Detection method

Abstract

To provide a detection method that allows single-stranded nucleic acids and double-stranded nucleic acids to be distinguished and quantified with improved accuracy.SOLUTION: A detection method comprising a step of introducing a target nucleic acid into wells of a device having a plurality of wells such that the amount is one molecule or less per well, a step of sealing the wells, a step of amplifying a signal derived from the target nucleic acid, and a step of detecting a signal emitted from the wells, wherein the target nucleic acid includes a first nucleic acid, a second nucleic acid, and a double-stranded nucleic acid, the double-stranded nucleic acid is sealed in the wells in a double-stranded state in the sealing step, in the signal amplification step a first specific binding substance binds to the first nucleic acid to emit a first signal and a second specific binding substance binds to the second nucleic acid to emit a second signal, the first signal and the second signal are different from each other, and a difference between a melting temperature Tm of the first specific binding substance and the first nucleic acid and a melting temperature Tm of the second specific binding substance and the second nucleic acid is 10°C or less.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a detection method. This application claims priority from Japanese Patent Application No. 2019-222153, filed on December 9, 2019, the contents of which are incorporated herein by reference. [Background technology]

[0002] Quantitative detection of target molecules in biological samples is used for early detection of diseases and prediction of the effectiveness of medication. Quantitative detection of nucleic acids such as DNA and RNA is performed using real-time PCR and other methods.

[0003] In recent years, there has been an increasing need for more accurate detection of target molecules for purposes such as earlier disease detection. As a method for detecting target molecules with high accuracy, for example, Non-Patent Document 1 describes a technology in which an enzyme reaction is carried out in a large number of microcompartments and a fluorescent signal is detected. This method is called digital measurement.

[0004] In digital measurement, the sample solution is divided into an extremely large number of micro-solutions. The signal from each micro-solution is then binarized, and the number of target molecules is measured by determining only whether or not the target molecule is present. Digital measurement can significantly improve detection sensitivity and quantitativeness compared to conventional methods such as real-time PCR.

[0005] In digital PCR, a type of digital measurement, a mixture of PCR reaction reagents and nucleic acids is diluted so that each microdroplet in a microcompartment contains zero or one template nucleic acid. In digital PCR, the volume of each microdroplet is preferably small in order to increase the sensitivity of nucleic acid amplification and to simultaneously amplify nucleic acids in multiple microdroplets. For example, Non-Patent Document 1 discloses a method using micro-sized droplets formed so that each well has a volume of several nanoliters.

[0006] Another digital measurement method is digital Invasive Cleavage Assay (ICA). Patent Document 1 discloses a method in which a DNA sample is amplified by a PCR reaction, the denatured PCR product is introduced into a device with microwells, and the DNA is detected by the Invader method without amplifying the DNA during detection. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2015 / 115635 [Non-patent literature]

[0008] [Non-Patent Document 1] Olmedillas-Lopez S., et al., Current and Emerging Applications of Droplet Digital PCR in Oncology. Mol Diagn Ther. 2017 Oct;21(5):493-510 Summary of the Invention [Problem to be solved by the invention]

[0009] FIG. 1 is a schematic diagram showing a method for detecting a target nucleic acid by digital PCR, a conventional detection method. When detecting a target nucleic acid by digital PCR, the target nucleic acid is amplified in the process of detecting the target nucleic acid. Therefore, as illustrated in FIG. 1, the target nucleic acid is contained in a microcompartment without undergoing an amplification process. The target nucleic acid, which is DNA or RNA purified from a biological sample such as blood or cells, includes a single-stranded nucleic acid (referred to as single-stranded nucleic acid 1), a single-stranded nucleic acid complementary to single-stranded nucleic acid 1 (referred to as single-stranded nucleic acid 2), and a double-stranded nucleic acid in which single-stranded nucleic acid 1 and single-stranded nucleic acid 2 are complementary bonded (referred to as double-stranded nucleic acid 3).

[0010] For example, as illustrated in FIG. 1 , a sample solution before being formed into droplets contains two molecules of single-stranded nucleic acid 1, two molecules of single-stranded nucleic acid 2, and four molecules of double-stranded nucleic acid 3. For example, when detecting a target nucleic acid using digital PCR, a conventional detection method, the single-stranded and double-stranded nucleic acids are confined in droplets, for example, droplets 4, 5, and 6, without distinction. The PCR products of the target nucleic acid in droplets 4, 5, and 6 are almost identical. In other words, as illustrated in FIG. 1 , eight detection signals are detected in digital PCR, as in droplet 7. From this detection result, it is not possible to distinguish whether the sample solution contained, for example, eight molecules of single-stranded nucleic acid 1, eight molecules of single-stranded nucleic acid 2, or eight molecules of double-stranded nucleic acid 3. In other words, digital PCR cannot distinguish and quantify single-stranded nucleic acid 1, single-stranded nucleic acid 2, and double-stranded nucleic acid 3 in the solution.

[0011] The present invention has been made in view of the above-mentioned circumstances, and aims to provide a technique that can distinguish and quantify single-stranded nucleic acids and double-stranded nucleic acids with higher accuracy. [Means for solving the problem]

[0012] The present invention includes the following aspects. [1] A method for detecting nucleic acids comprising the steps of: contacting a liquid containing a target nucleic acid with a device having a well array with a plurality of wells; introducing the target nucleic acid into the wells so that one molecule of the target nucleic acid is introduced into each well; sealing the wells so that the target nucleic acid does not move between the wells; amplifying a signal caused by the target nucleic acid in the well; and detecting the signal emitted from the well, wherein the target nucleic acid is a first nucleic acid, a second nucleic acid that is a complementary strand of the first nucleic acid, and a signal generated by complementarily binding to the first nucleic acid and the second nucleic acid. a double-stranded nucleic acid formed by binding to the first nucleic acid and emitting a double-stranded nucleic acid; in the sealing step, the double-stranded nucleic acid is sealed in the well in a double-stranded state; in the signal amplifying step, a first specific binding substance binds to the first nucleic acid to emit a first signal, and a second specific binding substance binds to the second nucleic acid to emit a second signal, the first signal and the second signal being different from each other; and the difference between the melting temperature Tm of the first specific binding substance to the first nucleic acid and the melting temperature Tm of the second specific binding substance to the second nucleic acid is 10°C or less. [2] The detection method according to [1], wherein the first signal and the second signal are luminescence signals, and the wavelength of the first signal is different from the wavelength of the second signal. [3] The detection method according to [1] or [2], wherein the step of amplifying the signal is carried out by an invasive cleavage assay. [4] The detection method according to any one of [1] to [3], wherein in the detecting step, a well in which only the first signal is detected is determined to have contained only the single-stranded first nucleic acid, a well in which only the second signal is detected is determined to have contained only the single-stranded second nucleic acid, and a well in which both the first signal and the second signal are detected is determined to have contained the double-stranded nucleic acid. [5] The detection method according to any one of [1] to [4], further comprising the step of counting the number of wells in which the signal is detected in the detecting step. [6] The detection method described in [5], wherein in the counting step, the number of wells in which only the first signal is detected, the number of wells in which only the second signal is detected, and the number of wells in which both the first signal and the second signal are detected are counted. [7] The detection method described in [6], wherein the ratio of the number of double-stranded nucleic acids to the total number of single-stranded first nucleic acids and double-stranded nucleic acids in the liquid is calculated from the number of wells in which only the first signal is detected and the number of wells in which both the first signal and the second signal are detected. [8] The detection method according to any one of [1] to [7], further comprising a step of denaturing the double-stranded nucleic acid after the sealing step.

[0013] Another aspect of the present invention includes the following embodiment.

[10] A method for detecting a target nucleic acid in a liquid, comprising: an introduction step of contacting the liquid with a device having a well array with a plurality of wells and introducing the target nucleic acid into the wells so that there is one molecule or less per well; a sealing step of sealing the wells to prevent the target nucleic acid from moving between the plurality of wells; a signal amplification step of amplifying a signal resulting from the target nucleic acid in the well; and a detection step of detecting a signal emitted from the well, wherein the target nucleic acid comprises a first nucleic acid and a second nucleic acid that is a complementary strand of the first nucleic acid, and in the signal amplification step, a first specific binding substance binds to the first nucleic acid to emit a first signal, and a second specific binding substance binds to the second nucleic acid to emit a second signal, and the first signal and the second signal are different.

[11] The detection method according to

[10] , wherein the first signal and the second signal are luminescence signals, and the wavelength of the first signal is different from the wavelength of the second signal.

[12] The detection method according to

[10] or

[11] , wherein the signal amplification step is carried out by an invasive cleavage assay.

[13] The detection method according to any one of

[10] to

[12] , wherein in the detection step, a well in which only the first signal is detected is determined to have contained only the single-stranded first nucleic acid, a well in which only the second signal is detected is determined to have contained only the single-stranded second nucleic acid, and a well in which both the first signal and the second signal are detected is determined to have contained a double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound.

[14] The detection method according to any one of

[10] to

[13] , further comprising a counting step of counting the number of wells in which the signal is detected in the detection step.

[15] The detection method described in

[14] , wherein in the counting step, the number of wells in which only the first signal is detected, the number of wells in which only the second signal is detected, and the number of wells in which both the first signal and the second signal are detected are counted.

[16] The detection method described in

[15] , wherein the proportion of double-stranded strands in which the first nucleic acid and the second nucleic acid are complementarily bound is calculated from the number of wells in which only the first signal is detected and the number of wells in which both the first signal and the second signal are detected, among the single-stranded first nucleic acid and double-stranded strands in which the first nucleic acid and the second nucleic acid are complementarily bound in the liquid.

[17] A method for detecting a target nucleic acid in a liquid, comprising: an introduction step of contacting the liquid with a device having a well array with a plurality of wells and introducing the target nucleic acid into the wells so that there is one molecule or less per well; a sealing step of sealing the wells to prevent the target nucleic acid from moving between the plurality of wells; a signal amplification step of amplifying a signal caused by the target nucleic acid in the well; and a detection step of detecting a signal emitted from the well, wherein the target nucleic acid comprises a first nucleic acid and a second nucleic acid that is a complementary strand of the first nucleic acid, and in the signal amplification step, a first specific binding substance binds to the first nucleic acid to emit a first signal. [Effects of the Invention]

[0014] According to the present invention, it is possible to provide a technique that can distinguish and quantify single-stranded nucleic acids and double-stranded nucleic acids with higher accuracy. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a diagram schematically illustrating a method for detecting a target nucleic acid by digital PCR. [Figure 2] 10A to 10C are schematic cross-sectional views illustrating a method for delivering a reagent solution to a device. [Figure 3] 10A to 10C are schematic cross-sectional views illustrating a method for delivering oil to a device. [Figure 4] FIG. 1 is a schematic cross-sectional view illustrating a method for detecting a signal from within a device. [Figure 5] 1A to 1C are diagrams schematically illustrating a detection method according to the present embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, embodiments of the present invention will be described in detail, with reference to the drawings where necessary. In the drawings, identical or corresponding parts are designated by identical or corresponding reference numerals, and redundant explanations will be omitted. The dimensional ratios in the drawings may be exaggerated for the purpose of explanation, and do not necessarily correspond to the actual dimensional ratios.

[0017] [Detection method] In one embodiment, the present invention provides a detection method comprising the steps of contacting a liquid containing a target nucleic acid with a device having a well array with a plurality of wells and introducing the target nucleic acid into the wells so that there is one molecule or less of the target nucleic acid per well; sealing the wells to prevent the target nucleic acid from moving between the multiple wells; amplifying a signal attributable to the target nucleic acid in the well; and detecting a signal emitted from the well, wherein the target nucleic acid comprises a first nucleic acid, a second nucleic acid that is a complementary strand of the first nucleic acid, and a double-stranded nucleic acid that is a double strand formed by complementarily binding the first nucleic acid and the second nucleic acid; in the sealing step, the double-stranded nucleic acid is sealed in the well in a double-stranded state; and in the signal amplification step, a first specific binding substance binds to the first nucleic acid to emit a first signal, and a second specific binding substance binds to the second nucleic acid to emit a second signal, wherein the first signal and the second signal are different.

[0018] According to the detection method of this embodiment, as described below, by detecting a signal resulting from the target nucleic acid, it is possible to distinguish whether each well contains only the first nucleic acid, only the second nucleic acid, or only a double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementary to each other. As a result, the number of molecules of each of the first nucleic acid, the second nucleic acid, and the double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementary to each other contained in the liquid can be more accurately quantified.

[0019] For example, when the target nucleic acid is derived from a biological sample, accurate quantification of the number of molecules of the first nucleic acid, the second nucleic acid, and the double-stranded nucleic acid may provide new insights into the status of cancer and diseases accompanied by gene mutations.

[0020] 5, the liquid containing the target nucleic acid contains a first nucleic acid 11, a single-stranded second nucleic acid 12 complementary to the first nucleic acid 11, and a double-stranded nucleic acid 13 in which the first nucleic acid 11 and the second nucleic acid 12 are complementarily bound. The sample solution before being formed into droplets contains two molecules of the single-stranded first nucleic acid 11, two molecules of the single-stranded second nucleic acid 12, and four molecules of the double-stranded nucleic acid 13. For example, according to the detection method of this embodiment, the introducing step and sealing step result in, for example, the double-stranded nucleic acid 13 being confined in droplet 14, the second nucleic acid 12 being confined in droplet 15, and the first nucleic acid 11 being confined in droplet 16.

[0021] Subsequently, in the signal amplification step, a first signal is emitted from droplet 18, a second signal is emitted from droplet 19, and both the first and second signals are emitted from droplet 17. Here, since the first and second signals are different, droplet 17, droplet 18, and droplet 19 can be distinguished from each other in the detection step. That is, according to the detection method of this embodiment, the first nucleic acid 11, the second nucleic acid 12, and the double-stranded nucleic acid 13 contained in each droplet can be distinguished from each other. As a result, it can be determined that the liquid containing the target nucleic acid contains two molecules of the first nucleic acid 11, two molecules of the second nucleic acid 12, and four molecules of the double-stranded nucleic acid 13.

[0022] (device) In the detection method according to this embodiment, a device having a well array with a plurality of wells is used. Examples of the device that can be used include, but are not limited to, the following:

[0023] The shape, dimensions, and arrangement of the wells are not particularly limited, but it is preferable to use a well array consisting of wells that can accommodate a liquid containing the target nucleic acid used in the method of the present invention and a certain amount of reagent solution used in the detection step. The wells may be used as is without any treatment, or at least one of an extraction reagent, a detection reagent such as an antibody, and a specific binding substance may be immobilized on the inner wall of the well in advance depending on the purpose. Pretreatment such as covering the well opening with a lipid bilayer membrane may also be performed.

[0024] The device may have a flow path, and a liquid containing dispersed target nucleic acid may be delivered through the flow path. The shape, structure, and capacity of the flow path are not particularly limited, but it is preferable to use a device with a flow path. When such a device is used, the target nucleic acid is introduced into each well of the well array when a liquid containing the target nucleic acid is delivered, and each well is individually sealed when a sealing liquid is inserted into the flow path, forming microdroplets.

[0025] (Example of a device) Figure 2 is a schematic cross-sectional view of a device according to one embodiment of the present invention. As shown in Figure 2, device 100 comprises a substrate 104 and a lid 101. Lid 101 has a convex portion. The convex portion is connected to substrate 104. Lid 101 is provided with a liquid supply port 102 and a liquid waste port 103, each having a hole penetrating through lid 101. Substrate 104 has a plurality of wells 105. A flow path 106 is located between lid 101 and the plurality of wells 105.

[0026] The substrate 104 may be made of a light-transmitting resin. The substrate 104 of this embodiment may be substantially transparent.

[0027] The wells 105 of the substrate 104 are open on the surface of the substrate 104. The shape, dimensions, and arrangement of the wells 105 are not particularly limited. In the example shown in FIG. 2, in the device 100, a plurality of wells 105 of the same shape and size that can contain a reagent solution 107 (a liquid in which a target nucleic acid is dispersed) are formed in the substrate 104. Furthermore, when particles are used in the detection method according to this embodiment, the wells 105 of the same shape and size that can contain one or more particles and can contain a certain amount of reagent solution 107 containing the particles may be formed in the substrate 104.

[0028] In device 100, wells 105 may have a diameter of 100 nm to 30 μm, preferably 1 μm to 15 μm, and more preferably 3 μm to 15 μm. Wells may have a depth of 100 nm to 30 μm, preferably 1 μm to 15 μm, and more preferably 3 μm to 15 μm. For example, wells 105 may have a diameter of about 3 μm and a depth of about 4.5 μm. Wells 105 may be formed in substrate 104 aligned to form a triangular lattice or a square lattice.

[0029] The number of wells in device 100 is preferably 100,000 to 6,000,000, and the total volume of the wells is preferably 0.1 to 10 μL.

[0030] The region of the substrate 104 that includes the multiple wells 105 is a region that is filled with a reagent solution 107 to be analyzed. Inside this region, a flow path 106 is provided between the substrate 104 and the lid member 101.

[0031] The lid material 101 may be welded or glued to the base material 104. For example, the lid material 101 may be formed from a thermoplastic resin such as a cycloolefin polymer or a cycloolefin copolymer.

[0032] The substrate 104 is formed using, for example, a resin. The type of resin is not particularly limited, but it is preferable to use one that is resistant to the reagent and the sealing liquid used to form the droplets. Furthermore, when observing signals using fluorescence, it is preferable to select a resin that transmits light at the detection wavelength and has little autofluorescence. Examples of such resins include cycloolefin polymers, cycloolefin copolymers, silicone, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesins, and amorphous fluororesins. Note that these materials shown as examples of the substrate 104 are merely examples, and the material is not limited to these.

[0033] A plurality of wells 105 may be formed on one surface of the substrate 104 in the thickness direction. Formation methods using resin include injection molding, thermal imprinting, photoimprinting, and the like. When a fluororesin is used, for example, a layer of CYTOP (registered trademark) (Asahi Glass) may be provided on the substrate 104, and minute holes formed in the CYTOP (registered trademark) may serve as the wells 105.

[0034] The lid member 101 is molded to have a convex portion on the surface facing the substrate 104 during assembly. For example, a thermoplastic resin fluid may be molded into a plate shape having a convex portion by using a molding die. In the device 100 shown in FIG. 2, the lid member 101 is formed with a liquid supply port 102 and a waste liquid port 103, but this is not limiting, and at least one of the liquid supply port 102 and the waste liquid port 103 may not be formed.

[0035] After the lid member 101 and the base material 104 are formed as described above, the lid member 101 and the base material 104 are overlapped so that the convex portion of the lid member 101 contacts the surface of the base material 104 on the side where the well 105 opens. Furthermore, the lid member 101 and the base material 104 are welded together by laser welding or the like while overlapped as described above.

[0036] Each step will be described in detail below. (Introduction process) In this step, a liquid containing the target nucleic acid is brought into contact with the device, and the target nucleic acid is introduced into the wells at one molecule or less per well. "Introducing" the target nucleic acid into the wells refers to distributing the target nucleic acid to each well of the well array.

[0037] More specifically, when device 100 is used in this embodiment, as illustrated in Fig. 2, reagent solution 107 (i.e., a liquid containing target nucleic acid) diluted so that one or less molecule of target nucleic acid is contained in each well 105 of device 100 may be fed from liquid feed port 102 of lid member 101 to flow channel 106 between substrate 104 and lid member 101. Reagent solution 107 fed to flow channel 106 between substrate 104 and lid member 101 is contained inside multiple wells 105.

[0038] The target nucleic acid may be dispersed in a liquid. The liquid in which the target nucleic acid is dispersed can be a common liquid used in biochemical analysis performed using the above-mentioned device, and is preferably an aqueous solution. The aqueous solution may contain a surfactant or the like to facilitate sealing the liquid in the well. It may also contain reagents necessary for the steps of extracting the target nucleic acid and detecting the target nucleic acid, which will be described later. For example, when an ICA reaction is used to detect the target nucleic acid, the liquid in which the target nucleic acid is dispersed may contain ICA reaction reagents such as an allele probe, an ICA oligo, flap endonuclease-1 (FEN-1), and a fluorescent substrate.

[0039] The means for introducing the target nucleic acid into the well is not particularly limited, and an appropriate means can be selected depending on the selected target nucleic acid. Alternatively, the introduction efficiency can be improved by using a substance that captures the target nucleic acid (capture substance), binding the capture substance to the target nucleic acid that is difficult to precipitate under its own weight, and then delivering the target nucleic acid, or by immobilizing the capture substance in the well in advance and capturing the delivered target nucleic acid.

[0040] In the introduction step, the target nucleic acid is introduced so that there is one molecule or less per well. In other words, zero or one molecule of target nucleic acid is introduced into one well. "Introducing one molecule or less of target nucleic acid per well" means that all wells are either wells into which one of one molecule of the first nucleic acid, one molecule of the second nucleic acid, and one molecule of double-stranded nucleic acid is introduced, or wells into which none of the first nucleic acid, second nucleic acid, and double-stranded nucleic acid is introduced. This allows detection of target nucleic acids on a single unit (single molecule basis), i.e., digital measurement is possible. Furthermore, it is not necessary to introduce target nucleic acids into all wells of the well array.

[0041] Specific examples of target nucleic acids herein include DNA, RNA, miRNA, mRNA, and artificial nucleic acids. The target nucleic acid may be artificially synthesized or isolated from a biological sample. Examples of biological samples include human cells, blood, lymph, interstitial fluid, body cavity fluid, digestive fluid, sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, milk, and cultured cells.

[0042] The length of the target nucleic acid is not particularly limited, but is preferably 10 to 1000 bases, and more preferably 30 to 300 bases. When the target nucleic acid is cfDNA in blood, it is preferably 100 to 200 bases.

[0043] The target nucleic acid may be a fragment of a nucleic acid strand isolated from a biological sample. The fragmentation of the nucleic acid strand can be carried out using, for example, a DNA fragmentation device (e.g., Covaris, MS Equipment).

[0044] The target nucleic acid may be a nucleic acid sequence known to be associated with a disease. For example, it may be a nucleic acid sequence containing a region known to have a mutation in cancer patients. Specific examples include a partial base sequence of the human EGFR (epidermal growth factor receptor) locus and a partial base sequence of the human VEGF (vascular endothelial growth factor) locus. An example of a mutation occurring in a partial base sequence of the human EGFR (epidermal growth factor receptor) locus is T790M.

[0045] In this embodiment, the target nucleic acid includes a first nucleic acid and a second nucleic acid that is a complementary strand of the first nucleic acid. The entire sequence of the first nucleic acid and the sequence of the second nucleic acid may be complementary, or a portion of the sequence may not be complementary. For example, the sequence of the first nucleic acid and the sequence of the second nucleic acid may have 1% to 20% base mismatches relative to the entire base sequence of the longer strand.

[0046] The liquid containing the target nucleic acid may contain a first nucleic acid in a single-stranded state, a second nucleic acid in a single-stranded state, and a nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound in a double-stranded state.

[0047] In a double-stranded nucleic acid, the two strands may be completely complementary, or a portion of the sequence may not be complementary. For example, in a double-stranded nucleic acid, 1% to 20% of the bases may be mismatched relative to the entire base sequence of the longer strand. Furthermore, the double-stranded nucleic acid may be a duplex of the same kind, selected from DNA, RNA, miRNA, mRNA, and artificial nucleic acids, or may be a duplex of different kinds. Examples of heterogeneous duplexes include a duplex of a DNA strand and an RNA strand, and a duplex of a DNA strand and an artificial nucleic acid strand.

[0048] In this embodiment, "one molecule of a first nucleic acid" refers to one single-stranded first nucleic acid, "one molecule of a second nucleic acid" refers to one single-stranded second nucleic acid, and "one molecule of a double-stranded nucleic acid in which one molecule of a first nucleic acid and one molecule of a second nucleic acid are complementarily bound" refers to one double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound.

[0049] (Sealing process) In this step, the wells are sealed to prevent the target nucleic acid from moving between the wells. The sealing method is not particularly limited, and may be, for example, by forming a layer of sealing liquid on top of the liquid introduced into the well, sealing the liquid within the well, and forming microdroplets of the liquid in the well.

[0050] For example, more specifically, when device 100 is used in this embodiment, as shown in Fig. 3, sealing liquid 201, for example, an oil, is fed from liquid feed port 102 of lid member 101 to flow path 106 between substrate 104 and lid member 101 to individually seal multiple wells 105. In the sealing step, sealing liquid 201 replaces reagent liquid 107 that has been fed to flow path 106 between substrate 104 and lid member 101 in the above liquid feeding step and that is not contained in wells 105. As a result, sealing liquid 201 individually seals multiple wells 105, and the wells 105 become independent reaction spaces (microcompartments 202).

[0051] After the sealing step, one or less molecule of the target nucleic acid is present in each well 105. In wells 105 where double-stranded nucleic acids are present as target nucleic acids, the double-stranded nucleic acids are sealed in the wells in a double-stranded state without being dissociated into single strands by denaturation.

[0052] The sealing liquid 201 is a liquid that can form droplets (microdroplets) by individually sealing the liquids (reagent liquids 107) introduced into multiple wells so that they do not mix with each other, and is preferably an oily solution, more preferably an oil. Examples of oil that can be used include fluorine-based oil, silicone-based oil, hydrocarbon-based oil, and mixtures thereof, such as Sigma's "FC-40" product.

[0053] The device used in this embodiment does not need to have a flow path, as long as the wells can be sealed to prevent the target nucleic acid from moving between them. When using such a device, for example, oil may be dropped from above into each well containing a liquid containing the target nucleic acid to seal the liquid in each well.

[0054] Furthermore, the device used in this embodiment may be a microwell plate without a lid and a flow path, as long as it can prevent the target nucleic acid from moving between multiple wells. When using such a device, after introducing a liquid containing the target nucleic acid into the wells, a plate member may be tightly attached from above to seal the well openings.

[0055] Furthermore, when using a microwell plate that does not have a lid and a flow channel, after introducing a liquid containing the target nucleic acid into the wells, excess liquid on the well plate may be removed to prevent the target nucleic acid from moving between multiple wells. For example, excess liquid on the well plate may be removed with a squeegee or by suction with an aspirator.

[0056] After the sealing step and before the signal amplification step, the double-stranded nucleic acids present in the individually sealed wells may be denatured. Denaturation conditions can be appropriately set depending on the heat resistance of the enzyme used in the signal amplification step. The denaturation temperature may be 55°C to 99°C, preferably 70°C to 99°C. The time required to raise the temperature to the denaturation temperature may be 25 seconds to 90 seconds, preferably 30 seconds to 60 seconds. The time required to maintain the denaturation temperature may be 10 seconds to 40 seconds, preferably 30 seconds to 40 seconds.

[0057] The denaturation conditions, such as the denaturation temperature, the time required to raise the temperature to the denaturation temperature, and the time required to maintain the temperature, can be any combination of the above ranges. For example, the double-stranded nucleic acid may be denatured by raising the temperature from room temperature (e.g., 25°C) to the denaturation temperature of 55°C to 99°C over 25 to 90 seconds, and then maintaining the temperature at the denaturation temperature for 10 to 40 seconds.

[0058] In the case where the target nucleic acid is heated in the signal amplification step described below and the double-stranded nucleic acid can be denatured, the double-stranded nucleic acid does not need to be denatured before the signal amplification step.

[0059] (Signal amplification step) In this step, the signal resulting from the target nucleic acid is amplified in the well to a detectable level.

[0060] For example, when device 100 is used in this embodiment, as illustrated in Figure 4, a reaction that amplifies a signal proceeds within well 105, and a micro-solution 301 that emits a signal is sealed within well 105 containing the target nucleic acid.

[0061] The signal to be amplified in this step is not particularly limited, and examples thereof include fluorescence, chemiluminescence, color development, potential change, and pH change.

[0062] The signal amplification reaction may be, for example, a biochemical reaction, more specifically, an enzymatic reaction. For example, the signal amplification reaction is an isothermal reaction in which a device containing a reagent solution containing an enzyme for signal amplification in a well is maintained under a constant temperature condition that allows the desired enzymatic activity to be obtained. The constant temperature condition is, for example, between 60°C and 99°C, preferably about 66°C, for at least 10 minutes, preferably about 15 minutes.

[0063] Specific examples of signal amplification reactions include ICA reactions such as the Invader (registered trademark) method. The ICA reaction is particularly preferred as a signal amplification reaction (see, for example, International Publication No. 2009 / 054474). This is related to the principle of the ICA reaction, in which signal amplification proceeds through two reaction cycles: (1) complementary binding between nucleic acids and (2) enzymatic recognition and cleavage of the triplex structure. In such signal amplification reactions, the effect of reaction cycle inhibition by contaminants other than the target nucleic acid is minimal. Therefore, even when various components other than the target nucleic acid are present in the microcompartment, the ICA reaction can accurately detect the target nucleic acid. For example, when the ICA reaction is used for the signal amplification reaction, the liquid used to introduce the target nucleic acid into the well (the liquid in which the target nucleic acid is dispersed) contains the reaction reagents necessary for the ICA reaction and the target nucleic acid. When the biochemical reaction in the signal amplification step is an ICA reaction, if the target nucleic acid is present in the well, an isothermal enzymatic reaction liberates the fluorescent substance from the quencher, thereby emitting a predetermined fluorescent signal in response to excitation light.

[0064] Alternatively, the target nucleic acid can be detected by binding a substance that binds to the target nucleic acid in a sequence-specific manner (specific binding substance) to the target nucleic acid, and then detecting the bound specific binding substance.

[0065] The specific binding substance can be the same as the specific binding substance for the target nucleic acid described below, such as an antibody, an antibody fragment, a polypeptide, or an aptamer. When the signal amplification step is an ICA reaction, the specific binding substance can be a flap probe, an invasion probe, or the like. The flap probe and invasion probe can be prepared using a known method so that they can recognize the first nucleic acid and the second nucleic acid.

[0066] The difference between the Tm (also referred to as melting temperature) between the first specific binding substance and the first nucleic acid and the Tm between the second specific binding substance and the second nucleic acid is preferably 10° C. or less. When the difference between the Tm between the first specific binding substance and the first nucleic acid and the Tm between the second specific binding substance and the second nucleic acid is 10° C. or less, both the first nucleic acid and the second nucleic acid can be detected in an isothermal reaction such as an ICA reaction.

[0067] In this step, a first signal resulting from the first nucleic acid and a second signal resulting from the second nucleic acid are amplified. The first signal and the second signal are different. More specifically, when the biochemical reaction in the signal amplification step is an ICA reaction, the flap probe and invasive probe for the first nucleic acid bind to the single-stranded first nucleic acid or the first nucleic acid generated by dissociation of a double-stranded nucleic acid. Furthermore, the flap probe and invasive probe for the second nucleic acid bind to the single-stranded second nucleic acid or the second nucleic acid generated by dissociation of a double-stranded nucleic acid.

[0068] The first signal and the second signal may be luminescent signals. When the first signal and the second signal are luminescent signals, the wavelength of the first signal is different from the wavelength of the second signal. More specifically, when the signal amplification step is an ICA reaction, the wavelength of the first signal can be made different from the wavelength of the second signal by using two types of fluorescent substances that emit different fluorescence from each other. In other words, by using a first fluorescent substance for detecting the first nucleic acid and a second fluorescent substance for detecting the second nucleic acid that emits fluorescence at a wavelength different from that of the first fluorescent substance, the first signal and the second signal can each be amplified.

[0069] (Detection process) In this step, the signal amplified in the signal amplification step is detected. A known appropriate method can be selected as the signal detection method depending on the type of signal to be detected. For example, when detecting in bright field, white light is irradiated perpendicularly onto the substrate on which the well array is provided. When detecting a fluorescent signal, excitation light corresponding to the fluorescent substance is irradiated into the well from the bottom side, and the fluorescence emitted by the fluorescent substance is detected. In this step, for example, an image of the entire or part of the well array may be captured and saved, and image processing may be performed using a computer system.

[0070] In this embodiment, as described above in the introduction step, the target nucleic acid contained in each well is either one molecule of the first nucleic acid, one molecule of the second nucleic acid, or one molecule of a double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementary to each other. Also, there may be wells that do not contain a target nucleic acid.

[0071] Therefore, from each well, either only the first signal, only the second signal, both the first and second signals, or no signal is detected.

[0072] In other words, a well in which only the first signal is detected can be determined to have contained only a single-stranded first nucleic acid; a well in which only the second signal is detected can be determined to have contained only a single-stranded second nucleic acid; and a well in which both the first and second signals are detected can be determined to have contained a double-stranded nucleic acid in which the first and second nucleic acids are complementarily bound.

[0073] (Counting process) The detection method according to this embodiment may further include a counting step of counting the number of wells in which a signal is detected.

[0074] More specifically, in the counting step, the number of wells in which only the first signal was detected, the number of wells in which only the second signal was detected, and the number of wells in which both the first signal and the second signal were detected may be counted.

[0075] This allows the number of wells in which only the single-stranded first nucleic acid was present, the number of wells in which only the single-stranded second nucleic acid was present, and the number of wells in which double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid were complementarily bound was present to be calculated.

[0076] Therefore, among the single-stranded first nucleic acid and the double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound in a liquid containing the target nucleic acid dispensed into the well, the proportion of double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound can be calculated.

[0077] In the method for detecting a target nucleic acid in a liquid according to this embodiment, an integrated apparatus may be used that includes a device into which the target nucleic acid is introduced, a light source used in the detection step, and a detector for detecting the signal. This apparatus may further include a processing unit that processes an image of the detected signal and calculates the proportion of the target nucleic acid described above.

[0078] In the method for detecting a target nucleic acid in a liquid according to this embodiment, a system may be used that includes a container that holds a device into which the target nucleic acid is introduced, a light source used in the detection step, and a detection device that detects the detection signal. This system may further include a processing device that processes an image of the detection signal and calculates the proportion of the target nucleic acid.

[0079] In one embodiment, the present invention provides a detection method comprising the steps of contacting a liquid containing a target nucleic acid with a device having a well array with a plurality of wells and introducing the target nucleic acid into the wells at one molecule per well or less; sealing the wells to prevent the target nucleic acid from moving between the plurality of wells; amplifying a signal attributable to the target nucleic acid in the well; and detecting the signal emitted from the well, wherein the target nucleic acid comprises a first nucleic acid, a second nucleic acid that is a complementary strand of the first nucleic acid, and a double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound, and the double-stranded nucleic acid is sealed in the well in a double-stranded state in the sealing step, and a first specific binding substance binds to the first nucleic acid to emit a first signal in the signal amplifying step.

[0080] According to the detection method of this embodiment, as described below in the Examples, by detecting a signal resulting from the first nucleic acid, it is possible to distinguish whether each well contains the first nucleic acid or not. Wells containing the first nucleic acid either contain a single-stranded first nucleic acid or a double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementary to each other. As a result, as described below in the Examples, it is possible to accurately quantify the total number of molecules of the single-stranded first nucleic acid and the double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementary to each other in the liquid.

[0081] Another aspect of the present invention includes the following embodiment.

[18] A detection method comprising the steps of contacting a liquid containing a first nucleic acid, a second nucleic acid that is a complementary strand of the first nucleic acid, and a double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound, as target nucleic acids, with a device having a well array with a plurality of wells, and introducing the target nucleic acid into the wells so that there is one molecule of the target nucleic acid per well or less; sealing the wells to prevent the target nucleic acid from moving between the multiple wells; denaturing the double-stranded nucleic acid after the sealing step; amplifying a signal caused by the target nucleic acid in the well; and detecting the signal emitted from the well, wherein in the sealing step, the double-stranded nucleic acid is sealed in the well in a double-stranded state; and in the signal amplification step, a first specific binding substance binds to the first nucleic acid to emit a first signal, and a second specific binding substance binds to the second nucleic acid to emit a second signal, and the first signal and the second signal are different.

[19] The detection method according to [1], wherein the first signal and the second signal are luminescence signals, and the wavelength of the first signal is different from the wavelength of the second signal.

[20] The detection method according to [1] or [2], wherein the step of amplifying the signal is carried out by an invasive cleavage assay.

[21] The detection method according to any one of [1] to [3], wherein in the detecting step, a well in which only the first signal is detected is determined to have contained only the single-stranded first nucleic acid, a well in which only the second signal is detected is determined to have contained only the single-stranded second nucleic acid, and a well in which both the first signal and the second signal are detected is determined to have contained the double-stranded nucleic acid.

[22] The detection method according to any one of [1] to [4], further comprising the step of counting the number of wells in which the signal is detected in the detecting step.

[23] The detection method described in [5], wherein in the counting step, the number of wells in which only the first signal is detected, the number of wells in which only the second signal is detected, and the number of wells in which both the first signal and the second signal are detected are counted.

[24] The detection method described in [6], wherein the ratio of the number of double-stranded nucleic acids to the total number of single-stranded first nucleic acids and double-stranded nucleic acids in the liquid is calculated from the number of wells in which only the first signal is detected and the number of wells in which both the first signal and the second signal are detected.

[25] The detection method according to any one of [1] to [7], wherein the difference between the melting temperature Tm between the first specific binding substance and the first nucleic acid and the melting temperature Tm between the second specific binding substance and the second nucleic acid is 10°C or less.

[26] The detection method according to any one of

[18] to

[25] , wherein the step of amplifying the signal is an isothermal reaction.

[27] The detection method according to any one of

[18] to

[26] , wherein the denaturing step and the signal amplifying step are carried out simultaneously.

[28] The detection method according to any one of

[18] to

[27] , wherein the denaturing step is carried out at a temperature higher than that of the signal amplifying step.

[29] The detection method according to any one of

[18] to

[28] , wherein the denaturing step is carried out at a temperature higher than that of the signal amplifying step.

[30] The detection method according to any one of

[18] to

[29] , wherein the denaturing step comprises maintaining the temperature at 55°C to 99°C for 10 seconds to 40 seconds. [Example]

[0082] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0083] [Experimental Example 1] (Detection of intracellular nucleic acids by ICA method) Using a region containing a partial nucleotide sequence of the human EGFR (epidermal growth factor receptor) locus as a target nucleic acid, quantitative detection of the target nucleic acid was performed. Here, a partial region of human EGFR is a region known to have mutations in some cancer patients. In this experimental example, the sense strand (SEQ ID NO: 7) of the EGFR locus was used as the target nucleic acid.

[0084] <Preparation of DNA Sample> Genomic DNA was isolated from cultured human cells (HT29) using a DNA extraction kit (AllPrep, QIAGEN), and the isolated DNA was fragmented using a DNA fragmentation device (Covaris, MS Instruments) to obtain a mock specimen of circulating DNA in blood, which was used as the DNA sample of Preparation Example 1.

[0085] To measure the concentration of the fragmented DNA sample, the DNA sample was heat denatured at 95°C for 10 minutes and then rapidly cooled to make it single-stranded. Immediately after rapid cooling, almost all of the DNA sample was in a single-stranded state. Subsequently, the absorbance of the DNA sample after rapid cooling was measured using a ultra-micro spectrometer (NanoDrop, Thermo Fisher), and it was confirmed that the concentration in the single-stranded state was 1.1 fM. That is, the concentration when the DNA sample was all double-stranded nucleic acid was 0.55 fM.

[0086] Here, the concentration of the single-stranded target nucleic acid means the concentration of the total number of sense strands and the number of antisense strands at the EGFR locus. Also, the concentration of the double-stranded target nucleic acid means the concentration of the number of sets consisting of the sense strand and the antisense strand at the EGFR locus. For example, in one diploid cell, if all were in the single-stranded state, there would be 4 target nucleic acids, and if all were in the double-stranded state, there would be 2 target nucleic acids.

[0087] The concentration was calculated by first calculating the mass-volume concentration of DNA from the absorbance measurement of the Nanodrop. Next, the concentration of single-stranded target nucleic acid in the quenched DNA sample was calculated, assuming that a 3 pg DNA sample contained one sense strand of the EGFR locus and one antisense strand of the EGFR locus.

[0088] <Preparation of nucleic acid detection reagents> To detect nucleic acids by ICA reaction, an ICA reaction reagent was prepared as a nucleic acid detection reagent. The ICA reaction reagent in this example contains 0.5 μM Allele Probe 1 (SEQ ID NO: 1) (flap probe), 0.1 μM Invader Oligo 1 (SEQ ID NO: 2) (invasive probe, also referred to as ICA oligo) (FASMAC Corporation), 4 μM FRET Cassette (Alexa488-BHQ) (SEQ ID NO: 3) (Japan Bioservices Co., Ltd.) (fluorescent substrate), 50 mM Tris-HCl (pH 7.9), 20 mM MgCl 2 , and 0.05 mg / mL FEN-1. The concentration of each component in these ICA reaction reagents is the final concentration in the mixture of the ICA reaction reagent and DNA sample according to Experimental Example 1. Allele Probe 1 and Invader Oligo 1 are used in the ICA reaction, and both specifically recognize one nucleotide strand of the sense strand and antisense strand of the EGFR locus.

[0089] <Preparing the device> The device was fabricated by fabricating a substrate with numerous microscopic wells using COP (cycloolefin polymer) and attaching a COP lid. 2 The total volume per well was 0.93 μL. The total number of wells used for the measurement was 1,000,000.

[0090] <Transport of reaction mixture> 8 μL of a solution prepared by mixing the DNA sample of Preparation Example 1 and the ICA reaction reagent was delivered to each well of the device, followed by delivery of 200 μL of FC-40 (Sigma) as a sealing liquid to seal each well.

[0091] This allows one or less molecule of DNA to be sealed in each well. That is, each well contains either only one of the following target nucleic acids: a single-stranded first nucleic acid to which the allele probe 1 binds complementarily, a single-stranded second nucleic acid that is the complementary strand of the first nucleic acid, and a double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound, or no target nucleic acid.

[0092] <Nucleic acid detection reaction> After the reaction mixture was delivered, the device was placed on a hot plate and reacted for 25 minutes at 66°C. This resulted in the recognition of the EGFR gene region by the allele probe and Invader oligo, cleavage of the allele probe by FEN-1, binding of the released allele probe fragment to the FRET cassette, and cleavage of the FRET cassette by FEN-1, resulting in the emission of a fluorescent signal from Alexa488.

[0093] <Fluorescence observation of wells> After heating at 66°C for 25 minutes, fluorescent images of the fluorescent signals obtained by the nucleic acid detection reaction in each well of the device were taken using a 4x objective lens and a GFP fluorescent filter under a fluorescent microscope (BZ-700, KEYENCE). The exposure time was 3000 msec. In wells where target nucleic acids containing EGFR gene regions containing single nucleotide polymorphisms were present, fluorescent signals due to the target nucleic acids could be observed. As a result of fluorescent microscopy observation, 2 Fluorescence was confirmed in 13.7 wells per well.

[0094] <Calculation of concentration> From these results, the concentration of DNA that is complementary to the allele probe and contains a single nucleotide polymorphism can be calculated as follows: The liquid introduced into the wells of the device is a 20-fold diluted solution of the sample measured using the Nanodrop. Concentration=(13.7 pieces / cm 2 ) ÷ (6.02×10 23 ) ÷ 0.93μL / cm 2 × 106 × 20 =49×10 -17 (M) =0.49 (fM) That is, in the DNA sample of Preparation Example 1, the sum of the concentration of the single-stranded first nucleic acid to which Allele Probe 1 binds complementarily and the concentration of the double-stranded first nucleic acid and the second nucleic acid complementarily bound was calculated to be 0.49 fM. Based on the measurement results using Nanodrop, the measurement result was equivalent to the concentration (0.55 fM) of the target nucleic acid contained in the DNA sample of Preparation Example 1.

[0095] [Comparative Example 1] <Detection of intracellular nucleic acids using digital PCR method> The concentration of the DNA sample was measured using ddPCR QX100 and PrimePCR for ddPCR EGFR T790M (Bio-Rad) according to the procedure in the manufacturer's instructions. The DNA sample used was the same as in Experimental Example 1, prepared in Preparation Example 1.

[0096] As in the above-mentioned Experimental Example 1, one or less molecule of DNA was sealed in each well. That is, each well contained either only one target nucleic acid selected from a first nucleic acid, a second nucleic acid that is a single-stranded strand complementary to the first nucleic acid, and a double-stranded strand in which the first nucleic acid and the second nucleic acid are complementarily bound, or no target nucleic acid.

[0097] In digital PCR, signals are detected from all wells, including wells containing only a single-stranded first nucleic acid, wells containing only a single-stranded second nucleic acid that is the complementary strand of the first nucleic acid, and wells containing only a double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound.

[0098] As a result of measurement by digital PCR, the total concentration of the single-stranded first nucleic acid, the single-stranded second nucleic acid, and the double-stranded nucleic acid formed by complementarily bonding the single-stranded first nucleic acid and the second nucleic acid contained in the DNA sample of Preparation Example 1 was calculated to be 1 fM.

[0099] From the results using the nanodrop, it was confirmed that the total concentration of the target nucleic acid, which was almost entirely in single-stranded form, contained in the DNA sample of Preparation Example 1 was 1.1 fM. Also, from the results of Comparative Example 1, the total of the concentration of the single-stranded first nucleic acid, the single-stranded second nucleic acid, and the concentration of the double-stranded form in which the single-stranded first nucleic acid and the second nucleic acid were complementarily bound was 1 fM. From these results, it became clear that most of the target nucleic acid contained in the DNA sample of Preparation Example 1 was in a single-stranded state.

[0100] On the other hand, from the results of Experimental Example 1, it became clear that the concentration of the single-stranded first nucleic acid to which Allele Probe 1 binds complementarily was 0.49 fM. In the DNA sample of Preparation Example 1, it became clear that the concentration of the first nucleic acid was approximately half of the total concentration of the target nucleic acid, most of which was in a single-stranded state.

[0101] [Experimental Example 2] Using the sense strand of the EGFR locus described in Experimental Example 1 and the antisense strand of one EGFR locus as the target nucleic acid, quantitative detection of the target nucleic acid was performed.

[0102] [Preparation of DNA Sample] Genomic DNA was extracted from cultured human cells (HT29) using a DNA extraction kit (AllPrep, QIAGEN). Using the commissioned service (DNA Shearing Service) of MS Instruments, the extracted genomic DNA was fragmented to 150 bp. After evaporating the liquid from 1.3 ml of the fragmented genomic DNA solution using a centrifugal dryer (DNA SpeedVac, Thermo Fisher Scientific), 100 μl of distilled water was added to obtain the DNA sample of Preparation Example 2.

[0103] The absorbance of the DNA sample was measured, and it was confirmed that the concentration in the single-stranded state was 1.0 fM. That is, the concentration when the DNA sample was all double-stranded nucleic acid was 0.5 fM.

[0104] [Preparation of Nucleic Acid Detection Reagent] In order to perform nucleic acid detection by ICA reaction, an ICA reaction reagent was prepared as a nucleic acid detection reagent. The ICA reaction reagent in this example contains 0.5 μM allele probe 1 (SEQ ID NO: 1) (flap probe), 0.1 μM invader oligo 1 (SEQ ID NO: 2) (invasion probe, also referred to as ICA oligo), 0.2 μM allele probe 2 (SEQ ID NO: 4) (flap probe), 5 nM invader oligo 2 (SEQ ID NO: 5) (invasion probe) (all FASMAC Corporation), 4 μM FRET Cassette (Alexa488-BHQ) (SEQ ID NO: 3) (Japan Bioservices Co., Ltd.) (fluorescent substrate), 2 μM FRET Cassette (Redmond Red-Epoch Eclipse Quencher) (SEQ ID NO: 6) (Tsukuba Oligo Service Co., Ltd.) (fluorescent substrate) 50 mM Tris-HCl (pH 7.9), 20 mM MgCl 2 and 0.05 mg / mL FEN-1. The concentrations of each component in these ICA reaction reagents are the final concentrations in the mixture of the ICA reaction reagent and the DNA sample according to Experimental Example 2. Allele Probe 1, Invader Oligo 1, Allele Probe 2, and Invader Oligo 2 are used in the ICA reaction. Allele Probe 1 and Invader Oligo 1 specifically recognize one of the nucleotide strands of the sense strand and the antisense strand of the EGFR locus, and Allele Probe 2 and Invader Oligo 2 specifically recognize the other of the nucleotide strands of the sense strand and the antisense strand of the EGFR locus.

[0105] <Device preparation and delivery of reaction mixture> Using the device described in Experimental Example 1, the reaction mixture solution was transferred in the same manner as in Experimental Example 1.

[0106] <Nucleic acid detection reaction> After the reaction mixture was delivered, the device was placed on a hot plate and reacted for 25 minutes at 66°C. This resulted in the recognition of the EGFR gene region by the allele probe and Invader oligo, cleavage of the allele probe by FEN-1, binding of the released allele probe fragment to the FRET cassette, and cleavage of the FRET cassette by FEN-1, resulting in the emission of fluorescent signals from Alexa488 and Redmond Red.

[0107] <Fluorescence observation of wells> Under the same conditions as in Experimental Example 1, fluorescent images of the fluorescent signals obtained in the nucleic acid detection reaction in each well in the device were taken. As a result, the number of wells in which only fluorescent signals from Alexa488 were detected was 508, the number of wells in which only fluorescent signals from Redmond Red were detected was 429, and the number of wells in which fluorescent signals from both Alexa488 and Redmond Red were detected was 3.

[0108] Wells in which only the fluorescent signal from Alexa488 was detected indicate that only one of the sense strand and antisense strand of the EGFR gene locus was present. Wells in which only the fluorescent signal from Redmond Red was detected indicate that only the other of the sense strand and antisense strand of the EGFR gene locus was present. Wells in which fluorescent signals from both Alexa488 and Redmond Red were detected indicate that double-stranded nucleic acid of the sense strand and antisense strand of the EGFR gene locus was present at the time of sealing in the well.

[0109] Although it is possible that the wells in which the fluorescent signals of both Alexa488 and Redmond Red were detected were accidentally sealed with both the sense strand and the antisense strand of the EGFR locus in one well, considering that the total number of wells in the device was 924,890 and the number of wells in which the fluorescent signals were detected was the above-mentioned value, this possibility is considered to be extremely low. In other words, the wells in which the fluorescent signals of both Alexa488 and Redmond Red were detected are not accidentally sealed with both the sense strand and the antisense strand of the EGFR locus in one well, but are considered to be wells in which double-stranded nucleic acids of the sense strand and the antisense strand of the EGFR locus were present at the time of sealing in the well.

[0110] The results of Experimental Example 2 demonstrated that wells containing a single-stranded first nucleic acid, a single-stranded second nucleic acid, and a double-stranded nucleic acid in which the first and second nucleic acids are complementarily bound can be individually detected by appropriately designing and using a probe for the second nucleic acid in addition to a probe for the first nucleic acid. Based on these results, it was demonstrated that the number of molecules and the concentration can be quantified using the calculation method described in Experimental Example 1. [Industrial Applicability]

[0111] According to the present invention, it is possible to provide a technique that can distinguish and quantify single-stranded nucleic acids and double-stranded nucleic acids with higher accuracy. [Explanation of symbols]

[0112] 1...single-stranded nucleic acid, 2...single-stranded nucleic acid, 3...double-stranded nucleic acid, 4, 5, 6, 7...droplet, 11...first nucleic acid, 12...second nucleic acid, 13...double-stranded nucleic acid, 14, 15, 16, 17, 18, 19...droplet, 100...device, 101...lid material, 102...liquid delivery port, 103...waste port, 104...substrate, 105...well, 106...channel, 107...reagent solution, 108...reagent solution filled in well, 201...sealing solution, 202...microcompartment, 301...signal-emitting microsolution

Claims

1. a step of contacting a liquid containing a target nucleic acid with a device having a well array with a plurality of wells, and introducing the target nucleic acid into the wells so that one molecule or less of the target nucleic acid is introduced into each well; sealing the wells to prevent the target nucleic acid from migrating between the wells; amplifying a signal resulting from the target nucleic acid in the well; detecting the signal emitted from the well; the target nucleic acid comprises a first nucleic acid, a second nucleic acid that is a complementary strand of the first nucleic acid, and a double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound, In the sealing step, the double-stranded nucleic acid is sealed in the well in a double-stranded state, In the step of amplifying the signal, a first specific binding substance binds to the first nucleic acid to emit a first signal, and a second specific binding substance binds to the second nucleic acid to emit a second signal, the first signal and the second signal are different; A detection method, wherein the difference between the melting temperature Tm between the first specific binding substance and the first nucleic acid and the melting temperature Tm between the second specific binding substance and the second nucleic acid is 10°C or less.

2. the first signal and the second signal are luminescent signals; The detection method according to claim 1 , wherein the wavelength of the first signal is different from the wavelength of the second signal.

3. The method of claim 1 or 2, wherein the step of amplifying the signal is carried out by an invasive cleavage assay.

4. In the detecting step, determining that only the single-stranded first nucleic acid was present in the well in which only the first signal was detected; The well in which only the second signal is detected is determined to contain only the single-stranded second nucleic acid; The detection method according to any one of claims 1 to 3, wherein the double-stranded nucleic acid is determined to be present in the well in which both the first signal and the second signal are detected.

5. The detection method according to any one of claims 1 to 4, further comprising the step of counting the number of wells in which the signal is detected in the detecting step.

6. The detection method according to claim 5, wherein in the counting step, the number of wells in which only the first signal is detected, the number of wells in which only the second signal is detected, and the number of wells in which both the first signal and the second signal are detected are counted.

7. 7. The detection method according to claim 6, wherein a ratio of the number of the double-stranded nucleic acids to the total number of the single-stranded first nucleic acid and the double-stranded nucleic acids in the liquid is calculated from the number of wells in which only the first signal is detected and the number of wells in which both the first signal and the second signal are detected.

8. The detection method according to any one of claims 1 to 7, further comprising a step of denaturing the double-stranded nucleic acid after the sealing step.

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