of the device to easily understand the contents of the sample solution. Alternatively, as shown in FIG. 11B, the ratio of counted DNAs contained in the sample solution may be displayed for each type of cancer-related gene and each type of mutation. In the example of FIG. 11B, the ratio of mutant genes in the target genes is displayed.

[0068] The results displayed on the monitor may be the number or percentage of DNA in the specimen solution as shown in Figure 11, or a graph in which the measured values ​​of the specimen solution are plotted on two axes, the fluorescence intensity of the fluorescent-labeled probe and the measured melting temperature as shown in Figure 10, or may include both. Also, it may include a histogram in which the number of DNA in the specimen solution is plotted against the fluorescence intensity of the fluorescent-labeled probe or the measured melting temperature.

[0069] The range of the fluorescence intensity of the fluorescently labeled probe used when counting the number of DNAs may be freely changeable by the user. The range of the reference melting temperature used when counting the number of DNAs may also be freely changeable by the user. The DNA detection system may accept an operation for changing these ranges and change the corresponding ranges. In this way, the user can look at a graph or histogram of the measurement results, change the range of the fluorescence intensity and / or the reference melting temperature, and recount the number of DNAs in the sample solution that fall within the new range.

[0070] As described above, since the specimen solution is treated as a solution in a well or droplet, the number of wells or droplets may be expressed instead of the number of DNAs in the specimen solution.

[0071] Furthermore, if the number of microcompartments near the air bubble is equal to or greater than a certain number, a measurement error alert may be displayed on the monitor 706. This alert may, for example, inform the user that adjustments to the digital PCR system are necessary.

[0072] (4) Method for detecting multiple target genes In digital PCR using melting curve analysis, the melting temperature between the fluorescently labeled probe and DNA varies depending on the target gene, and thus it is possible to distinguish between multiple target genes. For example, the target gene may include multiple types of wild-type alleles and mutant alleles, but the multiple types of target genes are not limited to these.

[0073] For example, if the DNA solution to be tested contains target gene P and target gene Q, in the micro-compartment containing target gene P, a fluorescently labeled probe corresponding to target gene P hybridizes to DNA amplified by PCR and emits fluorescence. By analyzing the generated fluorescence, the melting temperature corresponding to the fluorescently labeled probe of target gene P can be calculated. In addition, in the micro-compartment containing target gene Q, a fluorescently labeled probe corresponding to target gene Q hybridizes to DNA amplified by PCR and emits fluorescence. By analyzing the generated fluorescence, the melting temperature corresponding to the fluorescently labeled probe of target gene Q can be calculated. In this way, the presence or absence of target gene P and the presence or absence of target gene Q can be determined based on the fluorescence intensity, the type of fluorescence (e.g., color), and the melting temperature.

[0074] Since the DNA melting temperature is not affected by PCR reaction efficiency or in-plane measurement variability during fluorescence measurement, the DNA melting temperature can be used to determine the genotype of the DNA in a microcompartment with high accuracy. For example, by determining the sequence of fluorescent probes so that the melting temperature (Tm) of each fluorescent probe for the target gene is different, measuring the change in fluorescence intensity associated with temperature change for the DNA in the microcompartment, performing melting curve analysis, and comparing the melting temperatures, it becomes possible to determine the genotype of the DNA in the microcompartment.

[0075] When multiple types of target genes are detected simultaneously, they can be distinguished based on the reference melting temperature and the measured melting temperature, but it is preferable to distinguish them based on the reference melting temperature range. For example, if the measured melting temperature for a certain well is within a predetermined range including the reference melting temperature recorded in the database 704 for a certain target gene (for example, within ±1°C of the reference melting temperature), it is determined that the target gene is located in that well. Using such a reference melting temperature range allows for a more accurate determination that appropriately takes into account the allowable range.

[0076] Alternatively, the ratio or difference of the fluorescence intensity at different temperatures may be used as the information of the fluorescence intensity. For example, the fluorescence intensity can be standardized by using the ratio or difference between the fluorescence intensity at a temperature lower than the reference melting temperature and the fluorescence intensity at a temperature higher than the reference melting temperature. For example, if this ratio or difference for a certain well is within a predetermined range, the well is determined to be positive, and if not, the well is determined to be negative.

[0077] For example, by subtracting the fluorescence intensity at 85°C from the fluorescence intensity at 50°C, the effect of the fluorescence of the fluorescently labeled probe itself, that is, the effect of the background, can be removed.

[0078] The method for determining the range of fluorescence intensity and the range of reference melting temperature can be selected arbitrarily. For example, a pilot experiment or the like may be performed in advance and the operator may statistically determine the range from the results, or the DNA detection system may automatically determine the range. In addition, the threshold value of the fluorescence intensity and the predetermined range of the reference melting temperature may be statistically determined using the measurement data of each well in the cartridge every time a digital PCR measurement is performed.

[0079] Data for statistically distinguishing DNA in a well may include any or all of the following items, or may include items other than these.

[0080] - Fluorescence intensity at temperatures lower than the reference melting temperature

[0081] - Fluorescence intensity at temperatures higher than the reference melting temperature

[0082] - the ratio of the fluorescence intensity at a temperature lower than the reference melting temperature to the fluorescence intensity at a temperature higher than the reference melting temperature

[0083] - The difference between the fluorescence intensity at a temperature lower than the reference melting temperature and the fluorescence intensity at a temperature higher than the reference melting temperature

[0084] - Feature value representing the reference melting temperature

[0085] - Features representing the shape of the melting curve

[0086] (5) Program An embodiment of the present disclosure is a program for causing a DNA detection system to perform a DNA detection method. Also, yet another embodiment of the present disclosure is a recording medium storing the program. EXAMPLES

[0087] In this example, the melting temperature of DNA in a well was measured using a fluorescently labeled probe, and the results of distinguishing the KRAS gene and its mutant forms G12A and G13D are shown.

[0088] First, genomic DNA (final concentration 133 molecules / μL) of the wild type, G12A and G13D mutants of the KRAS gene was prepared, and the forward primer (final concentration 0.25 μM), reverse primer (final concentration 2.0 μM), fluorescently labeled probe corresponding to the wild type (final concentration 0.5 μM), fluorescently labeled probe corresponding to the G12A mutant (final concentration 0.5 μM), and 1x master mix (containing DNA polymerase and dNTPs) required for PCR were added to prepare a PCR reaction solution. At this time, the primer pair was added so that the concentration was asymmetric so that the complementary DNA strand of the fluorescently labeled probe was excessively amplified. The G13D mutant was detected using a fluorescently labeled probe containing a mismatched base corresponding to the wild type. The sequences of the primers and probes are as follows. Note that all of the fluorescently labeled probes have complementary sequences near both ends and are designed to form a double strand within the molecule. In addition, HEX is bound to the 5' end as a fluorescent dye, and BHQ-1 is bound to the 3' end as a quencher.

[0089] Forward primer: 5'-GTCACATTTTCATTATTTTTATTATAAGG-3' (SEQ ID NO: 1)

[0090] Reverse primer: 5'-GTATCGTCAAGGCACTCTTGCC-3' (SEQ ID NO: 2)

[0091] Fluorescently labeled probe corresponding to the wild type: 5'-TTGGAGCTGGTGGCGT-3' (SEQ ID NO: 3)

[0092] Fluorescently labeled probe corresponding to the mutation type: 5'-TTGGAGCTGCTGGCGT-3' (SEQ ID NO: 4)

[0093] Then, 15μL of PCR reaction solution was added to each well so that either one DNA of wild type or G12A, G13D mutant type of KRAS gene or neither was added, and DNA was amplified by PCR. The PCR reaction was performed at 96℃ for 10 minutes, followed by 59 cycles of (60℃, 2 minutes → 98℃, 30 seconds), and finally at 60℃ for 2 minutes. After the reaction, the chip with the wells was cooled from 85℃ to 50℃ on the temperature control stage while observing the change in fluorescence intensity of each well, and the melting curve was measured and analyzed.

[0094] FIG. 12A shows the differential curve of the melting curve of each well when a sample containing a mixture of wild type and G12A and G13D mutant types of the KRAS gene was measured. The differential curve of the melting curve of each well had one peak, and the temperature at the peak position was calculated as the melting temperature. FIG. 12B shows a plot of the fluorescence intensity at 50°C of each well on the horizontal axis and the melting temperature on the vertical axis based on the results of FIG. 12A. Excluding the negative well 1207, the positive wells are divided into three distributions based on the difference in melting temperature: a group 1201 having a distribution around 69°C is a well containing only the wild type, a group 1202 having a distribution around 66°C is a well containing only the G12A mutant type, and a group 1203 having a distribution around 63°C is a well containing only the G13D mutant type. From these results, the melting temperature range 1204 of the wild type, the melting temperature range 1205 of the G12A mutant type, and the melting temperature range 1206 of the G13D mutant type were set.

[0095] Here, when a sample containing only the wild-type KRAS gene was measured, a population 1208 with a melting temperature outside the melting temperature range of the wild-type was observed as shown in Figure 12D. The melting curve of this population 1208 with a melting temperature outside the melting temperature range of the wild-type had many peaks as shown in Figure 12C, and when the position of the wells was mapped on the fluorescent image of the chip as shown in Figure 12E, it was found that they were concentrated locally. When the fluorescent image was checked, this location coincided with the location where the bubble was present. Therefore, by excluding the data of the wells near the presence of the bubble, it was possible to determine 16 wells that were erroneously detected as the G12A mutant type and 1 well that was erroneously detected as the G13D mutant type.

[0096] In this way, by detecting air bubbles from the number of peaks in the melting curve to distinguish genotypes in digital PCR and removing the data from wells near the bubbles, it is possible to exclude wells from which a different melting temperature has been mistakenly calculated, preventing erroneous judgments and improving measurement accuracy. [Explanation of symbols]

[0097] 101...DNA 102...Fluorescently labeled probe 103...Fluorescent dye 104…Quencher 201…Droplet 202…Recess for droplet capture 203...Oil 204…Cartridge 205...bubbles 301...PCR reaction mixture 302...Through hole tip 303...Oil 304... Cartridge 305...bubbles 401...Melting temperature 501…Melting temperature of wild type allele 502…Melting temperature of mutant allele 503,504…Incorrectly calculated melting temperature 601,602,611…Droplets 603...Microchannel 604...Light source 605...Fluorescent filter 607...Imaging device 608...Lens 609…Dichroic mirror 610...Droplet detection cartridge 612…Temperature control stage 613...Cartridge 614,615...well 701...Fluorescence measurement unit 702...Computer 703…Analysis department 704...Database 705…Memory 706…Monitor 1001…Chip in cartridge 1002: Location where air bubbles were suspected to exist 1003…Melting temperature range of wild type allele 1004…Mutant allele melting temperature range 1005... Plot of each microcompartment calculated by melting curve analysis 1201…wells containing wild type 1202…wells containing the G12A variant 1203…Wells containing the G13D variant 1204…Melting temperature range of wild type allele 1205…Melting temperature range of G12A mutant 1206…Melting temperature range of G13D variant 1207…Negative well 1208...Population with melting temperatures outside the wild-type melting temperature range 1209: Wells with multiple peaks in the differential melting curve

Claims

1. A first step of dividing a sample solution containing a fluorescently labeled probe or a DNA intercalator and a plurality of types of DNA to be detected into a plurality of minute fractions; a second step of carrying out a nucleic acid amplification reaction in the microcompartments containing the microfractions; a third step of measuring the fluorescence intensity from the fluorescently labeled probe or the DNA intercalator in each of the microcompartments in response to a temperature change; a fourth step of calculating a melting temperature of the DNA to be detected from each of the measured fluorescence intensities; a fifth step of identifying, as the micro-compartment, a micro-compartment in which the number of peaks in a differential curve of a melting curve created from each fluorescence intensity measured in the third step is equal to or greater than a predetermined value set in advance, and the number of peaks in two or more adjacent micro-compartments is equal to or greater than the predetermined value; a sixth step of discriminating the types of the plurality of types of detection target DNA based on the melting temperature in each of the micro-compartments calculated in the fourth step; a seventh step of outputting a first analysis result including the micro-compartment identified in the fifth step and a second analysis result not including the micro-compartment identified in the fifth step for the type of DNA determined in the sixth step; A method for detecting DNA comprising the steps of:

2. 2. The method for detecting DNA according to claim 1, further comprising displaying the positions of the micro-compartments identified in the fifth step among all of the plurality of micro-compartments.

3. 2. The method for detecting DNA according to claim 1, wherein in the seventh step, the results of the first and second analyses are displayed on a graph of fluorescence intensity and melting temperature.

4. 2. The DNA detection method according to claim 1, wherein in the seventh step, the number of the micro-compartments or the ratio to the total number of the micro-compartments for each type of DNA is output as the first and second analysis results.

5. 5. The DNA detection method according to claim 4, wherein the types of DNA are output for different genes and their wild type and mutant type.

6. 6. The DNA detection method according to claim 1, wherein in the first step, the DNA solution is subjected to limiting dilution.

7. 7. The DNA detection method according to claim 1, wherein in the fourth step, the melting temperature is calculated as an inflection point of a melting curve created from the fluorescence intensities measured in the third step.

8. A DNA detection system comprising a fluorescence measurement unit, a computer, and a display unit, The computer includes an analysis unit and a memory, The memory has information regarding the relationship between the type of DNA and the melting temperature, the fluorescence measurement unit divides a sample solution containing a fluorescently labeled probe or a DNA intercalator and a plurality of types of DNA to be detected into a plurality of micro-fractions, and measures a fluorescence intensity from the fluorescently labeled probe or the DNA intercalator in accordance with a temperature change in each of the micro-compartments in which a nucleic acid amplification reaction is carried out in the micro-compartments containing the micro-fractions; The computer acquires information regarding the fluorescence intensity measured in the fluorescence measurement unit, and the analysis unit calculates the melting temperature of the amplification product and stores the melting temperature in the memory. The analysis unit is creating a melting curve from the information on the fluorescence intensity stored in the memory and calculating the number of peaks in a differential curve of the melting curve; Identifying micro-compartments in which the number of peaks in the differential curve of the melting curve is equal to or greater than a predetermined value set in advance and the number of peaks in two or more adjacent micro-compartments is equal to or greater than the predetermined value; Identifying the type of the DNA based on the melting temperature of the amplification product; The display unit outputs a first analysis result for the type of DNA, the first analysis result including the identified microcompartment, and a second analysis result not including the identified microcompartment.

9. 9. The DNA detection system according to claim 8, wherein the display unit displays the position of the identified micro-compartment among all of the plurality of micro-compartments.

10. 9. The DNA detection system according to claim 8, wherein the display unit displays the first and second analysis results on a graph of fluorescence intensity and melting temperature.

11. 9. The DNA detection system according to claim 8, wherein the display unit outputs the number of the micro-compartments or the ratio of the number of the micro-compartments to the total number for each type of DNA as the first and second analysis results.

12. 9. The DNA detection system according to claim 8, wherein the types of DNA are output for different genes and their wild type and mutant type.

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