Digital assay method, and digital assay kit

Abstract

Provided, as a technique for increasing the sensitivity of a digital assay with a simpler operation, is a digital assay method for determining the concentration of a target DNA in a sample. The method comprises: (1) a procedure for providing a substrate having a chamber array region in which 10 thousand to 10 billion chambers in a volume of 1 attoliter to 50 nanoliters are arranged; (2) a procedure for introducing a solution containing a target DNA and a reaction reagent into the chamber and sealing the chamber in a liquid-tight manner to form a reactor; (3) a procedure for simultaneously carrying out, in the reactor, an isothermal amplification reaction for the target DNA and an RNA transcription reaction including RNA aptamers; (4) a procedure for detecting fluorescence emitted from a complex of a dye and the RNA aptamer in the reactor; and (5) a procedure for determining the concentration of the target DNA in the solution on the basis of the number of reactors in which the fluorescence has been detected. The concentration of the nucleoside triphosphate in the solution is 0.2-2.0 mM.

Description

Digital assay method and digital assay kit

[0001] The present disclosure relates to a digital assay method and a digital assay kit for determining the concentration of target DNA in a sample. More specifically, the present disclosure relates to a digital assay method and a digital assay kit that include a procedure in which a large number of ultra-small volume reactors are formed on a substrate and an isothermal amplification reaction of target DNA and detection using a fluorescent aptamer are performed in the reactors.

[0002] Digital bioassay is a technique for determining the absolute concentration of a substance in a solution. The measurement principle of digital bioassay is that when a solution containing a low concentration of a substance is introduced into each of a large number of reactors, each with a small volume, no substance is introduced into most of the reactors, while the remaining reactors are each introduced with a single molecule of the substance. For example, if the substance is an enzyme, a fluorogenic assay substrate that emits fluorescence upon reaction with the enzyme can be introduced into the reactor together with the enzyme. The fluorescent signal from the reactor generated by the enzymatic reaction can be binarized into "0" and "1." The absolute concentration of the enzyme can then be determined by counting the number of "1" signals.

[0003] Patent Document 1 discloses a technology applicable to digital bioassays: "A method for detecting microsubstances contained in a plurality of receptacles formed at intervals from one another, the method comprising: (1) introducing a solvent containing the microsubstance into a space between a lower layer in which the receptacles are formed and an upper layer in the lower layer opposite the surface on which the receptacles are formed; (2) introducing a gas into the space to form droplets of the solvent containing the microsubstance in the receptacle; and (3) optically, electrically, and / or magnetically detecting the microsubstance present in the droplets." This technology is characterized by the use of a gas when forming droplets containing the microsubstance in the receptacle to serve as a reactor.

[0004] International Publication No. 2018 / 181488 International Publication No. 2012 / 121310 International Publication No. 2016 / 006208

[0005] A primary object of the present disclosure is to provide a technique for increasing the sensitivity of digital assays with simpler operations.

[0006] In order to solve the above problems, the present disclosure provides the following [1] to

[14] . [1] A digital assay method for determining the concentration of target DNA in a sample, comprising: (1) providing a substrate having a chamber array region in which 10,000 to 10 billion chambers, each having a volume of 1 attoliter to 50 nanoliters, are arranged; (2) introducing a solution containing target DNA and reaction reagents into the chambers and sealing the chambers liquid-tightly to form a reactor, wherein the reaction reagents include at least: DNA polymerase, RNA polymerase, deoxynucleotide triphosphates, nucleoside triphosphates, a dye, primer 1 comprising a sequence in which an RNA polymerase promoter sequence is added to a complementary sequence of the target DNA, and primer 2 comprising a sequence in which a sequence encoding an RNA aptamer that forms a complex with the dye is added to a complementary sequence of the target DNA; (3) simultaneously proceeding an isothermal amplification reaction of the target DNA and a transcription reaction of RNA containing the RNA aptamer in the reactor; and (4) detecting fluorescence emitted from a complex of the RNA containing the RNA aptamer and the dye in the reactor. (5) determining the concentration of the target DNA in the solution based on the number of reactors in which the fluorescence is detected. [2] The method according to [1], wherein the concentration of the nucleoside triphosphate in the solution is 0.2 mM or more and 2.0 mM or less, preferably 0.5 mM or more and 2.0 mM or less. [3] The method according to [1] or [2], wherein the RNA aptamer and the dye are any one or more of the following combinations: mango and a thiazole orange derivative (TO1-Biotin); spinach or broccoli and 3'5'-difluoro-4-hydroxybenzylideneimidazolinone (DFHBI) or a derivative thereof; pepper and ((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenylacetonitrile (HBC). Corn and 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO).[4] The method according to any one of [1] to [3], wherein the isothermal amplification reaction is any one of RPA (Recombinase Polymerase Amplification), NASBA (Nucleic acid sequences based amplification), LAMP (Loop-mediated isothermal amplification), NEAR (Nicking enzyme amplification reaction), and HDA (Helicase-dependent amplification), preferably RPA or NASBA. [5] The method according to any one of [1] to [4], wherein the liquid-tight sealing of the chambers in step (2) is performed by covering the chamber array region of the substrate with an adhesive tape comprising a base layer and an adhesive layer, and then pressing the adhesive layer of the adhesive tape against the surface of the substrate, thereby draining the excess solution on the chamber array region to the outside of the chamber array region and liquid-tightly sealing the solution inside each of the chambers. [6] The method according to any one of [1] to [5], wherein the surface of the substrate is made of a fluororesin film and is hydrophobic, and the chamber recessed from the surface is defined by a hydrophobic partition wall made of the fluororesin film and a hydrophilic bottom surface not having the fluororesin film. [7] The method according to [6], wherein the surface of the fluororesin film constituting the surface of the substrate is hydrophilized. [8] The method according to any one of [1] to [7], wherein the chambers have a diameter of 0.05-1000 μm, a depth of 0.001-1000 μm, and are arranged at intervals of 0.1-10 μm.

[0007] [9] A digital assay kit for determining the concentration of target DNA in a sample, comprising: a substrate having a chamber array region in which 10,000 to 10 billion chambers, each having a volume of 1 attoliter to 50 nanoliters, are arranged; and reaction reagents, wherein the reaction reagents include at least: a DNA polymerase, an RNA polymerase, deoxynucleotide triphosphates, nucleoside triphosphates, a dye, primer 1 comprising a sequence in which an RNA polymerase promoter sequence is added to a complementary sequence of the target DNA, and primer 2 comprising a sequence in which a sequence encoding an RNA aptamer that forms a complex with the dye is added to a complementary sequence of the target DNA.

[10] The kit of [9], wherein the nucleoside triphosphates are used in a reaction solution at a concentration of 0.2 mM to 2.0 mM, preferably 0.5 mM to 2.0 mM.

[11] The kit of

[10] , further comprising an adhesive tape comprising a base layer and an adhesive layer, which is attached to the chamber array region of the substrate.

[12] The kit of any of [9] to

[11] , wherein the surface of the substrate is made of a fluororesin film and is hydrophobic, and the chamber recessed from the surface is defined by a hydrophobic partition wall made of the fluororesin film and a hydrophilic bottom surface that does not have the fluororesin film.

[13] The kit of

[12] , wherein the surface of the fluororesin film that forms the surface of the substrate is hydrophilized.

[14] The kit of any of [9] to

[13] , wherein the chambers have a diameter of 0.05-1000 μm, a depth of 0.001-1000 μm, and are arranged at intervals of 0.1-10 μm.

[0008] The present disclosure provides a technique for increasing the sensitivity of digital assays with simpler operations.

[0009] Fig. 1 is a diagram for explaining a chamber array chip. Fig. 2 is a diagram for explaining a chamber array chip. Fig. 3 is a diagram for explaining the procedure of a digital assay method. Fig. 4 is a fluorescent photograph of the chamber array region of a chamber array chip when virus-derived DNA is digitally assayed using a microfluidic flow cell (Example 1). Fig. 5 shows the change over time in the number of bright spots on a chamber array chip when virus-derived DNA is digitally assayed using a microfluidic flow cell (Example 2).

[0010] Preferred embodiments for carrying out the present disclosure will be described below with reference to the drawings. Note that the embodiments described below are examples of typical embodiments of the present disclosure, and should not be construed as narrowing the scope of the present disclosure.

[0011] The digital assay method according to the present disclosure is a method for determining the concentration of target DNA in a sample, and includes the following steps: (1) Substrate provision step, (2) Solution introduction step, (3) DNA isothermal amplification reaction and RNA aptamer transcription reaction, (4) Detection step, and (5) Concentration determination step.

[0012] The sample is not particularly limited, and may be a biological sample, such as blood, plasma, serum, urine, tears, saliva, or swabs (nose, throat, skin, etc.). The sample may also be water from rivers, oceans, tap water, sewage, wastewater, or soil. The target DNA may be derived from a living organism or artificially synthesized, and may be circular or linear. The target DNA is not particularly limited, and may be DNA that can be contained in the above-mentioned samples, such as DNA derived from viruses, bacteria, cells, or the like, or DNA reverse-transcribed from RNA derived from viruses, etc.

[0013] 1. Substrate Provision Procedure In this procedure, a substrate (hereinafter referred to as a "chamber array chip") having a chamber array region in which 10,000 to 10 billion chambers with a volume of 1 attoliter to 50 nanoliters are arranged is provided.

[0014] 1 and 2 show a chamber array chip. The chamber array chip 1 has a chamber array region 3 in which a large number of chambers 2 are arranged. The size and shape of the chamber array chip 1 are not particularly limited. For example, the chamber array chip 1 is rectangular, 32 mm wide, 24 mm deep, and 2 mm thick. The size of the chamber array chip 1 may be 1-100 mm wide, 1-100 mm deep, and 0.1-10 mm thick. The shape of the chamber array chip 1 may be rectangular, square, circular, elliptical, or the like. The size of the chamber array region 3 is not particularly limited as long as it is smaller than the chamber array chip 1. However, if the chamber array chip 1 is rectangular, 32 mm wide, 24 mm deep, and 2 mm thick, the chamber array chip 1 will be rectangular, 20 mm wide, and 20 mm deep. The size of the chamber array region 3 may be 0.1-30 mm wide and 0.1-30 mm deep. The shape of the chamber array region 3 may be rectangular, square, circular, elliptical, or the like.

[0015] The size and shape of the chamber 2 are not particularly limited, but an example is a cylindrical or approximately cylindrical shape with a diameter (d) of 4 μm and a depth (h) of 3 μm. The size of the chamber 2 may be a diameter (d) of 0.05-1000 μm and a depth (h) of 0.001-1000 μm. From the viewpoint of ease of molding, the size and shape of the chamber 2 are preferably cylindrical or prismatic. The volume of the chamber 2 may be in the range of 1 attoliter to 50 nanoliters, but may be, for example, 10 attoliters to 5 nanoliters, preferably 100 attoliters to 500 picoliters, more preferably 1 femtoliter to 50 picoliters, even more preferably 10 femtoliters to 5 picoliters, particularly preferably 10 femtoliters to 500 femtoliters, and most preferably 10 femtoliters to 100 femtoliters. The number and spacing (pitch) of the chambers 2 arranged in the chamber array region 3 may be set as appropriate. The number of chambers 2 to be arranged is, for example, 10,000 to 10 billion, preferably 100,000 to 10 million. The interval (pitch) between the chambers 2 is, for example, 0.05 to 30 μm, preferably 0.1 to 10 μm.

[0016] The chamber array chip 1 can be formed using known techniques such as wet etching or dry etching of a glass substrate, or nanoimprinting, injection molding, or cutting of a plastic substrate. The chamber array chip 1 is preferably obtained by patterning the chambers 2 on the substrate 11 using a general-purpose photolithography method. Specifically, for example, a fluororesin film 12 is formed on the glass substrate 11 by spin coating and baking, and a photoresist film (not shown) is then formed on the fluororesin film 12. After irradiating the photoresist film with ultraviolet light through a mask patterned with the shapes of the chambers 2, the substrate 11 is immersed in a resist developer to remove the UV-irradiated portions of the photoresist film. The fluororesin film 12 in the resist-removed portions is then removed by dry etching to expose the surface of the substrate 11 that will become the bottom surfaces 21 of the chambers 2. Finally, the substrate 11 is washed to completely remove the resist film. Through the above steps, a large number of chambers 2 can be formed on the substrate 11, with the remaining fluororesin film 12 serving as partition walls 22 and the surface of the substrate 11 serving as bottom walls 21. The outermost surface 13 of the resulting chamber array chip 1 is formed by the fluororesin film 12 remaining as partition walls 22 and is hydrophobic. The chambers 2 are recessed from the outermost surface 13, and the bottom surfaces 21 of the chambers 2 are the surface of the glass substrate 11 and are hydrophilic. The outermost surface 13 of the chamber array chip 1 is preferably made hydrophilic by subjecting the fluororesin film 12 constituting it to a hydrophilic treatment. The hydrophilic treatment can be performed, for example, by coating the fluororesin film 12 with a hydrophilic polymer such as polyvinylpyrrolidone or polyvinyl alcohol, or by modifying the functional groups of the fluororesin film 12 to hydrophilic ones by plasma treatment. Alternatively, a chamber array chip 1 having a hydrophilic outermost surface 13 can be obtained by using a hydrophilic polymer film instead of a fluororesin film to form the pattern of the chambers 2.

[0017] The substrate 11 is made of a light-transmitting material, such as glass or various plastics (PP, PC, PS, COC, COP, PDMS, PMMA, PET, PVC, etc.) It is preferable to select a material for the substrate 11 that has little autofluorescence, small wavelength dispersion, and therefore little optical error.

[0018] Commercially available fluororesins such as CYTOP (registered trademark), TEFLON (registered trademark) AF2400, and TEFLON (registered trademark) AF1600 can be used as the fluororesin for forming the fluororesin coating 12. CYTOP (registered trademark) is preferred because it is easy to microfabricate.

[0019] 2. Solution Introduction Procedure In this procedure, a solution containing target DNA and a reaction reagent is introduced into the chamber 2, and the chamber 2 is sealed liquid-tight to form a reactor (reaction field).

[0020] The reaction reagents include reagents for performing an isothermal amplification reaction of the target DNA and detection using a fluorescent aptamer. Here, the term "fluorescent aptamer" refers to an RNA aptamer that has the function of acquiring fluorescence as a complex by forming a complex with a dye that is not fluorescent by itself, or the function of enhancing the fluorescence of a fluorescent dye by forming a complex with the dye.

[0021] The reagents for the isothermal amplification reaction include at least DNA polymerase, deoxynucleotide triphosphates (dNTPs), primer 1, which contains a sequence in which an RNA polymerase promoter sequence is added to the complementary sequence of the target DNA, and primer 2, which contains a sequence in which a sequence encoding an RNA aptamer that forms a complex with a dye is added to the complementary sequence of the target DNA, and optionally buffers, salts, surfactants, etc. The DNA polymerase is an enzyme appropriate for the method used in the isothermal amplification reaction, as described below. Deoxynucleotide triphosphates (dNTPs) refer to a mixture of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP).

[0022] Reagents for fluorescent aptamer detection include at least RNA polymerase, nucleoside triphosphate (NTP), and dye, and optionally buffer, salt, surfactant, etc. The RNA polymerase promoter sequence of primer 1 and the sequence encoding the RNA aptamer of primer 2 are also involved in fluorescent aptamer detection. An appropriate RNA polymerase enzyme is used depending on the RNA polymerase promoter sequence. For example, a T7 promoter and T7 RNA polymerase are preferably used. Nucleoside triphosphate (NTP) refers to a mixture of adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP). The combination of RNA aptamer and dye is not particularly limited, as long as the RNA aptamer forms a complex with a non-fluorescent dye alone, resulting in the complex's fluorescence, or the RNA aptamer forms a complex with a fluorescent dye, resulting in enhanced fluorescence of the dye. Commercially available combinations include the following: Mango and a thiazole orange derivative (TO1-Biotin); spinach or broccoli and 3'5'-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) or its derivatives. Derivatives include DFHBI-1T, which has a 1,1,1-trifluoroethyl substitution on the imidazole ring; pepper and ((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenylacetonitrile (HBC); and corn and 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO).

[0023] The solvent for the reaction reagent is water or a buffer solution. The buffer solution is not particularly limited, but examples thereof include MOPS (3-(N-morpholino) propanesulfonic acid), MES (2-morpholinoethanesulfonic acid), ADA (N-(2-Acetamido)iminodiacetic acid), PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)), ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid), BES (N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), TES (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Tris (Tris(hydroxymethyl)aminomethane), and DEA (diethanolamine).

[0024] The introduction of a solution containing target DNA and reaction reagents into the chambers 2 and the sealing of the chambers 2 may be performed by conventionally known techniques, such as those described in Patent Documents 1 to 3. Alternatively, the introduction of a solution into the chambers 2 and the sealing of the chambers 2 may be performed by introducing a solution containing target DNA and reaction reagents into the chamber array region 3 of the chamber array chip 1 and then pressing adhesive tape onto the chamber array region 3, as described below. This will be explained with reference to FIG. 3. A solution containing target DNA 4 (hereinafter referred to as "sample solution S") is introduced into the chamber array region 3 of the chamber array chip 1 (see FIG. 3(A)). The sample solution S also contains a dye 5 and the other reaction reagents described above. The introduction of the sample solution S into the chamber array region 3 may be performed, for example, by dripping the sample solution S onto the chamber array region 3. If the outermost surface 13 of the chamber array chip 1 is hydrophilized, the dripped sample solution S can be introduced into each of the chambers 2. On the other hand, if the outermost surface 13 of the chamber array chip 1 is hydrophobic, the dropped sample liquid S may be held by the hydrophobic outermost surface 13 and form droplets as shown in Fig. 3(A). In this case, in the procedure of Fig. 3(A), the sample liquid S does not need to be introduced completely into the chamber 2.

[0025] Next, the chamber array region 3 onto which the sample liquid S has been dropped is covered with adhesive tape 6 (see FIG. 3(B)). The adhesive tape 6 comprises a base layer 61 and an adhesive layer 62, and during this covering, the adhesive layer 62 of the adhesive tape 6 is prevented from coming into contact with the outermost surface 13 of the chamber array chip 1. The sample liquid S is pressed and spread by the adhesive tape 6 into the space formed between the adhesive layer 62 of the adhesive tape 6 and the outermost surface 13 of the chamber array chip 1 (left and right in the figure), and a portion of the sample liquid S is introduced into the chamber 2, filling the interior of the chamber 2 with the sample liquid S. The excess sample liquid S that does not fit into the interior of the chamber 2 is pushed out of the chamber array region 3.

[0026] Next, the adhesive layer 62 of the adhesive tape 6 is pressed onto the outermost surface 13 of the chamber array chip 1 (see FIG. 3(C)). This completely discharges excess sample liquid S that did not fit inside the chambers 2 to the outside of the chamber array region 3, and liquid-tightly seals the sample liquid S inside each chamber 2 to form a reactor. Each reactor is configured as an independent space (reaction field) partitioned by the bottom surface 21, partition wall 22, and adhesive tape 6. Pressing of the adhesive tape 6 can be performed, for example, by placing the chamber array chip 1 on a stable, flat workbench and applying an appropriate pressure to the adhesive tape 6 while moving a roller back and forth.

[0027] The concentration of the target DNA 4 in the sample solution S is usually in the range of 1 zM-1 mM. When the concentration of the target DNA 4 in the sample solution S is this low, either one molecule of the target DNA 4 or none at all is introduced into each chamber 2. On the other hand, a reaction reagent such as the dye 5 is contained in the sample solution S in a sufficient number of moles, and is introduced into all chambers 2 in an amount sufficient for the reaction to proceed.

[0028] The adhesive tape 6 is preferably optically transparent. However, optical transparency is not essential depending on the configuration of the optical detection system used in the detection procedure. Specifically, when irradiation with excitation light and detection of fluorescence are both performed from below the chamber array chip 1, the adhesive tape 6 may be optically opaque.

[0029] The adhesive tape 6 can be obtained by applying a solution of an adhesive composition to a base layer 61 and volatilizing the solvent to form an adhesive layer 62. Dissolving the adhesive composition in a solvent, applying the adhesive composition solution to the base layer 61, and drying can be performed by conventionally known methods. The solvent (e.g., toluene) can also be selected appropriately. The thickness of the base layer is not particularly limited, but is, for example, 1-100 μm, preferably 2-50 μm, and more preferably 4-25 μm. The thickness of the adhesive layer is, for example, greater than 2 μm and not more than 1000 μm, preferably 5-50 μm, and more preferably 5-20 μm. If the thickness is 2 μm or less, the elasticity of the adhesive layer 62 cannot absorb slight irregularities caused by dirt or the like on the outermost surface 13 of the chamber array chip 1, significantly reducing the assay success rate. If the thickness exceeds 1000 μm, it becomes difficult to form the adhesive layer 62 with a uniform thickness, and the compression of the adhesive layer often becomes uneven when pressed, which may result in localized incomplete sealing of the chamber 2 within the chamber array region 3.

[0030] The material of the base layer 61 may be a light-transmitting material, such as a film made of polyester, PP, polyimide, PET, cellophane, PVC, or polyolefin.

[0031] The adhesive composition for forming the adhesive layer 62 is not particularly limited and can be appropriately selected from conventional, commonly used adhesives such as rubber-based, silicone-based, acrylic-based and urethane-based adhesives. A rubber-based adhesive composition or a silicone-based adhesive composition is preferred, and a rubber-based adhesive composition is more preferred.

[0032] 3. DNA Isothermal Amplification Reaction and RNA Aptamer Transcription Reaction In this procedure, an isothermal amplification reaction of target DNA 4 and a transcription reaction of RNA containing an RNA aptamer proceed simultaneously in chamber 2. The isothermal amplification reaction and the RNA transcription reaction can be performed under the same temperature conditions (e.g., from room temperature to 42°C). The isothermal amplification reaction is not particularly limited and any conventionally known method can be used. Examples of isothermal amplification reactions include the Recombinase Polymerase Amplification (RPA) method, the Nucleic Acid Sequence Based Amplification (NASBA) method, the Loop-Mediated Isothermal Amplification (LAMP) method, the Nicking Enzyme Amplification Reaction (NEAR) method, and the Helicase-Dependent Amplification (HDA) method. The isothermal amplification reaction can be, in particular, the RPA method or the NASBA method.

[0033] When the concentration of target DNA 4 in sample solution S is low, either one or no target DNA 4 molecule is introduced into each chamber 2 (see Figure 3(C)). In chamber 2 containing target DNA 4, the target DNA 4 is amplified by an isothermal amplification reaction, producing an amplification product having an RNA polymerase promoter sequence (derived from primer 1) and a sequence encoding the RNA aptamer (derived from primer 2). A transcription reaction then produces RNA containing the RNA aptamer from the amplification product. Furthermore, the RNA containing the RNA aptamer interacts with the dye at its RNA aptamer portion, forming a complex.

[0034] When DNA isothermal amplification and RNA transcription reactions are carried out simultaneously in a very small reactor, the NTP concentration in sample solution S is 0.2 mM to 2.0 mM, 0.3 mM to 2.0 mM, or 0.4 mM to 2.0 mM, preferably 0.5 mM to 2.0 mM, more preferably 0.6 mM to 1.8 mM, 0.7 mM to 1.6 mM, 0.8 mM to 1.4 mM, or 0.9 mM to 1.2 mM, and particularly preferably about 1.0 mM. The NTP concentration in the reaction solution of commercially available RNA transcription kits using T7 polymerase is typically about 4 mM. For example, Takara Bio Inc.'s "In Vitro Transcription T7 Kit" uses an NTP concentration of 5 mM, while JENA BIOSCIENCE GMBH's "High Yield T7 RNA Synthesis Kit" uses an NTP concentration of 7.5 mM. The NTP concentration is the total concentration of adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP). NTPs usually contain equal concentrations of ATP, CTP, GTP, and UTP.

[0035] It was found that if the NTP concentration in sample solution S is lower or higher than this range, complex formation between the RNA containing the RNA aptamer and the dye does not occur, and a fluorescent signal may not be detected even when target DNA 4 is introduced into chamber 2. If the NTP concentration is too low, synthesis of RNA containing the RNA aptamer may not proceed sufficiently. On the other hand, if the NTP concentration is too high, NTP may be incorporated into DNA strand synthesis in place of dNTP, inhibiting DNA strand elongation during the isothermal amplification reaction. Optimizing the NTP concentration enables the application of a highly sensitive detection system using isothermal amplification and fluorescent aptamers to detection in a very small reactor volume, enabling digital assays to be performed with a simple operation of simultaneously conducting two reactions, DNA isothermal amplification and RNA transcription, under the same temperature conditions. The concentrations of reaction reagents other than NTP are not particularly limited and can be set appropriately within the scope of conventional technology.

[0036] 4. Detection Procedure In this procedure, the fluorescence emitted from the complex of the target DNA 4 and the dye 5 inside each of the chambers 2 is detected.

[0037] By optically detecting the fluorescence emitted from the complex in each chamber 2, the fluorescence signal can be binarized into "0" and "1." In the next concentration determination procedure, the absolute concentration of the target DNA 4 in the sample solution S is determined by counting the number of "1" signals.

[0038] 5. Concentration Determination Procedure In this procedure, the concentration of the target DNA 4 in the sample solution S is determined based on the number of chambers 2 in which fluorescence is detected.

[0039] Specifically, the volume of chamber 2 is V (unit: liter), and the ratio of the number of chambers 2 in which the presence of target DNA 4 is detected to the total number of chambers 2 is λ. When λ is sufficiently small, the molar concentration C (unit: M) of target DNA 4 can be calculated using the following formula, where N is Avogadro's number: C = λ / (N x V). Theoretically, λ is between 0 and 1, but to determine the concentration of target DNA 4, λ should be 0.5 or less, preferably 0.1 or less.

[0040] Example 1 (1) Preparation of a Chamber Array Chip A chamber array chip was prepared according to the method described in the literature ("A microreactor sealing method using adhesive tape for digital bioassays," Lab Chip, 2022, 22, 2001-2010). The procedure is outlined below. CYTOP (CTL-816AP, manufactured by AGC Chemicals) was spin-coated onto a glass cover slip, followed by photolithographic chamber patterning to obtain a chamber array chip. Double-sided adhesive tape (760H, manufactured by Teraoka Seisakusho) was cut using an automatic sheet cutter, ScanNCut CM300 (Brother Industries), to create a microchannel structure on the adhesive tape. The adhesive tape was attached to the chamber array chip at the microchannel-formed section, and a 5 mm-thick piece of glass was then attached to the opposite side of the adhesive tape. The glass piece had holes for the inlet and outlet of the liquid into the microchannel, forming a microfluidic flow cell. The inner surface of the glass piece (the surface facing the microchannel) was spin-coated with an M-type Cytop (CTL-809A, manufactured by AGC Chemicals). The resulting chamber array chip in the microfluidic flow cell contained approximately 5.7 million chambers, each 4 μm in diameter and 3 μm in height, arranged at a 9 μm pitch in a 20 x 20 mm chamber array area.

[0041] (2) Digital Assay (2-1) Preparation of Digital Assay Reaction Solution The T7-Mango assay premix was prepared at room temperature. The composition of 45 μL of the assay premix was as follows: For the DNA isothermal amplification reaction (RPA), a commercially available kit (TwistAmp Basic, Nippon Genetics) containing all necessary reagents except for primers and target DNA (template) was used. The kit is based on the RPA (Recombinase Polymerase Amplification) method.

[0042] [Assay premix (45 μl)] TwistAmp Basic, 1 pellet of TT7 polymerase (1,000,000 U / mL, TRL-252, Toyobo), 0.75 μL NTP mix (R0481, Thermo Scientific), 2 μL TO-1 Biotin (G955, abm), 1 μL Mango-tagged forward primer (10 μM), 2.1 μL T7 promoter-tagged reverse primer (10 μM), 2.1 μL 200 μM Alexa Fluor 647 (A20347, Invitrogen), 1.25 μL 1% (v / v) S-386 (AGC Seimi Chemical), 0.5 μL rehydration buffer from the TwistAmp Basic kit (TwistDX), 29.5 μL RNase-free water, 5.8 μL

[0043] 1 μL of sample containing DNA consisting of a reverse-transcribed partial sequence of the N1 sequence of the COVID-19 genome was added to 18 μL of assay premix. 1 μL of magnesium acetate (280 mM) from the TwistAmp Basic Kit was then added to prepare the sample solution, which was then immediately used in the subsequent digital assay.

[0044] (2-2) Digital Counting. The sample solution was introduced into a microfluidic flow cell, and the chambers of the chamber array chip were sealed liquid-tightly according to the method described in Patent Document 2. The procedure is outlined below. The sample solution was passed through the microfluidic flow cell. The microfluidic flow cell was then placed on an aluminum block half-immersed in a bath sonicator, and the sonicator was operated for 10 seconds to remove air bubbles from the chambers. A sealing oil mix (2% AE-3000 (AGC Chemicals), 98% Fomblin Y-25 (Solvay), 0.1% S-386) was then passed through the microfluidic flow cell. The oil flushed out excess sample solution within the microchannel and sealed each chamber into which the sample solution had been introduced. The microfluidic flow cell was placed on a 39°C heat block and incubated for 30 minutes to allow the DNA isothermal amplification reaction and RNA transcription reaction to proceed. The microfluidic flow cell was then placed under a fluorescence microscope, and the mango fluorescence was imaged and detected. The images were opened using the free software ImageJ / Fiji (National Institutes of Health) for image analysis, and the number of bright spots was counted. The number of bright spots was counted for 31,757–39,021 chambers. The Alexa Fluor 647 images were used to determine the presence or absence of air bubbles in the chambers. The results are shown in Table 1.

[0045]

[0046] At an NTP concentration of 1 mM, Mango fluorescent signals were detected in 5.687% of the chambers. Figure 4 shows a fluorescent photograph of the chamber array area of ​​the chamber array chip. On the other hand, at NTP concentrations of 3 mM or higher or 0.1 mM or lower, the percentage of chambers in which Mango fluorescent signals were detected decreased dramatically, even though the target DNA concentration in the sample solution was the same. If the number of positive signals at an NTP concentration of 1 mM is taken as 100%, the number of positive signals at an NTP concentration of 3 mM decreased to 2.1%, and the number of positive signals at an NTP concentration of 0.1 mM or lower decreased to 2.5%. This result indicates that the presence of target DNA that would have been detected at an NTP concentration of 1 mM was not detected (false negatives) at NTP concentrations of 3 mM or higher or 0.1 mM or lower. The number of positive signals at an NTP concentration of 0.2 mM was 6.8% of the number of positive signals at an NTP concentration of 1 mM, an improvement over the number of positive signals at an NTP concentration of 0.1 mM or lower. It was shown that by setting the NTP concentration at 0.2 mM-2 mM, preferably 0.5 mM-2 mM, it is possible to perform an accurate digital assay of target DNA that combines an isothermal DNA amplification reaction and an RNA transcription reaction.

[0047] [Example 2] Digital assay was performed using the NASBA (Nucleic acid sequences based amplification) method as the DNA isothermal amplification reaction instead of the RPA method used in Example 1. The chamber array chip prepared in Example 1 was used.

[0048] (1) Digital Assay (1-1) Preparation of Digital Assay Reaction Solution A T7-Mango assay mix was prepared at room temperature. The composition of 20 μL of the assay mix was as follows:

[0049] Assay mix composition: Tris-HCl (Invitrogen) pH 8.0, 40 mM Magnesium chloride (Invitrogen), 13.2 mM Potassium chloride (Invitrogen), 150 mM Dithiothreitol (Sigma), 10 mM DMSO (Sigma-Aldrich), 11% (v / v) dNTP (Invitrogen), 1 mM NTP (Thermo-Fisher), 1 mM RNase H (NEB), 37.5 U / mL ProtoScript RT (NEB), 50,000 U / mL TT7 polymerase (TOYOBO), 15,000 U / mL, 25 nM Mango-sequenced forward primer, 25 nM T7-sequenced reverse primer, TO-1 Biotin (G955, abm), 1 μM Alexa Fluor 647 (A20347, Invitrogen), 5 μM S-386 (AGC Seimi Chemical) 0.01% (v / v) Template DNA consisting of a reverse-transcribed sequence of a portion of the COVID-19 N1 sequence, 3.6 pM

[0050] (1-2) Digital Counting Following the method described in Example 1, the sample solution was introduced into the microfluidic flow cell, and the chambers of the chamber array chip were sealed liquid-tight. The microfluidic flow cell was placed on a microscope stage maintained at 41°C and incubated to allow the DNA isothermal amplification reaction and RNA transcription reaction to proceed. The mango fluorescence was photographed every 10 minutes for 3 hours, and the number of bright spots was counted.

[0051] Figure 5 shows the change in the number of bright spots over time. Thirty minutes after the start of imaging, chambers showing positive fluorescence were identified. Three hours after imaging began, the rate of positive bright spots was 18%, and the target DNA concentration was calculated to be 7.2 pM, which roughly matched the amount of DNA added to the assay mix (3.6 pM). By using an NTP concentration of 1 mM, we were able to demonstrate that a digital assay of target DNA combining an isothermal DNA amplification reaction and an RNA transcription reaction can be performed with high accuracy.

[0052] 1: Chamber array chip 11: Substrate 12: Fluororesin coating 13: Outermost surface 2: Chamber 21: Bottom surface 22: Partition wall 3: Chamber array area 4: Target substance 5: Dye 6: Adhesive tape 61: Base layer 62: Adhesive layer S: Sample liquid

Claims

1. A digital assay method for determining the concentration of target DNA in a sample, comprising: (1) providing a substrate having a chamber array region in which 10,000 to 10 billion chambers, each having a volume of 1 attoliter to 50 nanoliters, are arranged; (2) introducing a solution containing target DNA and reaction reagents into the chambers and sealing the chambers liquid-tightly to form a reactor, wherein the reaction reagents include at least: DNA polymerase, RNA polymerase, deoxynucleotide triphosphates, nucleoside triphosphates, dyes, primer 1 comprising a sequence in which an RNA polymerase promoter sequence is added to a complementary sequence of the target DNA, and primer 2 comprising a sequence in which a sequence encoding an RNA aptamer that forms a complex with the dye is added to a complementary sequence of the target DNA, and the concentration of the nucleoside triphosphates in the solution is 0.2 mM or more and 2.0 mM or less; and (3) simultaneously carrying out an isothermal amplification reaction of the target DNA and a transcription reaction of RNA containing the RNA aptamer in the reactor. (4) detecting fluorescence emitted from a complex between the RNA containing the RNA aptamer and the dye in the reactor; and (5) determining the concentration of the target DNA in the solution based on the number of reactors in which the fluorescence is detected.

2. The method of claim 1, wherein the concentration of the nucleoside triphosphate in the solution is 0.5 mM or more and 2.0 mM or less.

3. The method according to claim 2, wherein the RNA aptamer and the dye are any one or more of the following combinations: Mango and a thiazole orange derivative (TO1-Biotin). Spinach or broccoli with 3'5'-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) or its derivatives, pepper with ((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenylacetonitrile (HBC), and corn with 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO).

4. The method according to any one of claims 1 to 3, wherein the isothermal amplification reaction is performed by any one of the following methods: RPA (Recombinase Polymerase Amplification), NASBA (Nucleic acid sequences based amplification), LAMP (Loop-mediated isothermal amplification), NEAR (Nicking enzyme amplification reaction), and HDA (Helicase-dependent amplification).

5. A digital assay kit for determining the concentration of target DNA in a sample, comprising: a substrate having a chamber array region in which 10,000 to 10 billion chambers, each having a volume of 1 attoliter to 50 nanoliters, are arranged; and reaction reagents, wherein the reaction reagents include at least: a DNA polymerase, an RNA polymerase, deoxynucleotide triphosphates, nucleoside triphosphates, a dye, primer 1 comprising a sequence in which an RNA polymerase promoter sequence is added to a complementary sequence of the target DNA, and primer 2 comprising a sequence in which a sequence encoding an RNA aptamer that forms a complex with the dye is added to a complementary sequence of the target DNA, wherein the nucleoside triphosphates are used in a reaction solution at a concentration of 0.2 mM to 2.0 mM.

6. The kit according to claim 5, wherein the nucleoside triphosphate is used at a concentration of 0.5 mM to 2.0 mM in the reaction solution.

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