Detection Methods

Abstract

The present invention relates to a detection method, which comprises: a step of introducing a target nucleic acid into a hole of a device in a manner of less than one molecule per hole, wherein the hole array has a large number of holes; a step of sealing the holes in a manner so that the target nucleic acid does not move between the large number of holes, a step of amplifying a signal from the target nucleic acid in the hole, and a step of detecting a signal emitted from the hole; the target nucleic acid contains a first nucleic acid, a second nucleic acid which is a complementary chain of the first nucleic acid, and a double-stranded nucleic acid 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 hole in a double-stranded state; 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 is different from the second signal.

Description

Technical Field

[0001] The present invention relates to a detection method.

[0002] This application claims priority from Japanese Patent Application No. 2019-222153 filed in Japan on December 9, 2019, the contents of which are incorporated herein by reference. Background Art

[0003] By quantitatively detecting target molecules in biological samples, early detection of diseases and prediction of drug administration effects can be performed. Quantification of nucleic acids such as DNA and RNA is performed by real-time PCR and the like.

[0004] In recent years, for the purpose of earlier disease detection, the demand for more accurate detection of target molecules has increased. As a method for detecting target molecules with high accuracy, for example, a technique for detecting fluorescent signals by performing enzyme reactions in a large number of micro-partitions is described in Non-Patent Document 1. This method is called digital measurement.

[0005] In digital measurement, the sample solution is divided into a large number of tiny solutions. Then, the signal from each tiny solution is converted into a binary value, and only the presence or absence of target molecules is determined to measure the number of target molecules. Compared with the previous real-time PCR method, digital measurement can significantly improve the detection sensitivity and quantitativeness.

[0006] In digital PCR, which is a type of digital measurement, a mixture of PCR reaction reagents and nucleic acids is diluted in such a way that the number of nucleic acids serving as templates in one microdroplet in a micropartition is from 0 to 1. In digital PCR, in order to increase the sensitivity of nucleic acid amplification and to simultaneously amplify nucleic acids in a large number of microdroplets, it is preferred that the volume of each microdroplet is small. For example, non-patent document 1 discloses a method using micro-sized droplets formed in such a way that the volume of each well reaches several nanoliters.

[0007] In addition, as another method of digital measurement, there is a digital invasive cleavage assay (ICA). Patent document 1 discloses the following method: a sample containing DNA, i.e., a PCR product obtained by amplifying and denaturing a DNA sample by a PCR reaction, is introduced into a device having micropores, and the DNA is detected by an invader method without amplifying the DNA during detection.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: International Publication No. 2015 / 115635

[0011] Non-patent literature

[0012] Non-patent literature 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

[0013] Problems to be solved by the invention

[0014] Figure 1 Schematically shows a method for detecting a target nucleic acid by a conventional detection method, namely, digital PCR. When a target nucleic acid is detected by digital PCR, the target nucleic acid is amplified in the process of detecting the target nucleic acid. Figure 1 As shown, the target nucleic acid is contained in the micropartition without undergoing the amplification process. The target nucleic acid, i.e., DNA or RNA purified from biological samples such as blood and cells, includes a single-stranded nucleic acid (referred to as single-stranded nucleic acid 1), a single-stranded nucleic acid complementary to the single-stranded nucleic acid 1 (referred to as single-stranded nucleic acid 2), and a double-stranded nucleic acid formed by the complementary bonding of the single-stranded nucleic acid 1 and the single-stranded nucleic acid 2 (referred to as double-stranded nucleic acid 3).

[0015] For example, Figure 1 As shown, the sample solution before being dropletized 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 a conventional detection method, namely digital PCR, the sample solution is closed in a droplet state, such as droplet 4, droplet 5, and droplet 6, without distinguishing between single-stranded and double-stranded nucleic acids. The PCR products of the target nucleic acid in droplet 4, droplet 5, and droplet 6 are substantially the same. That is, Figure 1 As illustrated, in the digital PCR, eight detection signals are detected as in the droplet 7. Based on the detection results, it is impossible to distinguish whether, 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 are present in the sample solution. That is, in the digital PCR method, it is impossible to distinguish and quantify the single-stranded nucleic acid 1, the single-stranded nucleic acid 2, and the double-stranded nucleic acid 3 in the solution.

[0016] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a technique capable of distinguishing and quantifying single-stranded nucleic acids and double-stranded nucleic acids with higher accuracy.

[0017] Means for solving problems

[0018] The present invention includes the following aspects.

[0019] [1] A detection method comprising: bringing a liquid containing a target nucleic acid into contact with a device having a well array having a large number of wells, introducing the target nucleic acid into the wells at a rate of one molecule or less per well, sealing the wells so that the target nucleic acid does not move between the large number of wells, amplifying a signal from the target nucleic acid in the wells, and detecting the signal emitted from the wells; the target nucleic acid comprises a first nucleic acid, a second nucleic acid which is a complementary strand to the first nucleic acid, and a double-stranded nucleic acid 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 wells in a double-stranded state; 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 being different from the second signal.

[0020] [2] The detection method according to [1], wherein the first signal and the second signal are luminescent signals, and the wavelength of the first signal is different from the wavelength of the second signal.

[0021] [3] The detection method according to [1] or [2], wherein the step of amplifying the signal is performed by an invader cleavage reaction.

[0022] [4] The detection method according to any one of [1] to [3], wherein, in the detection step, for the hole in which only the first signal is detected, it is determined that only the first nucleic acid is present in a single-stranded state, for the hole in which only the second signal is detected, it is determined that only the second nucleic acid is present in a single-stranded state, and for the hole in which both the first signal and the second signal are detected, it is determined that the double-stranded nucleic acid is present.

[0023] [5] The detection method according to any one of [1] to [4], further comprising a step of counting the number of the wells in which the signal is detected in the step of performing the detection.

[0024] [6] The detection method according to [5], wherein, in the counting process, the number of the holes in which only the first signal is detected, the number of the holes in which only the second signal is detected, and the number of the holes in which both the first signal and the second signal are detected are counted respectively.

[0025] [7] The detection method according to [6], wherein the ratio of the number of double-stranded nucleic acids in the liquid to the total number of single-stranded first nucleic acids and double-stranded nucleic acids is calculated based on the number of holes in which only the first signal is detected and the number of holes in which both the first signal and the second signal are detected.

[0026] [8] The detection method according to any one of [1] to [7], wherein the difference between the melting temperature Tm of the first specific binding substance and the first nucleic acid and the melting temperature Tm of the second specific binding substance and the second nucleic acid is 10°C or less.

[0027] [9] A detection method comprising: bringing a liquid containing a target nucleic acid into contact with a device having a hole array having a large number of holes, a step of introducing the target nucleic acid into the holes in a manner of one molecule or less per hole, a step of sealing the holes in a manner so that the target nucleic acid does not move between the large number of holes, a step of amplifying a signal from the target nucleic acid in the holes, and a step of detecting a signal emitted from the holes; the target nucleic acid comprises a first nucleic acid, a second nucleic acid which is a complementary chain to the first nucleic acid, and a double-stranded nucleic acid formed by complementarily binding the first nucleic acid to the second nucleic acid; in the sealing step, the double-stranded nucleic acid is sealed in the hole in a double-stranded state; in the signal amplification step, a first specific binding substance binds to the first nucleic acid to emit a first signal.

[0028] Another aspect of the present invention includes the following aspects.

[0029]

[10] A method for detecting a target nucleic acid in a liquid, comprising: an introduction step of bringing the liquid into contact with a device having a hole array, the hole array having a large number of holes, and introducing the target nucleic acid into the holes in a manner of less than one molecule per hole; a sealing step of sealing the holes in a manner that prevents the target nucleic acid from moving between the large number of holes; a signal amplification step of amplifying the signal from the target nucleic acid in the holes; and a detection step of detecting the signal emitted from the holes; the target nucleic acid contains a first nucleic acid and a second nucleic acid that is a complementary chain to the first nucleic acid; 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 being different from the second signal.

[0030]

[11] The detection method according to

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

[0031]

[12] The detection method according to

[10] or

[11] , wherein the signal amplification step is performed by an invader cleavage reaction.

[0032]

[13] The detection method according to any one of

[10] to

[12] , wherein, in the detection step, for the hole in which only the first signal is detected, it is determined that only the first nucleic acid is present in a single-stranded state, for the hole in which only the second signal is detected, it is determined that only the second nucleic acid is present in a single-stranded state, and for the hole in which both the first signal and the second signal are detected, it is determined that a double strand formed by complementarity between the first nucleic acid and the second nucleic acid is present.

[0033]

[14] The detection method according to any one of

[10] to

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

[0034]

[15] The detection method according to

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

[0035]

[16] The detection method according to

[15] , wherein the ratio of the double strand formed by the complementarity binding of the first nucleic acid and the second nucleic acid in the liquid to the single-stranded first nucleic acid and the double strand formed by the complementarity binding of the first nucleic acid and the second nucleic acid is calculated based on the number of the holes in which only the first signal is detected and the number of the holes in which both the first signal and the second signal are detected.

[0036]

[17] A method for detecting a target nucleic acid in a liquid, comprising: an introduction step of bringing the liquid into contact with a device having a hole array, the hole array having a large number of holes, and introducing the target nucleic acid into the holes in a manner of less than one molecule per hole; a sealing step of sealing the holes in a manner that prevents the target nucleic acid from moving between the large number of holes; a signal amplification step of amplifying the signal from the target nucleic acid in the holes; and a detection step of detecting the signal emitted from the holes; the target nucleic acid comprises a first nucleic acid and a second nucleic acid which is a complementary chain to the first nucleic acid; in the signal amplification step, a first specific binding substance binds to the first nucleic acid to emit a first signal.

[0037] Effects of the Invention

[0038] According to the present invention, it is possible to provide a technique capable of distinguishing and quantifying single-stranded nucleic acids and double-stranded nucleic acids with higher accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a diagram schematically showing a method for detecting a target nucleic acid by digital PCR.

[0040] Figure 2 It is a schematic cross-sectional view for explaining a method of injecting a reagent solution into a device.

[0041] Figure 3 It is a schematic cross-sectional view for explaining a method of injecting oil into a device.

[0042] Figure 4 Schematic cross-sectional view for explaining a method of detecting a signal from within a device.

[0043] Figure 5 It is a diagram schematically showing the detection method according to the present embodiment. DETAILED DESCRIPTION

[0044] Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings as appropriate. In the accompanying drawings, the same or corresponding reference numerals are used to mark the same or corresponding parts, and repeated descriptions are omitted. It should be noted that the dimensional ratios in the drawings are exaggerated for the purpose of explanation and may not be consistent with the actual dimensional ratios.

[0045] [Detection method]

[0046] In one embodiment, the present invention provides a detection method, which comprises: bringing a liquid containing a target nucleic acid into contact with a device having a hole array, wherein the hole array has a large number of holes, introducing the target nucleic acid into the holes in a manner of less than one molecule per hole, sealing the holes in a manner so that the target nucleic acid does not move between the large number of holes, amplifying the signal from the target nucleic acid in the holes, and detecting the signal emitted from the holes; the target nucleic acid contains a first nucleic acid, a second nucleic acid which is a complementary chain of the first nucleic acid, and a double-stranded nucleic acid which is a double-stranded chain 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 hole in a double-stranded state, in the signal amplification step, the first specific binding substance binds to the first nucleic acid to emit a first signal, the second specific binding substance binds to the second nucleic acid to emit a second signal, and the first signal is different from the second signal.

[0047] According to the detection method of this embodiment, as described later, by detecting the signal 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 the double-stranded nucleic acid formed by the complementarity of the first nucleic acid and the second nucleic acid. As a result, the number of molecules of the first nucleic acid, the second nucleic acid, and the double-stranded nucleic acid formed by the complementarity of the first nucleic acid and the second nucleic acid contained in the liquid can be quantified more accurately.

[0048] For example, when the target nucleic acid is derived from a biological sample, accurate quantification of the number of molecules of each of the first nucleic acid, the second nucleic acid, and the double-stranded nucleic acid may provide new insights into the state of cancer and diseases involving gene mutations.

[0049] More specifically, if Figure 5 As shown, the target nucleic acid-containing liquid 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 formed by the complementarity between the first nucleic acid 11 and the second nucleic acid 12. The sample solution before being dropletized 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, through the introduction step and the blocking step, the double-stranded nucleic acid 13 is blocked in the droplet 14, the second nucleic acid 12 is blocked in the droplet 15, and the first nucleic acid 11 is blocked in the droplet 16.

[0050] Next, in the signal amplification step, a first signal is emitted from the droplet 18, a second signal is emitted from the droplet 19, and the first signal and the second signal are emitted simultaneously from the droplet 17. Here, since the first signal is different from the second signal, the droplets 17, 18, and 19 can be distinguished 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. As a result, it can be determined that 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 are contained in the liquid containing the target nucleic acid.

[0051] (Device)

[0052] In the detection method of this embodiment, a device having a hole array having a large number of holes is used. As the device, for example, the following devices can be used, but the device is not limited thereto.

[0053] The shape, size and arrangement of the wells are not particularly limited, but it is preferred to use a well array consisting of wells that can accommodate the 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 can be used directly without treatment, or at least one of an extraction reagent, a detection reagent such as an antibody, and a specific binding substance can be fixed in advance on the inner wall of the well according to the purpose. In addition, pretreatment such as covering the well opening with a lipid bilayer membrane can also be performed.

[0054] The device may have a flow path, or a liquid dispersed with the target nucleic acid may be infused through the flow path. The shape, structure and capacity of the flow path are not particularly limited, and a device having a flow path is preferably used. If such a device is used, the target nucleic acid can be introduced into each hole of the hole array when the liquid containing the target nucleic acid is infused, and each hole is respectively sealed to form a micro droplet when the sealing liquid is inserted into the flow path.

[0055] (An example of a device)

[0056] Figure 2Schematic cross-sectional view of a device in one embodiment of the present invention. Figure 2 As shown, the device 100 includes a substrate 104 and a cover material 101. The cover material 101 has a convex portion. The convex portion is connected to the substrate 104. The cover material 101 is provided with an infusion port 102 and a waste liquid port 103 having a hole penetrating the cover material 101. The substrate 104 has a large number of holes 105. The flow path 106 is located between the cover material 101 and the large number of holes 105.

[0057] The substrate 104 may be formed of a light-transmitting resin. The substrate 104 of this embodiment may be substantially transparent.

[0058] The holes 105 of the substrate 104 are open on the surface of the substrate 104. The shape, size and arrangement of the holes 105 are not particularly limited. Figure 2 In the example shown, in the device 100, a large number of holes 105 of the same shape and size capable of accommodating a reagent solution 107 (a liquid in which a target nucleic acid is dispersed) are formed on a substrate 104. In addition, when particles are used in the detection method of this embodiment, holes 105 of the same shape and size capable of accommodating one or more particles and capable of accommodating a certain amount of a reagent solution 107 containing particles may be formed on the substrate 104.

[0059] In the device 100, the diameter of the hole 105 can be 100nm to 30μm, preferably 1μm to 15μm, and more preferably 3μm to 15μm. The depth of the hole can be 100nm to 30μm, preferably 1μm to 15μm, and more preferably 3μm to 15μm. As an example, the diameter of the hole can be about 3μm, and the depth of the hole 105 can be, for example, about 4.5μm. In addition, the holes 105 can also be arranged in a triangular lattice or a square lattice and formed on the substrate 104.

[0060] The number of wells included in the device 100 is preferably 100,000 to 6,000,000. The total volume of the wells is preferably 0.1 to 10 μL.

[0061] The region of the substrate 104 including a large number of holes 105 is a region filled with a reagent solution 107 to be analyzed. Inside this region, a flow path 106 is provided between the substrate 104 and the cover member 101 .

[0062] The cover material 101 may be melt-bonded or adhesively bonded to the base material 104. For example, the cover material 101 may be formed of a thermoplastic resin such as a cycloolefin polymer or a cycloolefin copolymer.

[0063] The substrate 104 is formed, for example, using a resin. The type of resin is not particularly limited, and preferably a resin that is tolerant to the confining liquid when forming reagents and droplets is used. In addition, when the signal is observed by fluorescence, it is preferred to select a resin that transmits light of the detection wavelength and has little autofluorescence. For example, cycloolefin polymers, cycloolefin copolymers, silicones, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesins, and amorphous fluororesins can be cited. It should be noted that these materials shown as examples of the substrate 104 are only examples, and the materials are not limited thereto.

[0064] A large number of holes 105 may be formed on one surface of the substrate 104 in the thickness direction. As a forming method using a resin, in addition to injection molding, hot stamping, photo stamping, etc. may be mentioned. In addition, when a fluororesin is used, for example, a layer of CYTOP (registered trademark) (Asahi Glass) may be provided on the substrate 104, and the microscopic holes formed in CYTOP (registered trademark) may become the holes 105.

[0065] The cover material 101 is molded so that the surface facing the base material 104 during assembly has a convex portion. For example, a thermoplastic resin fluid may be molded into a plate-like shape having a convex portion by using a molding die. Figure 2 In the device 100 shown, the infusion port 102 and the waste liquid port 103 are formed in the cover member 101 , but the present invention is not limited thereto, and at least one of the infusion port 102 and the waste liquid port 103 may not be formed.

[0066] After the lid material 101 and the base material 104 are formed as described above, the lid material 101 and the base material 104 are overlapped in such a manner that the convex portion of the lid material 101 contacts the surface on the side where the hole 105 in the base material 104 opens. Furthermore, in the state where the lid material 101 and the base material 104 are overlapped as described above, they are melt-bonded by laser melting bonding or the like.

[0067] Hereinafter, each step will be described in detail.

[0068] (Introduction process)

[0069] In this step, a liquid containing a target nucleic acid is brought into contact with the device, and the target nucleic acid is introduced into the wells in a manner of one molecule or less per well. "Introducing" the target nucleic acid into the wells means distributing the target nucleic acid to each well of the well array.

[0070] More specifically, in the case of using the device 100 in this embodiment, as Figure 2As illustrated, the reagent solution 107 (i.e., liquid containing the target nucleic acid) diluted in such a manner that less than one molecule of the target nucleic acid per hole enters the hole 105 of the device 100 can be infused from the infusion port 102 of the cover material 101 to the flow path 106 between the substrate 104 and the cover material 101. The reagent solution 107 infused into the flow path 106 between the substrate 104 and the cover material 101 is contained inside a large number of holes 105.

[0071] The target nucleic acid can also be dispersed in a liquid. As the liquid for dispersing the target nucleic acid, a common liquid used in the biochemical analysis performed using the above-mentioned device can be used, preferably an aqueous solution. In order to facilitate the sealing of the liquid in the hole, the aqueous solution can also contain a surfactant, etc. In addition, it can also contain reagents required in the process of extracting the target nucleic acid and the process of detecting the target nucleic acid described later. For example, when the detection of the target nucleic acid uses an ICA reaction, the liquid for dispersing the target nucleic acid can contain ICA reaction reagents such as an allele probe, an ICA oligonucleotide, a Flap endonuclease-1 (FEN-1) and a fluorescent substrate.

[0072] The means for introducing the target nucleic acid into the hole is not particularly limited, and an appropriate means corresponding to the selected target nucleic acid can be selected. Alternatively, a substance (capture) capable of capturing the target nucleic acid is used to bind the capture to the target nucleic acid that is difficult to settle by its own weight and then infuse the capture, or the capture is fixed in the hole in advance to capture the infused target nucleic acid, thereby also being able to improve the introduction efficiency.

[0073] In the introduction process, the target nucleic acid is introduced in a manner of less than 1 molecule per hole. That is, 0 molecules or 1 molecule of target nucleic acid is introduced into 1 hole. "Introducing the target nucleic acid in a manner of less than 1 molecule per hole" means making all holes into a hole into which 1 molecule of the first nucleic acid, 1 molecule of the second nucleic acid and 1 molecule of the double-stranded nucleic acid are introduced, and any state in the hole into which none of the first nucleic acid, the second nucleic acid and the double-stranded nucleic acid is introduced. Thus, the detection of the target nucleic acid can be performed in 1 unit (1 molecule unit), that is, digital measurement can be performed. In addition, it is not necessary to introduce the target nucleic acid into all holes of the hole array.

[0074] In this specification, specific examples of target nucleic acids include DNA, RNA, miRNA, mRNA, and artificial nucleic acids. The target nucleic acid may be an artificially synthesized nucleic acid or a nucleic acid separated 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, breast milk, and cultured cells.

[0075] The length of the target nucleic acid is not particularly limited, but is preferably 10 to 1000 bases, more preferably 30 to 300 bases. When the target nucleic acid is cfDNA in blood, it is preferably 100 to 200 bases.

[0076] The target nucleic acid may be a nucleic acid obtained by fragmenting a nucleic acid chain separated from a biological sample. The nucleic acid chain can be fragmented using, for example, a DNA fragmentation device (eg, Covaris, M&S Instruments).

[0077] The target nucleic acid may also be a nucleic acid sequence known to be associated with a disease. For example, it may also be a nucleic acid sequence containing a region known to have a mutation in a cancer patient. Specifically, a portion of the base sequence of the human EGFR (epidermal growth factor receptor) gene locus, a portion of the base sequence of the human VEGF (vascular endothelial growth factor) gene locus, etc. may be cited. As an example of a mutation in a portion of the base sequence of the human EGFR (epidermal growth factor receptor) gene locus, T790M may be cited.

[0078] In this embodiment, the target nucleic acid contains a first nucleic acid and a second nucleic acid that is a complementary strand to the first nucleic acid. The sequence of the first nucleic acid and the sequence of the second nucleic acid may be complementary in all sequences or may be partially non-complementary in sequences. For example, in the sequence of the first nucleic acid and the sequence of the second nucleic acid, 1% to 20% of the bases relative to the entire base sequence of the longer strand may be mismatched.

[0079] The liquid containing the target nucleic acid may contain a single-stranded first nucleic acid, a single-stranded second nucleic acid, and a double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound to each other.

[0080] Double-stranded nucleic acids may be completely complementary to each other, or may have a part of the sequence not complementary. For example, in a double-stranded nucleic acid, 1% to 20% of the bases relative to the entire base sequence of the longer chain may be mismatched. In addition, double-stranded nucleic acids may be double-stranded of the same type among DNA, RNA, miRNA, mRNA and artificial nucleic acids, or double-stranded of different types. Examples of different types of double-stranded nucleic acids include double-stranded DNA chains and RNA chains, and double-stranded DNA chains and artificial nucleic acid chains.

[0081] In this embodiment, "one molecule of the first nucleic acid" refers to one single-stranded first nucleic acid, and "one molecule of the second nucleic acid" refers to one single-stranded second nucleic acid. In addition, "a double-stranded nucleic acid formed by complementarily binding one molecule of the first nucleic acid and the second nucleic acid" refers to one double-stranded nucleic acid formed by complementarily binding one molecule of the first nucleic acid and the second nucleic acid.

[0082] (Sealing process)

[0083] In this step, the wells are sealed in such a way that the target nucleic acid does not move between a large number of wells. The means for sealing is not particularly limited, and for example, a layer of sealing liquid can be formed on the upper layer of the liquid introduced into the wells, the liquid is sealed in the wells, and tiny droplets of the liquid are formed in the wells.

[0084] For example, more specifically, in the case of using the device 100 in this embodiment, as Figure 3 As shown, a sealing liquid 201, such as oil, is injected from the injection port 102 of the cover material 101 to the flow path 106 of the substrate 104 and the cover material 101 to seal a large number of holes 105. In the sealing step, the sealing liquid 201 replaces the reagent solution 107 that is not contained in the hole 105 in the reagent solution 107 injected into the flow path 106 of the substrate 104 and the cover material 101 in the above-mentioned injection step. Thus, the sealing liquid 201 seals a large number of holes 105 respectively, and the holes 105 become independent reaction spaces (micro-partitions 202).

[0085] After the blocking step, one or less target nucleic acid molecules exist in each well 105. In the well 105 where double-stranded nucleic acid exists as the target nucleic acid, the double-stranded nucleic acid is not dissociated into single strands by denaturation but is blocked in the well in a double-stranded state.

[0086] The above-mentioned sealing liquid 201 is a liquid that can be introduced into a large number of holes in a manner that the liquid (reagent solution 107) is not mixed with each other and is sealed separately to form droplets (microdroplets). It is preferably an oily solution, and more preferably oil. As the oil, fluorine oil, silicone oil, hydrocarbon oil, or a mixture thereof can be used, for example, the trade name "FC-40" made by Sigma can be used.

[0087] As long as the holes can be sealed in a manner that the target nucleic acid does not move between a large number of holes, the device used in this embodiment may not have a flow path. When such a device is used, for example, oil may be dripped from above into each hole containing a liquid containing the target nucleic acid to seal the liquid in each hole.

[0088] In addition, the device used in the present embodiment can be a microplate without a cover material and a flow path as long as the target nucleic acid is not moved between a large number of holes. When using such a device, after the liquid containing the target nucleic acid is introduced into the hole, the plate components can be tightly fitted from above and the hole opening can be closed.

[0089] In addition, when using a microplate without a cover material and a flow path, in order to prevent the target nucleic acid from moving between a large number of wells, the remaining liquid on the well plate can be removed after the liquid containing the target nucleic acid is introduced into the well. For example, the remaining liquid on the well plate can be removed with a scraper or by suction with an aspirator.

[0090] It is also possible to denature the double-stranded nucleic acid present in the respectively sealed holes after the sealing step and before the signal amplification step. The denaturation conditions can be appropriately set according to the heat resistance of the enzyme used in the signal amplification step. The denaturation temperature can be 55°C to 99°C, preferably 70°C to 99°C. The heating time to reach the denaturation temperature can be 25 seconds to 90 seconds, preferably 30 seconds to 60 seconds. The holding time at the denaturation temperature can be 10 seconds to 40 seconds, preferably 30 seconds to 40 seconds.

[0091] The denaturation temperature, the heating time to reach the denaturation temperature and the holding time under the denaturation conditions may be any combination of the above ranges. For example, the double-stranded nucleic acid may be denatured by heating from room temperature (e.g., 25° C.) to a denaturation temperature of 55° C. to 99° C. in 25 to 90 seconds and holding at the denaturation temperature for 10 to 40 seconds.

[0092] When the target nucleic acid is heated in the signal amplification step described later to allow denaturation of the double-stranded nucleic acid, denaturation of the double-stranded nucleic acid may not be performed before the signal amplification step.

[0093] (Signal Amplification Process)

[0094] In this step, the signal from the target nucleic acid is amplified in the well to a detectable level.

[0095] For example, in the case of using the device 100 in this embodiment, Figure 4 As illustrated, a reaction that amplifies the signal proceeds in the well 105 , and a minute solution 301 that generates the signal is sealed in the well 105 containing the target nucleic acid.

[0096] The signal amplified in this step is not particularly limited, and examples thereof include fluorescence, chemiluminescence, color development, potential change, and pH change.

[0097] The signal amplification reaction may be, for example, a biochemical reaction, more specifically, an enzyme reaction. As an example, the signal amplification reaction is an isothermal reaction in which a device containing a reagent solution containing an enzyme for signal amplification is maintained in a hole under a certain temperature condition that can obtain the desired enzyme activity. The certain temperature condition refers to, for example, maintaining at least 60°C and below 99°C, preferably at about 66°C, for at least 10 minutes, preferably about 15 minutes.

[0098] Specific examples of the signal amplification reaction include ICA reactions such as the Invader (registered trademark) method.

[0099] As a signal amplification reaction, the ICA reaction is particularly preferably used (for example, refer to International Publication No. 2009 / 054474). This is related to the principle of the ICA reaction, that is, the signal is amplified by a cycle of two reactions: (1) complementary binding of nucleic acids to each other and (2) recognition and shearing of a triple-chain structure by an enzyme. In such a signal amplification reaction, the influence of the reaction cycle obstruction caused by inclusions other than the target nucleic acid is small. Therefore, even in the case where various components other than the target nucleic acid are present in the micro-partition, the target nucleic acid can be detected with high precision by using the ICA reaction. For example, when the ICA reaction is used for the signal amplification reaction, the liquid used to introduce the target nucleic acid into the hole (the liquid that disperses the target nucleic acid) contains the reaction reagent and the target nucleic acid required for the ICA reaction. In the case where the biochemical reaction in the signal amplification process is an ICA reaction, through the enzyme reaction performed by the isothermal reaction, when the target nucleic acid is present in the hole, the fluorescent substance is freed from the extinguishing substance, thereby emitting a specific fluorescent signal corresponding to the excitation light.

[0100] Alternatively, the target nucleic acid can be detected by allowing a substance that specifically binds to the target nucleic acid sequence (specific binding substance) to bind to the target nucleic acid and detecting the bound specific binding substance.

[0101] As the specific binding substance, the same substance as the specific binding substance for the target nucleic acid described later can be used, such as an antibody, an antibody fragment, a polypeptide or an aptamer, etc. In the case where the signal amplification step is an ICA reaction, as the specific binding substance, a FLAP probe and an invasion probe, etc. can be used. As the FLAP probe and the invasion probe, a probe prepared by a known method in a manner capable of recognizing the first nucleic acid and the second nucleic acid can be used.

[0102] The difference between the Tm (also called melting temperature) of the first specific binding substance and the first nucleic acid and the Tm of the second specific binding substance and the second nucleic acid is preferably 10° C. or less. If the difference between the Tm of the first specific binding substance and the first nucleic acid and the Tm of 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.

[0103] In this process, the first signal from the first nucleic acid and the second signal from the second nucleic acid are amplified. The first signal is different from the second signal. More specifically, when the biochemical reaction in the signal amplification process is an ICA reaction, the FLAP probe and the invader probe for the first nucleic acid bind to the single-stranded first nucleic acid or the first nucleic acid generated by the dissociation of the double-stranded nucleic acid. In addition, the FLAP probe and the invader probe for the second nucleic acid bind to the single-stranded second nucleic acid or the second nucleic acid generated by the dissociation of the double-stranded nucleic acid.

[0104] 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 process is an ICA reaction, the wavelength of the first signal can be different from the wavelength of the second signal by using two fluorescent substances that emit different fluorescence. That is, when a first fluorescent substance for detecting a first nucleic acid and a second fluorescent substance for detecting a second nucleic acid that emits fluorescence of a different wavelength from the first fluorescent substance are used, the first signal and the second signal can be amplified respectively.

[0105] (Inspection process)

[0106] In this process, the signal amplified in the signal amplification process is detected. The signal detection method can select a known appropriate method according to the type of the detected signal. For example, in the case of detection in a bright field, white light is irradiated in a vertical direction to a substrate provided with a hole array. In the case of detecting a fluorescent signal, for example, an excitation light corresponding to a fluorescent substance is irradiated into the hole from the bottom side of the hole, and the fluorescence emitted by the fluorescent substance is detected. In this process, for example, an image of the entire or a part of the hole array can also be captured and saved, and image processing based on a computer system can be performed.

[0107] In this embodiment, as described above, in the introduction step, the target nucleic acid contained in each well is any one of only one molecule of the first nucleic acid, only one molecule of the second nucleic acid, and only one molecule of double-stranded nucleic acid formed by complementarity between the first nucleic acid and the second nucleic acid. In addition, there may be a well that does not contain a target nucleic acid.

[0108] Therefore, from each well, only the first signal is detected, only the second signal is detected, both the first signal and the second signal are detected, or no signal is detected.

[0109] In other words, for the hole where only the first signal is detected, it can be determined that only the single-stranded first nucleic acid exists, for the hole where only the second signal is detected, it can be determined that only the single-stranded second nucleic acid exists, and for the hole where the first signal and the second signal are detected at the same time, it can be determined that a double-stranded nucleic acid formed by the complementarity of the first signal and the second signal exists.

[0110] (Counting process)

[0111] The detection method of this embodiment may further include a counting step of counting the number of wells in which signals are detected.

[0112] More specifically, 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 may be counted separately.

[0113] Thus, the number of pores containing only the single-stranded first nucleic acid, the number of pores containing only the single-stranded second nucleic acid, and the number of pores containing a double-stranded nucleic acid in which the first nucleic acid and the second nucleic acid are complementarily bound can be calculated.

[0114] Therefore, the ratio of the double-stranded nucleic acid formed by complementarily binding the first nucleic acid and the second nucleic acid in the liquid containing the target nucleic acid dispensed into the well to the single-stranded first nucleic acid and the double-stranded nucleic acid formed by complementarily binding the first nucleic acid and the second nucleic acid can be calculated.

[0115] In the detection method of the target nucleic acid in the liquid of the present embodiment, a device having a device for introducing the target nucleic acid, a light source used in the detection process, and a detector for detecting the signal can be used. The device can also further have an image of the detection signal and a processing unit for calculating the ratio of the above-mentioned target nucleic acid.

[0116] In the detection method of the target nucleic acid in the liquid of the present embodiment, a system comprising a storage device for carrying a device for introducing the target nucleic acid, a light source device used in the detection process, and a detection device for detecting the detection signal can also be used. The system can further include a processing device for processing an image of the detection signal and calculating the ratio of the target nucleic acid.

[0117] In one embodiment, the present invention provides a detection method, comprising: contacting a liquid containing a target nucleic acid with a device having a hole array having a large number of holes, introducing the target nucleic acid into the holes in a manner of less than one molecule per hole, sealing the holes in a manner that the target nucleic acid does not move between the large number of holes, amplifying the signal from the target nucleic acid in the holes, and detecting the signal emitted from the holes; the target nucleic acid contains a first nucleic acid, a second nucleic acid that is a complementary chain to the first nucleic acid, and a double-stranded nucleic acid formed by complementarily binding the first nucleic acid to the second nucleic acid; in the sealing step, the double-stranded nucleic acid is sealed in the hole in a double-stranded state; in the signal amplification step, a first specific binding substance binds to the first nucleic acid to emit a first signal.

[0118] According to the detection method of this embodiment, as described later in the examples, by detecting the signal from the first nucleic acid, it is possible to distinguish whether each well contains the first nucleic acid or does not contain the first nucleic acid. The well containing the first nucleic acid is either a single-stranded first nucleic acid or a double-stranded nucleic acid formed by complementarily binding the first nucleic acid and the second nucleic acid. As a result, as described later in the examples, the total number of molecules of the single-stranded first nucleic acid contained in the liquid and the double-stranded nucleic acid formed by complementarily binding the first nucleic acid and the second nucleic acid can be accurately quantified.

[0119] Another aspect of the present invention includes the following aspects.

[0120]

[18] A detection method comprising: bringing a liquid containing a first nucleic acid, a second nucleic acid as a complementary strand of the first nucleic acid, and a double-stranded nucleic acid formed by complementarily binding the first nucleic acid and the second nucleic acid as target nucleic acids into contact with a device having a well array, the well array having a large number of wells, introducing the target nucleic acid into the wells at a rate of one molecule or less per well, sealing the wells so that the target nucleic acid does not move between the large number of wells, denaturing the double-stranded nucleic acid after the sealing step, amplifying a signal from the target nucleic acid in the wells, and detecting the signal emitted from the wells; in the sealing step, the double-stranded nucleic acid is sealed in the wells in a double-stranded state; 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 being different from the second signal.

[0121]

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

[0122]

[20] The detection method according to [1] or [2], wherein the step of amplifying the signal is performed by an invader cleavage reaction.

[0123]

[21] A detection method according to any one of [1] to [3], wherein, in the detection step, for the hole in which only the first signal is detected, it is determined that only the single-stranded first nucleic acid exists, for the hole in which only the second signal is detected, it is determined that only the single-stranded second nucleic acid exists, and for the hole in which both the first signal and the second signal are detected, it is determined that the double-stranded nucleic acid exists.

[0124]

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

[0125]

[23] The detection method according to [5], wherein, in the counting process, the number of the holes in which only the first signal is detected, the number of the holes in which only the second signal is detected, and the number of the holes in which both the first signal and the second signal are detected are counted respectively.

[0126]

[24] According to the detection method described in [6], wherein, based on 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, the ratio of the number of double-stranded nucleic acids in the liquid to the total number of single-stranded first nucleic acids and double-stranded nucleic acids is calculated.

[0127]

[25] According to the detection method described in any one of [1] to [7], wherein the difference in 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.

[0128]

[26] According to the detection method described in any one of

[18] to

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

[0129]

[27] According to the detection method described in any one of

[18] to

[26] , wherein the denaturation step and the signal amplification step are performed simultaneously.

[0130]

[28] According to the detection method described in any one of

[18] to

[27] , wherein the denaturation step is performed at a temperature higher than the signal amplification step.

[0131]

[29] According to the detection method described in any one of

[18] to

[28] , wherein the denaturation step is performed at a temperature higher than the signal amplification step.

[0132]

[30] According to the detection method described in any one of

[18] to

[29] , wherein the denaturation step includes holding at 55°C to 99°C for 10 seconds to 40 seconds.

[0133] Examples

[0134] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[0135] [Experimental Example 1]

[0136] (Detection of intracellular nucleic acids based on the ICA method)

[0137] A region containing a partial base sequence of the human EGFR (epidermal growth factor receptor) gene locus was used as the target nucleic acid, and quantitative detection of the target nucleic acid was performed. Here, a partial region of human EGFR is known to have mutations in some cancer patients. In this experimental example, the sense strand (SEQ ID NO: 7) of the EGFR gene locus was used as the target nucleic acid.

[0138] <Preparation of DNA sample>

[0139] Genomic DNA was isolated from cultured human cells (HT29) using a DNA extraction kit (AllPrep, QIAGEN) to simulate a sample of circulating DNA in the blood. The isolated DNA was fragmented using a DNA fragmentation device (Covaris, M&S Instruments) to prepare the DNA sample for Example 1.

[0140] In order to measure the concentration of the fragmented DNA sample, the DNA sample was thermally denatured at 95°C for 10 minutes and then quenched to make it single-stranded. Almost all of the DNA sample just after quenching was in a single-stranded state. Next, the absorbance of the DNA sample after quenching was measured using an ultra-micro spectrophotometer (NanoDrop, Thermo Fisher Company), and it was confirmed that the concentration in the single-stranded state was 1.1 fM. That is, the concentration of the DNA sample when it was all double-stranded nucleic acid was 0.55 fM.

[0141] Here, the concentration of the target nucleic acid in the single-stranded state refers to the concentration of the total number of the number of the sense strand and the number of the antisense strand in the EGFR gene locus. In addition, the concentration of the target nucleic acid in the double-stranded state refers to the concentration of the number of the group consisting of the sense strand and the antisense strand in the EGFR gene locus. For example, in 1 diploid cell, there are 4 target nucleic acids when all are in the single-stranded state, and there are 2 target nucleic acids when all are in the double-stranded state.

[0142] In the calculation of the above concentration, first, the mass volume concentration of DNA is calculated based on the absorbance measurement value of NanoDrop. Then, assuming that 3pg of DNA sample contains a sense strand of 1 EGFR gene locus and an antisense strand of 1 EGFR gene locus, the concentration of the single-stranded target nucleic acid contained in the DNA sample after rapid cooling is calculated.

[0143] <Preparation of nucleic acid detection reagents>

[0144] In order to perform nucleic acid detection based on ICA reaction, an ICA reaction reagent was prepared as a nucleic acid detection reagent. The ICA reaction reagent in this embodiment contains 0.5μM allele probe 1 (sequence number 1) (FLAP probe), 0.1μM InvaderOligo 1 (sequence number 2) (invasion probe, also known as ICA oligonucleotide) (the above are from Fasmac), 4μM FRETCassette (Alexa488-BHQ) (sequence number 3) (Japan Bio Services) (fluorescent substrate), 50mM Tris-HCl (pH 7.9), 20mM MgCl2 and 0.05mg / mL FEN-1. It should be noted that the concentration of each component in these ICA reaction reagents is the final concentration in the mixed solution of the ICA reaction reagent and DNA sample of Experimental Example 1. Allele probe 1 and Invader Oligo 1 are probes used for ICA reaction, both of which specifically recognize one of the nucleotide chains in the sense chain and antisense chain of the EGFR gene site.

[0145] <Device Preparation>

[0146] A substrate with a large number of tiny holes is made of COP (cycloolefin polymer), and a COP cover is attached to produce a device. 2 The total volume of the wells was 0.93 μL, and the total number of wells used for measurement was 1,000,000.

[0147] <Infusion of reaction mixture solution>

[0148] 8 μl of a solution in which the DNA sample of Preparation Example 1 and the above-mentioned ICA reaction reagent were mixed was poured into the device and introduced into each well. Then, 200 μl of FC-40 (Sigma) was poured as a blocking solution to block each well.

[0149] Thus, less than one molecule of DNA is enclosed in each well. That is, each well contains, as a target nucleic acid, only one selected from a first nucleic acid that is a single-stranded nucleic acid complementary to the allele probe 1, a second nucleic acid that is a single-stranded complementary chain to the first nucleic acid, and a double-stranded nucleic acid that is complementary to the first nucleic acid, or does not contain any of the target nucleic acids.

[0150] <Nucleic acid detection reaction>

[0151] The device after the reaction mixture solution was infused was placed on a hot plate and reacted at 66°C for 25 minutes. Thus, the EGFR gene region was identified by the allele probe and Invader Oligo, the allele probe was cleaved by FEN-1, the released allele probe fragments were bound to the FRET Cassette, and the cleavage of the FRET Cassette by FEN-1 progressed, and a fluorescent signal was emitted from Alexa488.

[0152] <Fluorescence Observation of Holes>

[0153] After heating at 66°C for 25 minutes, a fluorescence image of the fluorescence signal obtained by the nucleic acid detection reaction in each hole in the device was taken using a fluorescence microscope (BZ-700, KEYENCE) using a 4x objective lens and a fluorescence filter for GFP. The exposure time was set to 3000msec. In the hole where there is a target nucleic acid with an EGFR gene region containing a single nucleotide polymorphism, a fluorescence signal from the target nucleic acid can be observed. The result of the fluorescence microscopy observation was that every 1cm 2 13.7 wells emitted fluorescence.

[0154] <Calculation of concentration>

[0155] Based on this result, the concentration of DNA complementary to the allele probe and containing the single nucleotide polymorphism can be calculated as follows: The liquid introduced into the well of the device is a solution obtained by diluting the sample measured using NanoDrop by 20 times.

[0156] Concentration = (13.7 / cm 2 )÷(6.02×10 23 )÷0.93μL / cm 2 ×10 6 ×20

[0157] =49×10 -17 (M)

[0158] =0.49(fM)

[0159] That is, in the DNA sample of Preparation Example 1, the total concentration of the single-stranded first nucleic acid to which the allele probe 1 is complementary and the concentration of the double-stranded nucleic acid formed by the complementary binding of the first nucleic acid and the second nucleic acid is calculated to be 0.49 fM. Based on the measurement results obtained by NanoDrop, it is a measurement result equivalent to the concentration of the target nucleic acid contained in the DNA sample of Preparation Example 1 (0.55 fM).

[0160] [Comparative Example 1]

[0161] <Detection of intracellular nucleic acids by digital PCR>

[0162] Using ddPCR QX100 and PrimePCR for ddPCR EGFR T790M (Biolad), the concentration of the DNA sample was measured according to the steps described in the instruction manual. The DNA sample used was the DNA sample of Preparation Example 1 that was the same as in Experimental Example 1.

[0163] Similar to Experimental Example 1 above, DNA with less than one molecule per well was enclosed in the well. That is, in each well, as the target nucleic acid, it contained only one of the single-stranded first nucleic acid selected, the second nucleic acid that was the complementary strand of the first nucleic acid, and the double-stranded molecule formed by complementary binding of the first nucleic acid and the second nucleic acid, or did not contain any of the target nucleic acids.

[0164] In digital PCR, signals were detected from all of the wells containing only the single-stranded first nucleic acid, the wells containing only the single-stranded second nucleic acid that was the complementary strand of the first nucleic acid, and the wells containing only the double-stranded molecule formed by complementary binding of the first nucleic acid and the second nucleic acid.

[0165] Based on the measurement results of digital PCR, the total concentration of the single-stranded first nucleic acid, the single-stranded second nucleic acid, and the double-stranded molecule formed by complementary binding of 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.

[0166] Based on the results using NanoDrop, it was confirmed that the total concentration of almost all of the target nucleic acids contained in the DNA sample of Preparation Example 1 that were in a single-stranded state was 1.1 fM. In addition, based on the results of Comparative Example 1, the total concentration of the single-stranded first nucleic acid, the single-stranded second nucleic acid, and the double-stranded molecule formed by complementary binding of the single-stranded first nucleic acid and the second nucleic acid was 1 fM. From this result, it can be seen that most of the target nucleic acids contained in the DNA sample of Preparation Example 1 were in a single-stranded state.

[0167] On the other hand, from the results of Experimental Example 1, it was known that the concentration of the single-stranded first nucleic acid that was complementary bound by Allele Probe 1 was 0.49 fM. In the DNA sample of Preparation Example 1, it was known that the concentration of the first nucleic acid was approximately half of the total concentration of the target nucleic acids that were mostly in a single-stranded state.

[0168] [Experimental Example 2]

[0169] The sense strand of the EGFR gene locus and the antisense strand of 1 EGFR gene locus described in Experimental Example 1 were used as target nucleic acids, and quantitative detection of the target nucleic acids was performed.

[0170] [Preparation of DNA Sample]

[0171] Genomic DNA was extracted from cultured human cells (HT29) using a DNA extraction kit (AllPrep, QIAGEN). The extracted genomic DNA was broken into 150 bp using the DNA Shearing Service of M&S Instruments. 1.3 ml of the broken genomic DNA solution was evaporated using a centrifugal dryer (DNA SpeedVac, Thermo Fisher Scientific), and 100 μl of distilled water was added to prepare the DNA sample of Example 2.

[0172] The absorbance of the DNA sample was measured, and the concentration in the single-stranded state was confirmed to be 1.0 fM. In other words, assuming that all the DNA sample was double-stranded nucleic acid, the concentration was 0.5 fM.

[0173] <Preparation of nucleic acid detection reagents>

[0174] In order to perform nucleic acid detection based on ICA reaction, an ICA reaction reagent was prepared as a nucleic acid detection reagent. The ICA reaction reagent in this embodiment contains 0.5 μM allele probe 1 (sequence number 1) (FLAP probe), 0.1 μM InvaderOligo 1 (sequence number 2) (invasion probe, also known as ICA oligonucleotide), 0.2 μM allele probe 2 (sequence number 4) (FLAP probe), 5nM Invader Oligo 2 (sequence number 5) (invasion probe) (the above are from Fasmac), 4 μM FRETCassette (Alexa488-BHQ) (sequence number 3) (Japan Bio Services) (fluorescent substrate), 2 μM FRETCassette (Redmond Red-Epoch Eclipse Quencher) (sequence number 6) (TSUKUBA OLIGO SERVICE) (fluorescent substrate) 50 mM Tris-HCl (pH 7.9), 20 mM MgCl2 and 0.05 mg / mL FEN-1. It should be noted that the concentration of each component in these ICA reaction reagents is the final concentration in the mixed solution of the ICA reaction reagent and the DNA sample of Experimental Example 2. Allele probe 1, Invader Oligo 1, Allele probe 2 and Invader Oligo 2 are probes used for ICA reaction. Allele probe 1 and Invader Oligo 1 specifically recognize one of the nucleotide chains of the sense chain and the antisense chain of the EGFR gene locus, and allele probe 2 and Invader Oligo 2 specifically recognize the other nucleotide chain of the sense chain and the antisense chain of the EGFR gene locus.

[0175] <Device Preparation and Infusion of Reaction Mixture>

[0176] Using the device described in Experimental Example 1, the reaction mixture solution was infused according to the same procedure as in Experimental Example 1.

[0177] <Nucleic acid detection reaction>

[0178] The device after the reaction mixture solution was infused was placed on a hot plate and reacted at 66°C for 25 minutes. Thus, the EGFR gene region was identified by the allele probe and Invader Oligo, the allele probe was cleaved by FEN-1, the released allele probe fragments were bound to the FRET Cassette, and the cleavage of the FRET Cassette by FEN-1 progressed, and fluorescent signals were emitted from Alexa488 and Redmond Red.

[0179] <Fluorescence Observation of Holes>

[0180] Under the same conditions as in Experimental Example 1, fluorescent images of the fluorescent signals obtained through the nucleic acid detection reaction in each well in the device were captured. 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.

[0181] The hole where only the fluorescence signal from Alexa488 is detected indicates that it is a hole where only one of the sense strand and antisense strand of the EGFR gene locus is present. The hole where only the fluorescence signal from Redmond Red is detected indicates that it is a hole where only the other of the sense strand and antisense strand of the EGFR gene locus is present. The hole where the fluorescence signals of both Alexa488 and Redmond Red are detected indicates that it is a hole where the double-stranded nucleic acid of the sense strand and antisense strand of the EGFR gene locus is present at the time point enclosed in the hole.

[0182] It should be noted that, for the wells where the fluorescent signals of both Alexa488 and Redmond Red were detected, the possibility that both the sense chain and the antisense chain of the EGFR gene locus were accidentally enclosed in one well was also considered, but considering that the total number of wells of the device is 924890 and the number of wells where the fluorescent signals were detected is the above value, it is considered that the possibility is significantly low. That is, it can be considered that the wells where the fluorescent signals of both Alexa488 and Redmond Red were detected are not wells where both the sense chain and the antisense chain of the EGFR gene locus were accidentally enclosed in one well, but wells where the double-stranded nucleic acid of the sense chain and the antisense chain of the EGFR gene locus existed at the time of being enclosed in the well.

[0183] From the results of Experimental Example 2, it can be seen that by appropriately designing and using a probe for the second nucleic acid in addition to the probe for the first nucleic acid, it is possible to detect a single-stranded first nucleic acid, a single-stranded second nucleic acid, and a double-stranded pore formed by the complementarity of the first nucleic acid and the second nucleic acid. Based on this result, it can be seen that the calculation method described in Experimental Example 1 can be used to quantify the number of molecules and the concentration.

[0184] Industrial Applicability

[0185] According to the present invention, it is possible to provide a technique capable of distinguishing and quantifying single-stranded nucleic acids and double-stranded nucleic acids with higher accuracy.

[0186] Explanation of symbols

[0187] 1 Single-stranded nucleic acid

[0188] 2 Single-stranded nucleic acid

[0189] 3 Double-stranded nucleic acids

[0190] 4, 5, 6, 7 droplets

[0191] 11 First Nucleic Acid

[0192] 12 Second Nucleic Acid

[0193] 13 Double-stranded nucleic acid

[0194] 14, 15, 16, 17, 18, 19 Droplets

[0195] 100 devices

[0196] 101 Covering Material

[0197] 102 Infusion port

[0198] 103 Wastewater outlet

[0199] 104 Substrate

[0200] 105 holes

[0201] 106 flow path

[0202] 107 Reagent Solution

[0203] 108 Reagent solution filled in the well

[0204] 201 Blocking Solution

[0205] 202 Micro-partitions

[0206] 301 Tiny Solution Sending Signals

Claims

1. A detection method, comprising: The step of bringing a liquid containing a target nucleic acid into contact with a device having a well array having a large number of wells, and introducing the target nucleic acid into the wells in a manner of one molecule or less per well, a step of sealing the pores in such a manner that the target nucleic acid does not migrate between a large number of the pores, a step of amplifying the signal from the target nucleic acid in the pore, and a step of detecting the signal emitted from the hole; The target nucleic acid includes a first nucleic acid, a second nucleic acid which is a complementary strand to the first nucleic acid, and a double-stranded nucleic acid formed by complementarily binding the first nucleic acid to the second nucleic acid. In the step of sealing the pore, the double-stranded nucleic acid of the target nucleic acid is sealed in the pore in a double-stranded state. In the step of amplifying the signal from the target nucleic acid, the first specific binding substance binds to the first nucleic acid to emit a first signal, and the second specific binding substance binds to the second nucleic acid to emit a second signal. The first signal is different from the second signal.

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

3. The detection method according to claim 1 or 2, wherein: The step of amplifying the signal from the target nucleic acid is performed by an invader cleavage reaction.

4. The detection method according to claim 1 or 2, wherein: In the step of detecting the signal emitted from the hole, For the pore in which only the first signal is detected, it is determined that only the single-stranded first nucleic acid exists. For the pore in which only the second signal is detected, it is determined that only the second nucleic acid in a single-stranded state exists. For the well in which the first signal and the second signal are detected simultaneously, it is determined that the double-stranded nucleic acid exists.

5. The detection method according to claim 1 or 2, further comprising: A step of counting the number of the wells in which the signal is detected in the step of detecting the signal emitted from the wells.

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

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

8. The detection method according to claim 1 or 2, wherein: The difference between the melting temperature Tm of the first specific binding substance and the first nucleic acid and the melting temperature Tm of the second specific binding substance and the second nucleic acid is 10° C. or less.

Images