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, and in particular to digital PCR. [Background technology]

[0002] Conventional genetic testing methods include PCR (Patent Documents 2-4) and real-time PCR (Non-Patent Document 1). These methods have poor measurement reproducibility when the amount of the gene to be detected (referred to as the "target gene" in this specification) is very small.

[0003] Digital PCR (Patent Document 1) was developed as a method to solve this problem. Digital PCR uses limiting diluted samples to determine whether DNA is 0 (absent) or 1 (present), allowing 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. The DNA polymerase, primers, and fluorescently labeled probe required for PCR are then added to prepare the PCR reaction solution. The resulting PCR reaction solution is then divided into micro-aliquots as described above.

[0005] Next, the target gene in each micro-fraction is amplified by PCR. After PCR is complete, the fluorescence intensity of each micro-fraction is measured and the number of micro-compartments 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] This type of digital PCR uses limiting diluted samples, which can reduce the influence of sample-derived components that can inhibit PCR reactions. In addition, since no calibration curve is required, the absolute amount of target DNA can be measured directly.

[0007] In conventional PCR, the reaction efficiency is reduced due to factors such as 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 either 0 or 1 at the end point of the reaction, so it has been thought that the PCR reaction efficiency itself does not have a significant effect on the measurement results. However, in reality, even when measuring at the end point, the fluorescence intensity varies greatly due to uneven reaction efficiency between each micro-fraction. This has been the cause of reduced measurement reproducibility and accuracy in digital PCR.

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

[0010] [Patent Document 1] Publication of Special Publication No. 2013-521764 [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, digital PCR using melting curve analysis poses a problem: if air bubbles occur near the microcompartments containing the sample during fluorescence image acquisition for Tm calculation, the calculated Tm value can be incorrect. In digital PCR using melting curve analysis, the sample is divided into microcompartments, and the target gene in each microcompartment is amplified within the microcompartments arranged on a flat surface. Then, fluorescence images of the microcompartments are acquired while varying the temperature, and the melting temperature of the gene within each microcompartment is calculated from the change in fluorescence intensity for each microcompartment. During this process, air bubbles are likely to occur in locations where the temperature rises during fluorescence image acquisition. In particular, when using through-holes in a substrate as microcompartments and the PCR reaction solution added to the through-holes is blocked with oil or when droplets in oil are arranged on a flat surface within a flow channel, air bubbles can occur in the oil. The movement of the generated air bubbles during fluorescence image acquisition causes fluctuations in the fluorescence intensity of each microcompartment, resulting in 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, the object of the present invention is to provide a novel DNA detection method and a DNA detection system that detects microcompartments located near generated 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 microcompartments in which melting temperatures different from the genotype contained in the added sample were calculated in digital PCR using melting curve analysis, and found that the locations of such microcompartments are unevenly distributed, and that the cause is the presence of air bubbles. They then found that erroneous determination of genotypes can be reduced by excluding microcompartments located near air bubbles from the analysis data, leading to the completion of the present invention.

[0015] One embodiment of the present invention comprises 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 minute fractions, and a second step of dividing a sample solution containing the minute fractions. MultipleThis DNA detection method includes a second step of performing a nucleic acid amplification reaction in each microcompartment, 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 temperature changes, a fourth step of calculating the melting temperature of the DNA to be detected from the measured fluorescence intensities, a fifth step of identifying the microcompartments affected by air bubbles, and a sixth step of removing data for the microcompartments affected by the air bubbles from the total data obtained for the multiple microcompartments. In the fifth step, the microcompartments affected by the air bubbles may be identified as those having a predetermined number of peaks or more in a differential curve of a melting curve created from the fluorescence intensities measured in the third step. Furthermore, if two or more adjacent microcompartments are identified as microcompartments affected by the air bubble in the fifth step, the data of the two or more adjacent microcompartments may be removed in the sixth step, and if a single microcompartment is identified as a microcompartment affected by the air bubble in the fifth step, the data of the single microcompartment may not be removed in the sixth step. Furthermore, in the fifth step, the microcompartment affected by the air bubble may be identified by analyzing an image of the microcompartment. Furthermore, in the fifth step, a microcompartment having a number of peaks equal to or greater than a predetermined value in a differential curve of a melting curve created from the fluorescence intensities measured in the third step may be selected, and the image of the selected microcompartment may be analyzed to confirm whether it is affected by the air bubble, thereby identifying the microcompartment affected by the air bubble. In the fifth step, two or more adjacent micro-divisions may be selected in which the number of peaks of the differential curve is equal to or greater than a predetermined value, and in the fifth step, a single micro-division in which the number of peaks of the differential curve is equal to or greater than a predetermined value may not be selected. specimenThe solution may be limiting diluted. In a fourth step, the types of the multiple target DNAs may be identified in each of the microcompartments based on the melting temperatures, and in a fifth step, the microcompartments may be identified for each type of DNA whose type has been identified based on information representing a predetermined reference melting temperature. The microcompartments may be composed of wells arranged in an array or droplets dispersed in oil. In the fourth step, the melting temperature may be calculated as the 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 images 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 imaging device to capture images in the microcompartments to obtain fluorescence intensities that change with temperature changes, and a fifth device for detecting the occurrence of bubbles from the obtained fluorescence intensities of the microcompartments.

[0016] A further embodiment of the present invention comprises 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 micro-fractions; a second step of carrying out a nucleic acid amplification reaction in micro-compartments containing the micro-fractions; 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 micro-compartments; 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 the melting temperature of the DNA to be detected from each of the micro-compartments created from the fluorescence intensities measured in the third step. a fifth step of identifying microcompartments in which the number of peaks in a differential curve of the melting curve calculated is equal to or greater than a predetermined value and the number of peaks in two or more adjacent microcompartments is equal to or greater than the predetermined value; a sixth step of distinguishing the types of DNA to be detected based on the melting temperatures of each microcompartment 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 excluding the microcompartment identified in the fifth step for the types of DNA distinguished in the sixth step. The positions of the microcompartments identified in the fifth step among the entire plurality of 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 microcompartments or the ratio of each type of DNA to the total number may be output as the first and second analysis results. The types of DNA may be output for different genes and their wild-type and mutant types. 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 comprising a fluorescence measurement unit, a computer, and a display unit, wherein the computer includes an analysis unit and a memory, the memory having information on the relationship between the type of DNA and the melting temperature, and the fluorescence measurement unit divides a sample solution containing a fluorescently labeled probe or a DNA intercalator and multiple types of DNA to be detected into multiple minute fractions, and displays the minute fractions. Multiple In each of the microcompartments in which a nucleic acid amplification reaction is carried out, the fluorescence intensity from the fluorescently labeled probe or the DNA intercalator is measured in accordance with a temperature change, the computer acquires information about the fluorescence intensity measured in the fluorescence measurement unit, and the analysis unit DNA to be detected the analysis unit calculates the melting temperature of the microcompartment and stores it in the memory, the analysis unit creates a melting curve from the information about the fluorescence intensity stored in the memory and calculates the number of peaks in the differential curve of the melting curve, identifies microcompartments 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 microcompartments is equal to or greater than the predetermined value, Kiyong The DNA detection system determines the type of DNA from the melting temperature, and the display unit outputs a first analysis result for the DNA type that includes the identified microcompartment and a second analysis result that does not include the identified microcompartment. The display unit may display the position of the identified microcompartment among all of the multiple microcompartments. The display unit may display the first and second analysis results on a graph of fluorescence intensity and melting temperature. The display unit may output the number of microcompartments or their proportion to the total number for each DNA type as the first and second analysis results. The DNA type may be output for different genes and their wild type and mutant type. [Effects of the Invention]

[0017] The present invention provides a novel DNA detection method and system that can detect microcompartments located near generated bubbles in digital PCR using melting curve analysis, thereby enabling more accurate quantification of target genes. [Brief explanation 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. [Figure 2] FIG. 1 is a schematic diagram showing the relationship between droplets and 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. [Figure 3] FIG. 2 is a schematic diagram showing the relationship between wells and bubbles when wells are used as microcompartments in a DNA detection method according to one embodiment of the present invention. [Figure 4] FIG. 10 is a diagram showing 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. [Figure 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 measurement unit in one embodiment of the present invention. [Figure 7] FIG. 1 is a schematic diagram of a digital PCR system according to one embodiment of the present invention. [Figure 8] FIG. 1 is a diagram showing 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. 10 is a diagram showing measurement results displayed on a monitor in one embodiment of the present invention. [Figure 11] FIG. 10 is a diagram showing measurement results displayed on a monitor in one embodiment of the present invention. [Figure 12] FIG. 10 is a diagram showing measurement results obtained in an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] The objectives, features, advantages, and ideas relating to the present invention will be apparent to those skilled in the art from the description in this specification. Those skilled in the art can easily reproduce the present invention from the description in this specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are presented for illustration or explanation purposes, and are not intended to limit the present invention thereto. It will be apparent to those skilled in the art that various changes and modifications can be made based on the description in this specification without departing from the spirit and scope of the present invention disclosed herein.

[0020] (1) DNA detection method One embodiment disclosed herein is a DNA detection method comprising the following steps: a first step of dividing a DNA solution containing a fluorescently labeled probe or DNA intercalator and multiple types of target DNA into multiple micro-fractions; a second step of performing a nucleic acid amplification reaction in micro-compartments containing the micro-fractions; a third step of measuring the fluorescence intensity from the fluorescently labeled probe or DNA intercalator in each micro-compartment in response to temperature changes during the nucleic acid amplification reaction; a fourth step of calculating the melting temperature of the target DNA from the measured fluorescence intensity; a fifth step of identifying micro-compartments affected by air bubbles; and a sixth step of removing data from the micro-compartments affected by air bubbles from the total data obtained from the multiple micro-compartments. This DNA detection method will be specifically described below with reference to the schematic diagrams shown in 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 minute fractions.

[0022] First, a sample solution containing DNA derived from a biological sample is prepared. The sample solution may contain multiple types of target genes. This sample solution is added to a PCR reaction solution. The PCR reaction solution contains DNA polymerase, primers, a DNA intercalator or a fluorescently labeled probe, deoxyribonucleotides, and a buffer solution. As a result, the sample 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 that can hybridize 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 either 0 or 1 DNA molecule.

[0024] The biological sample used is not particularly limited, but may be any sample containing the target DNA. Examples include individual samples (body fluids, tissues, cells, excrement, etc. of animals and plants) and soil samples (containing 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 disease-affected tissues (e.g., cancer tissues from the breast, liver, etc.) obtained by surgery or biopsy. Tissues may be fixed tissues, such as formalin-fixed, paraffin-embedded tissue sections (FFPE). Examples of cells include cells collected by biopsy (from the affected area or its vicinity) and circulating tumor cells circulating in the blood.

[0025] There are no particular limitations on the pretreatment of these specimens; 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 in which the nucleic acids contained therein have been extracted or purified.

[0026] (Second step) In this step, a nucleic acid amplification reaction is carried out in each microcompartment containing a 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 microcompartment as the temperature changes. 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 (e.g., 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 used for PCR, or a different fluorescently labeled probe or DNA intercalator may be used.

[0028] FIG. 1 is a schematic diagram showing the behavior of a fluorescently labeled probe when the temperature of the reaction solution is changed. Examples of fluorescently labeled probes include molecular beacons and Taqman probes; however, FIG. 1 will be explained using a molecular beacon as an example. A molecular beacon is composed of an oligonucleotide and has a sequence complementary to the sequence between a primer pair used in a nucleic acid amplification reaction to amplify a target gene. A molecular beacon also has complementary sequences at both ends, with a fluorescent dye 103 at one end and a quencher dye 104 at the other end. In a nucleic acid amplification reaction, in the initial state, as shown in FIG. 1B, the molecular beacon 102 exists independently and free. At this time, the molecular beacon 102 forms a stem-loop, and the fluorescent dye 103 and quencher 104 are in close proximity, so no fluorescence is emitted. In the first denaturation step, when the sample solution is heated, it takes on a highly flexible structure as shown in Figure 1C. However, the fluorescent dye and quencher dye do not always separate, so the fluorescence remains quenched. In the annealing step, the temperature is lowered to approximately room temperature, and the loop portion of the molecular beacon 102 anneals to the amplified DNA 101 in the sample solution as shown in Figure 1A. This causes the fluorescent dye 103 and quencher 104 to constantly separate, causing the fluorescently labeled probe 102 to emit strong fluorescence. In the next extension step, the molecular beacon 102 is released, returning to the state shown in Figure 1B, and the fluorescence is quenched. In the next denaturation step, the structure returns to the state shown in Figure 1C, and the fluorescence remains quenched. This process is repeated during nucleic acid amplification reactions, so the fluorescence intensity can be measured at any stage, either during heating or cooling. The same method can be used to measure fluorescence intensity after the nucleic acid amplification reaction is complete. When a DNA intercalator is used, if the sample DNA is double-stranded, the DNA intercalator intercalates between the double strands and emits fluorescence, but if the sample DNA is single-stranded, the DNA intercalator is released and the fluorescence is quenched. Therefore, in a nucleic acid amplification reaction, the fluorescence intensity can be measured at some stage, during heating or cooling, as with molecular beacon 102. After the nucleic acid amplification reaction is completed, the sample may be heated or cooled and the fluorescence intensity measured just for that purpose.

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

[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, 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 its fluorescence intensity increases upon binding to double-stranded DNA and it can be used to detect double-stranded DNA. 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, and the like can be used. If the DNA intercalator is heat-resistant, it can be added to the reaction solution before the nucleic acid amplification reaction.

[0032] (Fourth step) In this step, the melting temperature of the DNA to be detected is calculated from the measured fluorescence intensities. The method for calculating the melting temperature is not particularly limited. The melting temperature may be determined by obtaining a function of fluorescence intensity versus temperature, calculating the derivative of that function, and calculating the temperature at which the function reaches its maximum value. Alternatively, the function of fluorescence intensity versus temperature may be plotted on a graph to create a melting curve (i.e., a curve showing the change in fluorescence intensity relative to temperature). The fluorescence intensity may then be differentiated 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 the 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 a fluorescently labeled probe for detecting a target gene can be adjusted based on known techniques during design of the fluorescently labeled probe. For example, it can be adjusted by changing the probe sequence or strand length. 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 microcompartments 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 bubbles generated near microcompartments in digital PCR using melting curve analysis.

[0035] The cartridge 204 shown in FIG. 2 has microcompartments, each of which has a recess for capturing a droplet of PCR reaction solution. As shown in FIG. 2A, the interior of the cartridge 204 is filled with oil 203, and a droplet 201 is captured in a droplet capture recess 202 provided within the cartridge 204. In digital PCR using melting curve analysis, fluorescent images of the droplets are acquired while changing the temperature, and a melting curve analysis is performed for each droplet. During this process, air bubbles 105 may form within the cartridge 104, and this occurs more frequently at higher temperatures. The presence of air bubbles 205 below the droplet 201 causes 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 below the droplet 201. However, if the droplets 201 are not evenly captured in the droplet capturing wells 202 as shown in Figure 2C, even if the cartridge 204 is tilted, bubbles 205 may be captured in empty droplet capturing wells 202, making it impossible to accurately measure the fluorescence intensity of the droplets 201 near the bubbles.

[0036] The cartridge 304 shown in FIG. 3 has wells for containing PCR reaction solution as microcompartments. As shown in FIG. 3A, the interior of the cartridge 304 is filled with oil 303, and PCR reaction solution 301 is added to the wells of a through-hole chip 302 provided inside the cartridge 304. When using wells, as with droplets, bubbles 305 may form inside the cartridge 304. Bubbles 305 are particularly likely to form at high temperatures. The presence of bubbles 305 below the through-hole chip 302 can cause noise in the fluorescence intensity measurement. Therefore, it is necessary to tilt the cartridge 304 as shown in FIG. 3B to allow the bubbles to escape from below the through-hole chip 302. However, if the PCR reaction solution is not evenly added to the through-hole chip 302 as shown in FIG. 3C, tilting the cartridge 304 may trap bubbles 305 below wells with a small amount of PCR reaction solution, making it difficult to accurately measure the fluorescence intensity of the wells near the bubbles.

[0037] Figure 4 shows examples of melting curves for microcompartments with and without bubbles. When no bubbles are generated, measuring the change in fluorescence intensity of the microcompartment with a change in temperature results in a smooth melting curve, as shown in Figure 4A. When its derivative curve is calculated, it has a single peak, as shown in Figure 4B, and the temperature of that peak is the melting temperature (Tm) 401. However, when bubbles are generated, measuring the change in fluorescence intensity of the microcompartment with a change in temperature results in the fluorescence intensity fluctuating with the movement of the bubbles, as shown in Figure 4C. Therefore, when the derivative curve is calculated, it has multiple peaks, as shown in Figure 4D, and the melting temperature of the target gene in the microcompartment may not be identified or may result in an incorrect melting temperature.

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

[0039] Figure 5 shows examples of melting curve analysis results for microcompartments with and without bubbles. As shown in Figure 5A, a melting temperature 501 corresponding to the fluorescently labeled probe for the wild-type allele is detected in a microcompartment containing a target gene having a wild-type allele, while a melting temperature 502 corresponding to the fluorescently labeled probe for the mutant allele is detected in a microcompartment containing a target gene having a mutant allele. However, when bubbles are generated, as shown in Figure 5B, a microcompartment containing a target gene having a wild-type allele may erroneously exhibit melting temperatures 503 and 504 that are different from the melting temperature of the wild-type allele. As a result, the distribution of melting temperatures 503, which are different from the melting temperature of the wild-type allele, overlaps with the distribution of melting temperatures 502 for the mutant allele, 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 microcompartment containing a target gene having a mutant allele may erroneously exhibit a melting temperature different from the melting temperature of the mutant allele, resulting in the microcompartment containing the target gene having the mutant allele being identified as a microcompartment containing a target gene having a wild-type allele. In this case, if the occurrence of an air bubble is detected and the position of the microcompartment near the air bubble is identified, it is possible to remove from the analysis data the microcompartment that exhibits a melting temperature 503 different from the melting temperature of the wild-type allele, thereby reducing erroneous determination of genes in the microcompartment and enabling more accurate gene detection.

[0040] (2) DNA detection system The DNA detection system disclosed herein is intended to detect target genes in a DNA solution and includes a first device having multiple microcompartments for containing a DNA solution containing a fluorescently labeled probe or a DNA intercalator, a second device for capturing images 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 imaging device to capture images in the microcompartments to obtain fluorescence intensities that change with temperature, and a fifth device for detecting the occurrence of bubbles from the obtained fluorescence intensities of the microcompartments. This DNA detection system executes the DNA detection method described above.

[0041] In addition to the above-mentioned devices, this DNA detection system may also 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 changes in fluorescence intensity with changes in the temperature of the DNA solution, and a monitor that displays 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 others exist independently.

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

[0043] Figure 6 is a schematic diagram of a fluorescence measurement unit for measuring the intensity of fluorescence emitted from a DNA solution, and Figure 7 is a schematic diagram of a digital PCR system. The fluorescence measurement unit in Figure 6 measures the color and fluorescence intensity of the fluorescent dye contained in limiting diluted droplets or wells. The digital PCR system in Figure 7 includes the fluorescence measurement unit illustrated in Figure 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 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 fluorescence lights with different wavelengths via multiple fluorescence filters 605.

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

[0046] The measurement procedure is, for example, as follows: First, excitation light is emitted from a light source 604 through a lens 608, a fluorescent filter 605, and a dichroic mirror 609 and irradiated onto each droplet 611. The excitation light excites a fluorescent substance contained in each droplet 611, 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 such as that 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. PCR is then performed in the well, and the cartridge is set on a temperature-controlled stage 612, which is a temperature control unit. The temperature of the cartridge 613 is changed by the temperature-controlled stage 612, and changes in the fluorescence intensity of the wells associated with the temperature change are 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 light source 604 through lens 608, fluorescence filter 605, and dichroic mirror 609. The excitation light excites the fluorescent substance contained in the reaction solution in the well, and the emitted fluorescence is detected by CCD camera 607 through dichroic mirror 609, fluorescence filter 605, and lens 608. When using wells as shown in Figure 6D, everything from PCR to melting curve analysis can be performed within cartridge 613 without the step of arranging droplets in a droplet detection cartridge.

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

[0050] As shown in Figure 7, fluorescence data detected by a fluorescence measurement unit 701 is sent to a computer 702. In the computer 702, an analysis unit 703 calculates the melting temperature of the amplified product and stores it in a memory 705. In this way, the memory 705 stores the melting temperature of the double-stranded DNA in each microcompartment. As will be described later, the melting temperature is obtained based on the change in fluorescence intensity accompanying a change in temperature in an image captured by an imaging device.

[0051] Furthermore, the relationship between gene type and melting temperature is prepared in advance in database 704. In particular, database 704 stores information indicating a predetermined reference melting temperature (hereinafter referred to as the reference melting temperature) for each wild-type and mutant type of the target gene. By referring to database 704, the genotype of the target gene is identified based on the measured melting temperature 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-described DNA detection system will be described below with reference to Figures 8 and 9. In this embodiment, the change in fluorescence intensity during 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 derivative curve.

[0056] FIG. 8 shows an example of a database containing information indicating the reference melting temperatures of genes, which is prepared prior to digital PCR measurement. The data shown in FIG. 8 can be measured in advance, for example, through a pilot experiment, and saved as database 704. In the example of FIG. 8, data on the reference melting temperatures for each gene, both for the wild-type and mutant forms of that gene, are stored in memory as a database. In this example, a reference melting temperature corresponds to each genotype. Multiple mutant forms may be defined for a single gene. In this example, the reference melting temperature is specified as a value representing a single temperature, but it may also 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 to prevent evaporation of the DNA solution divided into wells during the nucleic acid amplification reaction and melting curve analysis. The oil is preferably insoluble or poorly soluble in the PCR reaction solution and chemically inert, and is also preferably stable against temperature changes at high temperatures such as those encountered in 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 longer carbon chains reduce volatility. Examples of silicone-based oils include polyphenylmethylsiloxane and trimethylsiloxysilicate. Examples of hydrocarbon-based oils include mineral oil, liquid paraffin, and hexadecane. A surfactant may be added to the oil. The type of surfactant is not particularly limited, but examples include Tween 20, Tween 80, Span 80, and Triton X-100.

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

[0060] First, a nucleic acid amplification reaction is carried out in each well by controlling the temperature of a thermal cycler (S903). DNA is amplified by repeating cycles including denaturation, extension, and annealing steps. If a DNA intercalator is used, it intercalates into the amplified DNA, and if a molecular beacon is used, it hybridizes to the amplified DNA. This increases the fluorescence intensity as the DNA is amplified. Reaction conditions, including the temperature, time, and number of cycles for each step, can be easily determined 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, the temperature is changed by a temperature control device, and the fluorescence intensity, which 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-controlled stage 612 of the DNA detection system. While the temperature of the cartridge 613 is changed by the temperature-controlled 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 photomultiplier 606, or it may be obtained by acquiring a fluorescent image and analyzing the image. Specifically, for example, the computer 702 or another component functions as an imaging control unit and causes the imaging device to capture an image. The obtained fluorescent image is sent to the computer 702, and the analysis unit 703 calculates the fluorescence intensity of each microcompartment from the fluorescent image. The obtained fluorescence intensity data for each microcompartment is stored in memory 705.

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

[0063] Meanwhile, 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). The positions of the positive wells with a set number of peaks in the differential curve of the melting curve or more are identified (S907). If wells with a set number of peaks in the differential curve of the melting curve or more are located close to each other, it is determined that an air bubble is present at that position (S908). Here, information used for bubble detection may include the detection of round air bubbles by image recognition using the entire fluorescent image of the microcompartment. The melting temperature is calculated from the melting curve of each well and stored in memory 705 (S909). The melting temperature calculated based on the fluorescence intensity measurement in this way is called the "measured melting temperature" and is distinguished from a predefined reference melting temperature. The database 704 in memory 705 is referenced, and the type of DNA in the well is identified based on the fluorescence color and the measured melting temperature, and the reference melting temperature is referenced (S910).

[0064] The analysis results, such as a graph of the relationship between fluorescence intensity and melting temperature, including or excluding wells in the area determined to contain bubbles, are displayed (S911), and the user selects whether to include or exclude 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 Figure 10A, marks 1002 indicating the locations where bubbles are estimated to exist may be superimposed on an illustration or fluorescent image of a chip 1001 in a cartridge containing tiny wells. By displaying the locations of microcompartments near the bubbles and the number of compartments on the monitor in this way, this information can be used to control the accuracy of digital PCR. Furthermore, the melting temperature range 1003 of the wild type and the melting temperature range 1004 of the mutant type, as well as plots 1005 of each microcompartment calculated by melting curve analysis, may be superimposed on a graph of fluorescence intensity and melting temperature, and analysis results including wells in the region determined to contain bubbles (Figure 10B) and excluding them (Figure 10C) may be displayed. This allows the user to use both results.

[0067] Figure 11 shows an example of measurement results displayed on the monitor. This example shows the results of measuring wild-type and mutant forms of cancer-related genes A and B as target genes. After selecting either the analysis results including or excluding wells in the region determined to contain bubbles, the number of counted DNAs in the sample solution may be displayed for each type of cancer-related gene and each type of mutation, as shown in Figure 11A. In this case, the total number of genes may be calculated by adding up the wild-type and mutant forms 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 Figure 11B, the percentage of counted DNA in the sample solution may be displayed for each type of cancer-related gene and each type of mutation. In the example of Figure 11B, the percentage 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 plotting the measured values ​​of the specimen solution 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, the results may include a histogram plotting the number of DNA in the specimen solution 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 operations to change these ranges and change the corresponding ranges. In this way, the user can view 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, the specimen solution is treated as a solution in a well or droplet, and therefore 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 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 a fluorescently labeled probe and DNA differs depending on the target gene, allowing discrimination of 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 microcompartment containing target gene P, a fluorescently labeled probe corresponding to target gene P will hybridize to DNA amplified by PCR and emit fluorescence. By analyzing the emitted fluorescence, the melting temperature corresponding to the fluorescently labeled probe for target gene P can be calculated. Also, in the microcompartment containing target gene Q, a fluorescently labeled probe corresponding to target gene Q will hybridize to DNA amplified by PCR and emit fluorescence. By analyzing the emitted fluorescence, the melting temperature corresponding to the fluorescently labeled probe for target gene Q can be calculated. In this way, the presence or absence of target gene P and 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] Because the DNA melting temperature is not affected by PCR reaction efficiency or in-plane measurement variability during fluorescence measurement, it can be used to accurately determine the genotype of DNA within a microcompartment. For example, by determining the sequence of fluorescently labeled probes so that each probe has a different melting temperature (Tm) for the target gene, measuring the change in fluorescence intensity with temperature for the DNA within the microcompartment, performing melting curve analysis, and comparing the melting temperatures, it becomes possible to determine the genotype of the DNA within the microcompartment.

[0075] When multiple target genes are detected simultaneously, they can be distinguished based on the reference melting temperature and the measured melting temperature, but preferably based on the reference melting temperature range. For example, if the measured melting temperature for a given well is within a predetermined range including the reference melting temperature recorded in database 704 for a given target gene (e.g., within ±1°C of the reference melting temperature), it is determined that the target gene is located in that well. Using such a reference melting temperature range allows for more accurate determination by appropriately considering the tolerance range.

[0076] Alternatively, the ratio or difference between the fluorescence intensities at different temperatures may be used as information on the fluorescence intensity. For example, the fluorescence intensity can be normalized by using the ratio or difference between the fluorescence intensity at a temperature lower than the reference melting temperature and the fluorescence intensity at a temperature higher than the reference melting temperature. For example, if this ratio or difference for a certain well is within a predetermined range, the well is determined to be positive; otherwise, the well is determined to be negative.

[0077] 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, ie, the influence 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 can be conducted in advance and the operator can statistically determine the range based on the results, or the DNA detection system can automatically determine the range. Furthermore, the threshold value of fluorescence intensity and the predetermined range of reference melting temperature can be statistically determined using the measurement data of each well in the cartridge each time a digital PCR measurement is performed.

[0079] The data for statistically distinguishing the DNA in the wells 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 temperatures lower than the reference melting temperature to the fluorescence intensity at temperatures 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 representing the reference melting temperature

[0085] -Features that represent 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 invention is a recording medium storing the program. [Example]

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

[0088] First, genomic DNA (final concentration: 133 molecules / μL) of wild-type, G12A, and G13D mutant KRAS genes was prepared. The PCR reaction mixture was prepared by adding the forward primer (final concentration: 0.25 μM), reverse primer (final concentration: 2.0 μM), fluorescently labeled probes corresponding to the wild-type gene (final concentration: 0.5 μM), fluorescently labeled probes corresponding to the G12A mutant gene (final concentration: 0.5 μM), and 1x master mix (containing DNA polymerase and dNTPs). The primer pair concentrations were asymmetric to ensure excessive amplification of the complementary DNA strand of the fluorescently labeled probe. The G13D mutant gene was detected using a fluorescently labeled probe containing a mismatched base corresponding to the wild-type gene. The primer and probe sequences are as follows. Each fluorescently labeled probe contains complementary sequences near both ends, allowing them to form an intramolecular duplex. The 5'-terminus contains HEX as a fluorescent dye, and the 3'-terminus contains BHQ-1 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 mutant type: 5'-TTGGAGCTGCTGGCGT-3' (SEQ ID NO: 4)

[0093] Then, 15 μL of PCR reaction solution was added to each well, so that either one copy of wild-type KRAS DNA, one copy of G12A or G13D mutant KRAS DNA, or neither was added, and the DNA was amplified by PCR. The PCR reaction was performed at 96°C for 10 minutes, followed by 59 cycles of (60°C for 2 minutes, then 98°C for 30 seconds), and finally at 60°C for 2 minutes. After the reaction, the chip containing the wells was cooled from 85°C to 50°C on a temperature-controlled stage, and the change in fluorescence intensity in each well was observed, and melting curves were measured and analyzed.

[0094] Figure 12A shows the differential melting curves of each well when measuring a sample containing a mixture of wild-type and G12A and G13D mutant KRAS genes. Each well's differential melting curve had a single peak, and the temperature at the peak was calculated as the melting temperature. Figure 12B plots the fluorescence intensity at 50°C for each well on the horizontal axis and the melting temperature on the vertical axis based on the results of Figure 12A. Excluding negative well 1207, positive wells were divided into three distributions based on differences in melting temperature: population 1201, with a distribution around 69°C, contains wells containing only wild-type KRAS; population 1202, with a distribution around 66°C, contains wells containing only G12A mutant KRAS; and population 1203, with a distribution around 63°C, contains wells containing only G13D mutant KRAS. From these results, the melting temperature ranges 1204 for the wild type, 1205 for the G12A mutant type, and 1206 for the G13D mutant type were set.

[0095] When measuring a sample containing only the wild-type KRAS gene, we observed population 1208, which had a melting temperature outside the wild-type melting temperature range, as shown in Figure 12D. The melting curve for population 1208, which had a melting temperature outside the wild-type melting temperature range, had multiple peaks, as shown in Figure 12C. Mapping the well locations on the fluorescent image of the chip, as shown in Figure 12E, revealed that they were concentrated in a localized area. Confirmation of the fluorescent image revealed that this location coincided with the location of an air bubble. Therefore, by excluding the data from the wells near the air bubble, we were able to identify 16 wells that were erroneously detected as G12A mutant and 1 well that was erroneously detected as G13D mutant.

[0096] In this way, when determining genotypes in digital PCR, bubbles are detected from the number of peaks in the melting curve, and by excluding data from wells near the bubbles, wells for which a different melting temperature was mistakenly calculated can be excluded, 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...Droplet retrieval recess 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 bubbles are estimated 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 variant Melting temperature range of the 1206…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 multiple types of DNA to be detected into multiple minute fractions; a second step of carrying out a nucleic acid amplification reaction in a plurality of 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 accordance with a temperature change; a fourth step of calculating a melting temperature of the DNA to be detected from the measured fluorescence intensities; a fifth step of identifying, as the microcompartment, a microcompartment 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, and the number of peaks in two or more adjacent microcompartments is equal to or greater than the predetermined value; a sixth step of distinguishing the types of the plurality of types of DNA to be detected based on the melting temperatures in the respective microcompartments calculated in the fourth step; 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 type of DNA determined in the sixth step; A DNA detection method comprising:

2. 2. The DNA detection method according to claim 1, wherein the positions of the micro-compartments identified in the fifth step among all of the plurality of micro-compartments are displayed.

3. 2. The DNA detection method 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 microcompartments or the ratio of the number to the total number of the microcompartments 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 types.

6. 6. The DNA detection method according to claim 1, wherein in the first step, the sample 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 contains information about 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 multiple types of DNA to be detected into multiple micro-fractions, and performs a nucleic acid amplification reaction in multiple micro-compartments containing the micro-fractions, and measures the fluorescence intensity from the fluorescently labeled probe or the DNA intercalator in each of the micro-compartments in accordance with a temperature change; the computer acquires information about the fluorescence intensity measured by the fluorescence measurement unit, the analysis unit calculates the melting temperature of the DNA to be detected and stores the calculated temperature in the memory; The analysis unit creating a melting curve from the information about the fluorescence intensity stored in the memory and calculating the number of peaks in the 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, 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 DNA from the melting temperature; The display unit outputs a first analysis result for the type of DNA that includes the identified microcompartment, and a second analysis result that does not include 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 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 types.

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