Nucleic acid detection method, hydrogel capsule and dispersion thereof

Abstract

The present invention aims to provide a nucleic acid detection method that can be easily and inexpensively implemented, has high quantitative reliability, and exhibits excellent reactivity in PCR, as well as a hydrogel capsule and its dispersion used in the nucleic acid detection method. [Solution] The method is characterized by comprising: a dispersion formation step in which a core-shell type hydrogel capsule 1, in which an aqueous reaction solution containing at least nucleic acid, amplification components necessary for amplification of the nucleic acid, and detection components that emit a detection signal in response to the amplification of the nucleic acid is disposed in a core portion 2 and the outer circumference of the core portion 2 is covered with a heat-resistant gel film 3 is dispersed in a dispersion medium 4 to form a dispersion 10; a nucleic acid amplification step in which the dispersion 10 is subjected to a PCR thermal cycle to obtain an amplification product of the nucleic acid within the hydrogel capsule 1; and a detection step in which the detection signal is detected.

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid detection method applicable to the amplification and detection of nucleic acids by PCR, as well as to a hydrogel capsule and its dispersion. [Background technology]

[0002] qPCR (quantitative PCR) is a widely used nucleic acid detection method that utilizes PCR (Polymerase Chain Reaction) (see Non-Patent Documents 1 and 2 for representative qPCR methods). In the qPCR method described above, for example, a calibration curve of Cq values ​​(the number of amplification cycles when the PCR amplification product reaches a certain amount) is created using reference standard samples of known concentrations, and the amount of nucleic acid in the unknown sample is estimated by comparing the Cq values ​​of the unknown sample with the calibration curve.

[0003] However, this method is an expensive relative quantitative analog technique because it requires the development of a reference standard substance with the same sequence as the nucleic acid to be quantified, and the creation of the calibration curve and the measurement of the Cq value of the unknown sample are performed manually by the operator. Furthermore, the detection results are strongly affected by variability due to the operator's technique, as the Cq value takes an exponentially large value depending on the amplification cycle, and even a small error can cause a large discrepancy in the estimated amount of nucleic acid.

[0004] To address these problems, the droplet digital PCR (ddPCR) method has been developed (see Non-Patent Documents 3 and 4 for representative ddPCR methods). In the ddPCR method described above, the unknown sample is diluted to the limit (limit dilution) so that one nucleic acid is contained in each droplet, and the sample is divided into many droplets. By digitally detecting the presence or absence of a detection signal in each droplet, it becomes possible to absolutely quantify the amount of nucleic acid based on the detection signal without using the calibration curve. For example, if the detection signal is a fluorescence signal emitted after amplification of the nucleic acid, the amount of nucleic acid can be absolutely quantified by digitally counting the droplets that emit the fluorescence signal, taking advantage of the fact that droplets without the nucleic acid do not emit the fluorescence signal after amplification, while droplets containing the nucleic acid do.

[0005] However, in the ddPCR method, the droplets receive thermal energy during the PCR thermal cycling process, which destabilizes the interface between the droplet and its surroundings, causing adjacent droplets to fuse. Furthermore, the internal pressure increases due to the thermal energy, potentially causing the droplets to rupture. Additionally, fused or ruptured droplets may attract other droplets due to the accumulation of charge caused by the increase in volume, leading to further fusion. This increases the possibility that one droplet may contain two or more nucleic acids, compromising the reliability of the quantification.

[0006] Therefore, in order to avoid the fusion or rupture of the droplets, it has also been proposed to gel the droplets using a gelling material (see, for example, Patent Documents 1 and 2). In these proposals, the gelled droplets are sometimes referred to as gel capsules, but in reality, they are gel beads formed by gelling the entire droplet. These gel beads cause the nucleic acids and various PCR reagents encapsulated inside to lose their fluidity, leading to a problem of reduced reaction rates in the PCR process.

[0007] For these reasons, an encapsulation technique has been proposed in which a film-forming protein is used as the outer membrane, and a fluid containing the PCR reagent, etc., is sealed inside (see Patent Document 3). However, the film-forming protein used in this proposal either lacks thermal resistance to the PCR process or, if it does, is difficult to handle, thus complicating the implementation of the ddPCR method. Although there is a technique for encapsulating a fluid containing raw materials such as PCR reagents in a capsule (see Patent Document 4), this technique is performed for the purpose of appropriately preparing the reagents necessary for the PCR reaction before the PCR is carried out (furthermore, various reagents are prepared in smaller capsules, each containing a different type of reagent). When the PCR is carried out, the capsule is broken and the spilled reagents are reacted in a microwell (see paragraphs

[0005] ,

[0117] , Figures 4A, B, etc.). In other words, the aforementioned technology is a technology for appropriately distributing prepared reagents, etc., into the microwells. In fact, no encapsulation that takes into account the resistance to thermal cycling of the PCR performed after distribution or the reaction rate of the reagents is disclosed. When the outer membrane that forms the capsule is made of a polymer without relying on phase separation of the oil phase and the aqueous phase, it is disclosed that it is made of gel beads (see paragraphs

[0007] ,

[0011] ,

[0014] ,

[0049] to

[0051] , etc.). [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2020 / 218551 [Patent Document 2] Special Publication No. 2002-531056 [Patent Document 3] Japanese Patent Publication No. 2016-136954 [Patent Document 4] Special Publication No. 2015-528283 [Non-patent literature]

[0009] [Non-Patent Document 1] Holland et al., Proceedings of National Academy of Science USA, 88 (16): 7276, 1991. [Non-Patent Document 2] Higuchi et al., Nature Biotechnology, 10 (4): 413, 1992. [Non-Patent Document 3] Hindson et al., Analytical Chemistry, 83 (22): 8604, 2011. [Non-Patent Document 4] Hindson et al., Nature Methods, 10 (10): 1003, 2013. [Overview of the project] [Problems that the invention aims to solve]

[0010] The present invention aims to solve the aforementioned problems of the prior art, provide a nucleic acid detection method that can be implemented easily and at low cost, has high quantitative reliability, and exhibits excellent reactivity in PCR, as well as a hydrogel capsule and its dispersion used in the nucleic acid detection method. [Means for solving the problem]

[0011] The means to solve the aforementioned problem are as follows: <1> A nucleic acid detection method characterized by comprising: a dispersion formation step in which a core-shell type hydrogel capsule, in which an aqueous reaction solution containing at least nucleic acid, an amplification component necessary for the amplification of the nucleic acid, and a detection component that emits a detection signal in response to the amplification of the nucleic acid is disposed in the core portion and the outer circumference of the core portion is covered with a heat-resistant gel film, is dispersed in a dispersion medium to form a dispersion; a nucleic acid amplification step in which the dispersion is subjected to a PCR thermal cycle to obtain an amplification product of the nucleic acid within the hydrogel capsule; and a detection step in which the detection signal is detected. <2> The volume of the hydrogel capsule is 0.5 fL to 20 μL. <1> The nucleic acid detection method described above. <3> The nucleic acid detection method according to <1> or <2> above, wherein the hydrogel capsule has mechanical strength such that it does not rupture even when pressed from one direction and its diameter is increased by up to twice. <4> The nucleic acid detection method according to any one of <1> to <3> above, wherein the heat-resistant gel film can be solubilized while maintaining the nucleic acid sequence of the amplification product amplified within the core part. <5> The nucleic acid detection method according to any one of <1> to <4> above, wherein the material for forming the heat-resistant gel film is selected from any one of sodium alginate, poly(N-isopropylacrylamide-co-acrylic acid), polylactic acid, and photocurable chitosan. <6> The nucleic acid detection method according to any one of <1> to <5> above, wherein the specific gravity of the dispersion medium is 0.6 to 1.4, and the thermal conductivity of the dispersion medium is 0.05 W / m·K to 0.23 W / m·K. <7> The nucleic acid detection method according to any one of <1> to <6> above, wherein the viscosity of the dispersion medium is 0.5 mPa·s to 500 mPa·s. <8> The nucleic acid detection method according to any one of <1> to <7> above, wherein the thermal cycle in the nucleic acid amplification step is provided by replacing the dispersion medium in the dispersion liquid with a high-temperature body of the dispersion medium temperature-controlled on the high-temperature side and a low-temperature body of the dispersion medium temperature-controlled on the low-temperature side. <9> A hydrogel capsule having a core-shell type structure, wherein an aqueous reaction solution containing at least a nucleic acid, an amplification component necessary for amplification of the nucleic acid, and a detection component that emits a detection signal in response to amplification of the nucleic acid is disposed in the core part, and the outer periphery of the core part is covered with a heat-resistant gel film. <10> A dispersion liquid of a hydrogel capsule, characterized in that a core-shell type hydrogel capsule in which an aqueous reaction solution containing at least a nucleic acid, an amplification component necessary for amplification of the nucleic acid, and a detection component that emits a detection signal in response to amplification of the nucleic acid is disposed in the core part and the outer periphery of the core part is covered with a heat-resistant gel film is dispersed in a dispersion medium.

Advantages of the Invention

[0012] According to the present invention, it is possible to solve the above problems in the prior art, implement it simply at low cost, have high reliability in quantification, and also have excellent reactivity in the PCR. It is possible to provide a nucleic acid detection method, a hydrogel capsule used in the nucleic acid detection method, and a dispersion thereof.

Brief Description of Drawings

[0013] <C <C [Figure 1(a)] It is an explanatory diagram (1) showing a detection scheme in a nucleic acid detection method. <C <C [Figure 1(b)] It is an explanatory diagram (2) showing a detection scheme in a nucleic acid detection method. <C <C [Figure 1(c)] It is an explanatory diagram (3) showing a detection scheme in a nucleic acid detection method. <C <C [Figure 2] It is a diagram showing a fluorescence microscope image of a dispersion. <C <C [Figure 3] It is a diagram showing microscope images of a hydrogel capsule before and after a compression operation according to Reference Example 1. <C <C [Figure 4(a)] It is a diagram showing a microscope image of a hydrogel capsule before compression. <C <C [Figure 4(b)] It is a diagram showing a microscope image of a hydrogel capsule after compression. <C <C [Figure 5] It is a diagram showing microscope images of the hydrogel capsule before and after solation. <C <C [Figure 6] It is a diagram showing the results of applying statistical analysis by ANOVA (analysis of variance) and Tukey's HSD test (Tukey's range test) to the data of Cq values measured by each nucleic acid detection method according to Example 2 (gddPCR method), Comparative Example 1 (vddPCR method), and Comparative Example 2 (qPCR method). <C <C [Figure 7] It is a diagram showing the results of applying statistical analysis by ANOVA and Tukey's HSD test to the measurement data of fluorescence intensity for each reaction solution in Example 2 (gddPCR method), Comparative Example 1 (vddPCR method), and Comparative Example 2 (qPCR method). <C <C [Figure 8]This figure shows the results of applying statistical analysis using ANOVA and Tukey's HSD test to the fluorescence intensity measurement data for each reaction solution in Example 2 (fluorinated oil) and Example 3 (soybean oil). [Figure 9] This figure shows photographs of the reaction solutions from Example 2 (fluorinated oil) and Example 3 (soybean oil) stored in microtubes. [Figure 10] This figure shows an image illustrating the fluorescence behavior in the reaction solutions of Example 2 (fluorinated oil) and Example 3 (soybean oil). [Figure 11] This figure shows the relationship between specific gravity and thermal conductivity for typical oils and heat transfer fluids. [Modes for carrying out the invention]

[0014] (Method for detecting nucleic acids) The nucleic acid detection method of the present invention includes at least a dispersion formation step, a nucleic acid amplification step, and a detection step.

[0015] <Dispersion liquid formation process> The aforementioned dispersion formation step is a step of forming a dispersion of hydrogel capsules by dispersing the hydrogel capsules in a dispersion medium.

[0016] -Hydrogel Capsules- The hydrogel capsule has a core-shell structure.

[0017] =Core section= The core portion is a part in which an aqueous reaction solution is disposed, which contains at least nucleic acid, amplification components necessary for amplifying the nucleic acid, and detection components that emit a detection signal during the amplification of the nucleic acid. In other words, the aqueous reaction solution in which the PCR reaction components are dissolved or dispersed is enclosed within the shell portion of the hydrogel capsule.

[0018] The nucleic acid is DNA or RNA having a target nucleic acid sequence extracted from a subject. If it is RNA, complementary DNA is synthesized by reverse transcription PCR and subjected to subsequent PCR. There are no particular restrictions on the subject material, and it can be appropriately selected according to the purpose. Examples include biological samples such as bodily fluids, tissues, cells, and excretions of animals and plants, including humans, as well as liquid samples such as river water and treated water, and soil samples, which may contain fungi and bacteria. The nucleic acid is extracted from these samples and subjected to the PCR. The synthesis of the complementary DNA by the reverse transcription PCR method may be performed inside the hydrogel capsule, or it may be performed before the hydrogel capsule is formed, and the synthesized complementary DNA may be used as the nucleic acid.

[0019] The amplification components are not particularly limited and include known components used in PCR, such as DNA polymerase as a replication enzyme, the enzyme substrate of the DNA polymerase (four types of bases: dGTP, dCTP, dATP, dTTP), cofactors such as magnesium salts, and known components represented by primers that amplify the target nucleic acid sequence. In addition, a reverse transcriptase may be included in order to synthesize the complementary DNA in the reverse transcription PCR method within the hydrogel capsule. As the amplification components, premix reagents containing various components are commercially available and can be suitably used.

[0020] There are no particular restrictions on the detection component, and it can be appropriately selected depending on the detection method. The aforementioned detection methods include known methods such as fluorescence detection, absorbance detection, detection by microscopy, and detection by magnetic measurement. Among these, fluorescence detection is representative, and typical examples of the detection components include fluorescently labeled probe reagents such as TaqMan probe (registered trademark) and intercalator reagents such as SYBR Green I (registered trademark).

[0021] =Shell part (heat-resistant gel film)= The hydrogel capsule has its core portion coated with the heat-resistant gel membrane. In the nucleic acid detection method of the present invention, the nucleic acid is amplified by PCR within the hydrogel capsule, so the heat-resistant gel membrane is required to withstand the thermal cycling of the PCR. In this specification, "heat resistance" means that when the hydrogel capsule is subjected to the thermal cycle in the temperature range of 45°C to 98°C, the entire gel film does not transfer to a sol and the hydrogel capsule does not become a liquid droplet. Furthermore, the heat-resistant gel membrane is a membrane that covers only the outer periphery of the core portion, and the concept of the hydrogel capsule does not include the concept of "gel beads" in which the entire core portion is gelled. Such a hydrogel capsule ensures the fluidity of the reaction solution placed in the core portion, thereby enabling higher efficiency and faster PCR.

[0022] There are no particular limitations on the method for forming the heat-resistant gel film, and any known method of gelling a gel film-forming material by applying an external stimulus can be cited. For example, methods of gelling by applying a crosslinking agent, methods of gelling by applying a pH change, methods of gelling by applying a temperature change, methods of gelling by applying a pressure change, and methods of gelling by applying an electromagnetic field or plasma irradiation can be cited.

[0023] The gel film-forming material when the aforementioned crosslinking agent is provided to induce gelation is not particularly limited and includes sodium alginate, ethylenediamine, benzylamine, phenoxide monomers (for example, resorcinol, dihydroxyanthraquinone, 9,9-bis(4-hydroxyphenyl)fluorene, 5,5',6,6'-tetrahydroxy-3,3,3',3'-tetramethyl-1,1'-spirobindan, etc.). Examples of the crosslinking agent include divalent metal salts such as barium salts and calcium salts (sodium alginate), terephthaloyl dichloride (ethylenediamine), acetyl chloride (benzylamine), and benzenetricarbonyl trichloride (phenoxide). This type of gel film-forming material provides the hydrogel capsule with a core-shell structure, rather than the gel beads that are entirely gelled, by locally mixing the crosslinking agent at the interface.

[0024] The gel film-forming material used when gelation is induced by the aforementioned pH change is not particularly limited. For example, a polymer having properties in which hydrophilicity changes in response to pH and hydrophobicity forms a gel network by linking hydrophobic parts can be used. A specific example is a triblock copolymer based on polyacrylic acid with polystyrene-grad-acrylic acid at both ends. In this specific example, gelation occurs at pH < 7, and conversely, solification is induced at pH > 8. This type of gel film-forming material provides a hydrogel capsule with a core-shell structure, rather than a gel bead, by controlling the pH of the dispersion medium to the critical pH at which the gel film-forming material contained in droplets of the aqueous reaction solution undergoes sol-gelation, that is, a pH that can maintain a sol inside the gel.

[0025] The gel film-forming material used when gelation is performed by applying the aforementioned temperature change is not particularly limited, and examples include poly(N-isopropylacrylamide-co-acrylic acid). This type of gel film-forming material provides the hydrogel capsule with a core-shell structure, rather than the gel beads that are entirely gelled, by heating the droplet interface of the aqueous reaction solution.

[0026] The gel film-forming material used when gelation is induced by applying the aforementioned pressure change is not particularly limited, but examples include poly(N-isopropylacrylamide-co-acrylic acid). This type of gel film-forming material provides the hydrogel capsule with a core-shell structure, rather than the gel beads that are entirely gelled, by applying pressure to the droplet interface, for example, by applying the critical osmotic pressure necessary for gelation.

[0027] The gel film-forming material used when gelling is performed by applying the aforementioned electromagnetic field is not particularly limited, and examples include photocurable chitosan (light), photocurable acrylamide (light), azobenzene polyester (light), carboxystyrenepyrene (light), and a mixture of polyvinyl alcohol and methyl vinyl ether-maleic anhydride copolymer (microwave). This type of gel film-forming material provides hydrogel capsules with a core-shell structure, rather than gel beads, by concentrating an electromagnetic field, such as light or microwaves of a specific wavelength, onto the droplet interface of the aqueous reaction solution. Furthermore, there are no particular limitations on the gel film forming material when it is gelled by plasma irradiation, and examples include polylactic acid. This type of gel film-forming material provides hydrogel capsules with a core-shell structure, rather than gel beads, by concentrating and irradiating plasma at the droplet interface of the aqueous reaction solution.

[0028] Among these gel film-forming materials, sodium alginate, poly(N-isopropylacrylamide-co-acrylic acid), polylactic acid, and photocurable chitosan are preferred from the viewpoint of obtaining the hydrogel capsules that are low-cost, easy to handle, and have excellent mechanical strength, and among these, sodium alginate is particularly preferred. Sodium alginate gels when it reacts with divalent metal ions such as barium ions and calcium ions. However, since calcium ions are a substance that inhibits the reaction to PCR, it is preferable to use a barium salt as the crosslinking agent and react the sodium alginate with barium ions to form the heat-resistant gel film.

[0029] Furthermore, in addition to detection, the amplified product after the nucleic acid amplification step may be subjected to further analysis such as sequencing. Therefore, it is preferable that the heat-resistant gel film can be sol-formed while maintaining the nucleic acid sequence of the amplification product amplified within the core. In other words, if the gel film-forming material of the heat-resistant gel film is a material that can be converted into a sol, then by simply performing the operation of converting the heat-resistant gel film of the hydrogel capsule separated from the dispersion into a sol, an aqueous solution of the amplification product can be obtained with the nucleic acid sequence maintained without causing destruction or denaturation.

[0030] There are no particular limitations on the gel film-forming material that can be sol-formed. Examples of known gel-sol transition materials include materials that are sol-formed by chemical operations, materials that are sol-formed by pH changes, materials that are sol-formed by temperature changes, materials that are sol-formed by pressure changes, and materials that are sol-formed by electromagnetic fields.

[0031] There are no particular limitations on the material that is sol-formed by the aforementioned chemical operation, but examples include sodium alginate. The heat-resistant gel film formed using sodium alginate as the gel film-forming material, such as barium alginate or calcium alginate, is converted into a sol by reacting it with a chelating agent such as ethylenediaminetetraacetic acid (EDTA). There are no particular restrictions on the solvent used for the chelating agent when supplying it to the hydrogel capsule; aqueous solvents such as water or phosphate buffer, or oily solvents containing emulsifier components such as soybean oil can be used. However, from the viewpoint of avoiding dilution of the nucleic acids inside the hydrogel capsule when it is converted into a sol, it is preferable to use the oily solvent.

[0032] There are no particular limitations on the material that is sol-formed by the aforementioned pH change. For example, polymers that have the property of changing their hydrophilicity or hydrophobicity in response to pH and forming a gel network by linking hydrophobic parts can be cited. A specific example is a triblock copolymer based on polyacrylic acid with polystyrene-grad-acrylic acid at both ends. In this specific example, gelation occurs at pH < 7, and conversely, sol formation is induced at pH > 8.

[0033] There are no particular limitations on the material that is solubilized by the aforementioned temperature change, but examples include poly(N-isopropylacrylamide-co-acrylic acid). The heat-resistant gel film, formed from poly(N-isopropylacrylamide-co-acrylic acid), is sol-formed by lowering its temperature to a temperature below the lower critical solution temperature.

[0034] There are no particular limitations on the material that is solubilized by the aforementioned pressure change, but examples include poly(N-isopropylacrylamide-co-acrylic acid). The heat-resistant gel film, formed from poly(N-isopropylacrylamide-co-acrylic acid), is solified by reducing the pressure to a level that reaches the lower critical solution temperature.

[0035] There are no particular limitations on the material that is sol-formed by applying the aforementioned electromagnetic field, and examples include azobenzene polyester and carboxystyrenepyrene. The heat-resistant gel film, formed from azobenzene polyester, carboxystyrenepyrene, etc., is sol-formed by irradiation with light of a specific wavelength different from the wavelength required for gelation.

[0036] There are no particular limitations on the hydrogel capsule, but it is preferable that it has mechanical strength that prevents it from rupturing even when its diameter is increased by up to twice its original size by pressing from one direction. Having such mechanical strength makes the hydrogel capsule less likely to break even when subjected to disturbances during the thermal cycling process or when removed for further analysis, allowing for easy handling.

[0037] There are no particular restrictions on the volume of the hydrogel capsule, but it is preferably 0.5 fL to 20 μL. With such a volume, the amount of nucleic acid sample prepared for limiting dilution can be made into a practical amount. Furthermore, since a large number of hydrogel capsules are formed in the dispersion, the volume can be determined as the average value of 10 arbitrarily selected hydrogel capsules in the dispersion, and the volume is calculated based on the size of the hydrogel capsules measured from a microscope image.

[0038] -Dispersion medium- The dispersion medium is selected from a material that is insoluble or sparingly soluble in the heat-resistant gel film and the aqueous reaction solution, and provides the dispersion in which the hydrogel capsules are dispersed. There are no particular restrictions on the dispersion medium, and it can be appropriately selected from known non-water-soluble fluids such as fluorinated oils, vegetable oils, mineral oils, silicone oils, and hydrocarbon oils. However, vegetable oils, mineral oils, and silicone oils are preferred because they belong to a group of materials that have a specific gravity close to that of water and good thermal conductivity. In other words, if the thermal conductivity is good, heat is conducted from the dispersion medium to the hydrogel capsules with high responsiveness during the thermal cycle, thereby improving the reaction efficiency of the PCR. Furthermore, if the material has a specific gravity close to that of water, a phase separation of the dispersion medium does not occur from the suspension phase between the dispersion medium and the hydrogel capsules, and a dispersion is obtained in which the hydrogel capsules are uniformly suspended in the dispersion medium, thereby enabling efficient heat exchange during the thermal cycle. Specifically, it is preferable that the specific gravity of the dispersion medium is 0.6 to 1.4, and the thermal conductivity of the dispersion medium is 0.05 W / m·K to 0.23 W / m·K.

[0039] Furthermore, while there are no particular restrictions on the dispersion medium, it is preferable that its viscosity is between 0.5 mPa·s and 500 mPa·s. Such viscosity makes it easier to stabilize the dispersion state of the hydrogel capsules and to mix them. The viscosity of typical dispersion media is as follows: mineral oil is 0.6 mPa·s or higher, soybean oil is 65 mPa·s or lower, olive oil is 100 mPa·s or lower, and silicone oil (various types exist) is approximately 0.5 mPa·s to 10,000,000 mPa·s. These viscosities can also be adjusted by adding known surfactants. Furthermore, there are no particular restrictions on the dispersion medium, but when using the crosslinking agent or when performing sol formation by the chemical operation, it is preferable to use a dispersion medium capable of dissolving or emulsifying the corresponding crosslinking agent or chelating agent. Examples include soybean oil containing a large amount of lecithin capable of emulsifying barium chloride or EDTA.

[0040] There are no particular limitations on the method for preparing the dispersion, and one method is to mix and stir the aqueous reaction solution and the aqueous liquid containing the gel film forming material with the dispersion medium. The stirring can be performed using a known vortex mixer or the like. By performing such stirring, a dispersion can be easily obtained in which a large number of hydrogel capsules of a particle size corresponding to the limiting dilution are dispersed in the dispersion medium. Other methods for preparing the dispersion include droplet generation by membrane emulsification, droplet generation by microchannels, and gel cutting by piezoelectric elements.

[0041] <Nucleic acid amplification process> The nucleic acid amplification step involves subjecting the dispersion to the thermal cycle of the PCR and obtaining the amplified nucleic acid product within the hydrogel capsule.

[0042] The nucleic acid amplification step is not particularly limited and can be carried out according to known conditions applied to the conventional ddPCR method. Typical conditions include first raising the temperature of the dispersion to a relatively high temperature (around 92°C to 98°C) to dissociate the double-stranded nucleic acid into single strands (thermal denaturation step). Next, lowering the temperature of the dispersion to a lower temperature (around 45°C to 60°C) to bind the target nucleic acid sequence of the nucleic acid (a specific sequence having a sequence complementary to the primer sequence) to the primer (annealing step). Next, raising the temperature of the dispersion to a slightly higher temperature than the annealing temperature (around 70°C to 75°C) to extend the complementary strand of nucleotides complementary to the nucleic acid through the action of the DNA polymerase, thereby obtaining a DNA amplification product with the same sequence as the original double-stranded nucleic acid (extension step). These steps, from thermal denaturation to extension, are repeated to exponentially increase the number of amplified products. However, depending on the properties of the nucleic acid to be amplified (such as chain length) and the melting temperature of the primer, the extension step may be omitted, and DNA amplification products can be obtained in a two-step thermal cycle consisting of the thermal denaturation step and the annealing step. Alternatively, single-stranded DNA or RNA may be used as the nucleic acid, and if RNA is used, the synthesis of complementary DNA using the reverse transcriptase may be carried out in the hydrogel capsule before these thermal cycles. In this process, the detection signal (e.g., fluorescence intensity) is detected after the thermal cycle is completed or after each cycle, and the nucleic acid is quantitatively detected based on the obtained detection signal.

[0043] Furthermore, there are no particular restrictions on the method of carrying out the nucleic acid amplification step, and it can be carried out using a known PCR device having a thermal cycler and a signal detection unit, but the following method is preferred. In other words, since the hydrogel capsule has superior mechanical strength compared to the droplets in the conventional ddPCR method, the heating and cooling operations for the dispersion during the thermal cycle can be performed by replacing the dispersion medium in the dispersion with a high-temperature component of the dispersion medium that is conditioned to a high temperature and a low-temperature component of the dispersion medium that is conditioned to a low temperature, instead of directly heating the dispersion and allowing the dispersion to cool. Furthermore, if the thermal cycle consists of three steps from the thermal denaturation step to the extension step, it can also be performed by sequentially replacing the dispersion medium, which is conditioned to three different temperatures suitable for each step, step by step. The operation of applying the thermal cycle by replacing the dispersion medium with one of the dispersions heated to a different temperature can be carried out in a significantly shorter time compared to directly heating the dispersion or allowing the dispersion to cool. Furthermore, there is no overshoot or undershoot of the target temperature, and a dispersion can be easily obtained in which the hydrogel capsules are redispersed in the dispersion medium after replacement without being destroyed, thus enabling rapid and highly sensitive detection of the nucleic acid.

[0044] <Detection Process> The detection step is the step of detecting the detection signal. There are no particular restrictions on the method of carrying out the detection step, and examples include known detection methods applied in the conventional ddPCR method, which are carried out according to the detection component used. For example, when fluorescence detection is used as the detection method, the fluorescence signal generated in response to the amplification of the nucleic acid is detected by the PCR device.

[0045] The scheme of the nucleic acid detection method of the present invention will be briefly explained below, using fluorescence detection as an example, with reference to Figures 1(a) to (c). First, the hydrogel capsules 1 are dispersed in the dispersion medium 4 to form a dispersion 10. Here, the hydrogel capsule 1 has a core portion 2 whose outer circumference is covered with a heat-resistant gel film 3, and the core portion 2 contains an aqueous reaction solution containing the nucleic acid, the amplification component, and the fluorescently labeled probe reagent as the detection component (the above is the dispersion formation step. See Figures 1(a) and (b)). Next, the dispersion 10 is subjected to the thermal cycle of the PCR to obtain the amplified nucleic acid product in the hydrogel capsule 1. During amplification, the fluorescent substance in the fluorescently labeled probe reagent is separated from the quenching substance, and fluorescence may be generated. In the hydrogel capsule 1 containing the nucleic acid, unlike those without it, the amount of the fluorescent substance increases as the thermal cycle is repeated, increasing the fluorescence intensity, and the number of these substances is digitally counted (the above describes the nucleic acid amplification step and the detection step. See Figure 1(c)). As described above, the nucleic acid detection method can absolutely quantify the nucleic acid.

[0046] (Hydrogel Capsules) The hydrogel capsule of the present invention has a core-shell structure in which an aqueous reaction solution containing at least a nucleic acid, an amplification component necessary for amplifying the nucleic acid, and a detection component that emits a detection signal in response to the amplification of the nucleic acid is disposed in the core, and the outer circumference of the core is covered with a heat-resistant gel film. Specifically, the present invention is constructed by applying the matters relating to the hydrogel capsule described in the section on the nucleic acid detection method.

[0047] (Dispersion of hydrogel capsules) The hydrogel capsule dispersion of the present invention is composed of core-shell type hydrogel capsules dispersed in a dispersion medium, each having a core portion containing an aqueous reaction solution comprising at least a nucleic acid, an amplification component necessary for amplifying the nucleic acid, and a detection component that emits a detection signal in response to the amplification of the nucleic acid, with the outer periphery of the core portion covered by a heat-resistant gel film. Specifically, the present invention is constructed by applying the matters relating to the dispersion described in the section on the nucleic acid detection method of the present invention. [Examples]

[0048] (Example 1) 15.8 μL of an aqueous solution of sodium alginate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 2% by mass sodium alginate aqueous solution) as a material for forming a heat-resistant gel film, and a diluted solution of standard DNA (2 × 10) for quantifying influenza virus as nucleic acid. 10 follow mL -1 An aqueous sample was prepared by mixing 10 μL of (AIST) (manufactured by the National Institute of Advanced Industrial Science and Technology), 10 μL of (1) premix reagents such as enzymes as nucleic acid amplification components (Thermo Fisher Scientific, TaqMan Fast Virus 1-Step Master Mix), 1.60 μL of (2) primer reagents (Thermo Fisher Scientific, MP-39-67For [5'-CCMAGGTCGAAACGTAYGTTCTCTCTATC], MP-183-153Rev [5'-TGACAGRATYGGTCTTGTCTTTAGCCAYTCCA] 10 μM aqueous solution), and 1.00 μL of probe reagent as a detection component (Thermo Fisher Scientific, MP-96-75ProbeAs [5'-(FAM)ATYTCGGCTTTGAGGGGGCCTG(MGB)] 10 μM aqueous solution).

[0049] Next, 38.4 μL of the aqueous sample and 100 μL of soybean oil (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number: 190-03776) were mixed, and the mixture was stirred at 2,000 rpm for 10 minutes using a vortex mixer (manufactured by Vortexer, HS120318) to form a water-in-oil emulsion in which numerous droplets of the aqueous sample were generated in the soybean oil.

[0050] Next, barium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) is added to the water-in-oil emulsion as a gelling agent for sodium alginate to gel the surface of the droplets, forming hydrogel capsules containing the nucleic acid, amplification component, and detection component, and a dispersion of the hydrogel capsules is formed (dispersion formation step). Furthermore, the barium chloride was added to the water-in-oil emulsion under the condition that its concentration in the soybean oil was 20 mM, in order to gel only the surface side of the droplet.

[0051] 50 μL of the dispersion was placed in a microtube and subjected to a PCR thermal cycle using a PCR analyzer (Roche Diagnostics LightCycler 96, thermal cycler) (nucleic acid amplification step). The specific conditions for the thermal cycling described above are shown in Table 1 below, and the cycle was performed from top to bottom in the table.

[0052] [Table 1]

[0053] A portion of the dispersion after the thermal cycle was extracted, and fluorescence from the detection component was observed using a fluorescence microscope (Olympus Corporation, with a 10x objective lens) (detection step).

[0054] Figure 2 shows a fluorescence microscope image of the dispersion. Figure 2 shows both the hydrogel capsules that emit fluorescence (indicated by the thick arrows) and the hydrogel capsules that do not emit fluorescence (indicated by the thin arrows). Therefore, in the nucleic acid detection method according to Example 1, a highly quantitative ddPCR method can be achieved, for example, by digitally counting the absolute number of the fluorescent hydrogel capsules. In the following, the nucleic acid detection method using the hydrogel capsule will be referred to as the "gddPCR" (gel droplet digital PCR) method to distinguish it from the nucleic acid detection method that does not use the hydrogel capsule (ddPCR method).

[0055] (Reference example 1) As shown in Figure 2, the hydrogel capsule maintains its capsule shape even when exposed to disturbances such as heat, pressure, and external forces associated with fluid flow during the thermal cycle. However, there are concerns that the hydrogel capsules may have insufficient mechanical strength compared to gel beads that have gelled throughout, and their mechanical stability during the thermal cycle becomes a matter of concern.

[0056] Therefore, the sodium alginate hydrogel capsule was formed in the same manner as in Example 1, except that the aqueous sample was prepared by adding 32.6 μL of an aqueous solution of red food coloring (Food Red No. 102) (a 1% by mass aqueous solution of the red food coloring) instead of the nucleic acid, amplification component and detection component (32.6 μL) in Example 1. This will be referred to as the hydrogel capsule sample according to Reference Example 1.

[0057] One hydrogel capsule was taken from the hydrogel capsule sample according to Reference Example 1 using a micropipette, placed between two glass slides, and compressed by pressing. The state before and after the compression operation was observed with a microscope (Olympus BX51TF).

[0058] Figure 3 shows microscopic images of the hydrogel capsule according to Reference Example 1 before and after compression. The left side of Figure 3 shows the state before compression, and the right side shows the state after compression. As shown in Figure 3, even when a compressive force was applied to increase the diameter by approximately 10%, no leakage of the red food coloring enclosed in the hydrogel capsule according to Reference Example 1 was observed. In other words, no rupture or other damage to the hydrogel capsule according to Reference Example 1 was observed. Furthermore, it was confirmed that when the compressive force is released, it returns to its original diameter (see left side of Figure 3).

[0059] Furthermore, in order to more appropriately evaluate the effects of the compression operation, relatively large hydrogel capsules were extracted from the hydrogel capsule sample related to Reference Example 1, and their mechanical strength was confirmed by applying a stronger compressive force. Specifically, the mechanical strength was confirmed by applying a compressive force from a certain direction to crush the hydrogel capsule until its thickness was approximately 1 / 5 of its original thickness.

[0060] Figure 4(a) shows a microscopic image of the hydrogel capsule before compression, and Figure 4(b) shows a microscopic image of the hydrogel capsule after compression. As shown in Figure 4(b), after compression, the maximum diameter of the hydrogel capsule increased by more than double, but no fracture was observed. In this compression operation, the hydrogel capsule did not expand concentrically, but rather expanded in a somewhat distorted shape. However, the cross-sectional area S0 at the position with the largest cross-sectional area in the hydrogel capsule before compression was 2,471 μm². 2 Furthermore, since the cross-sectional area S1 at the position where the hydrogel capsule has the largest cross-sectional area after compression is 10,006, the average increase in diameter is (S1 / S0) 1 / 2 It is approximately 201%. In other words, the hydrogel capsule has mechanical strength that prevents it from rupturing even when pressed from one direction, causing its (average) diameter to increase by up to two times. Furthermore, it was confirmed that when the compressive force is released, the hydrogel capsule returns to approximately its original diameter (see Figure 4(a)) and returns to its original shape. As described above, contrary to the aforementioned concerns, the hydrogel capsule can be evaluated as having excellent mechanical strength and sufficient resistance to the thermal cycle.

[0061] Furthermore, the hydrogel capsule is useful not only for the quantitative detection of nucleic acids but also for the further analysis (sequencing) of the amplified products obtained in the nucleic acid amplification step. In other words, by solving the gel membrane of the hydrogel capsule, the amplification product generated within the hydrogel capsule can be obtained in its original state. That is, the amplification product can be removed from the hydrogel capsule without destruction or denaturation, while maintaining its nucleic acid sequence, and subjected to further analysis.

[0062] Specifically, as a solifying agent for the gel film, ethylenediaminetetraacetic acid (EDTA, manufactured by Nacalai Tesque, a chelating agent) at a concentration of 10 mg / mL was added to the hydrogel capsule according to Reference Example 1 to eliminate the crosslinking of the gel film and to solify the gel film.

[0063] Figure 5 shows microscopic images of the hydrogel capsule before and after solification. In Figure 5, the left side shows the microscopic image before solification, and the right side shows the microscopic image after solification. As shown in Figure 5, the addition of EDTA causes the gel film to become sol-like, and the hydrogel capsule can be converted into a liquid sample with the same components as the aqueous sample in the core.

[0064] (Example 2) 7.9 μL of the aforementioned 2% by mass sodium alginate aqueous solution and a diluted solution of standard DNA for influenza virus quantification (1 × 10⁻¹⁰ 7 follow mL -1 An aqueous sample was prepared by mixing 5 μL of (manufactured by the National Institute of Advanced Industrial Science and Technology), the amplification components ((1) 0.8 μL of the premix reagent, (2) 0.8 μL of the primer reagent), and 0.5 μL of the probe reagent.

[0065] Next, barium chloride was added to a fluorinated oil (Automated Droplet Generator Oil for Probes, manufactured by Bio-Rad) to prepare 30 μL of the fluorinated oil with barium chloride added to a concentration of 20 mM in the oil.

[0066] Next, 20 μL of the aqueous sample and 30 μL of the fluorine-based oil to which the barium chloride was added were mixed, and then stirred at 2,000 rpm for 10 minutes using the vortex mixer to form a dispersion in which numerous hydrogel capsules containing the aqueous sample were generated in the fluorine-based oil. (Dispersion formation step).

[0067] Next, 50 μL of the dispersion was placed in the microtube and subjected to a PCR thermal cycle using the PCR analyzer under the same conditions as in Example 1 (nucleic acid amplification step). Furthermore, the fluorescence intensity of each cycle was measured using the PCR measuring instrument, and the Cq value was determined (detection step). Based on the above, the nucleic acid detection method according to Example 2 was carried out.

[0068] (Comparative Example 1) The nucleic acid detection method according to Comparative Example 2 was carried out in the same manner as in Example 2, except that 5.0 μL of water was added instead of 7.9 μL of the 2% by mass sodium alginate aqueous solution to prepare the aqueous sample (without gel film forming material). In the nucleic acid detection method according to this Comparative Example 2, the hydrogel capsule is not formed; instead, a dispersion is formed in which droplets of the aqueous sample are dispersed in the oil phase, and these droplets correspond to the droplets in the conventional ddPCR method. In the following, the PCR method using droplets generated by vortex mixing may be referred to as the "vddPCR" (vortex droplet digital PCR) method to make it easier to distinguish it from the "gddPCR" method of the present invention.

[0069] (Comparative Example 2) 20 μL of the aqueous sample (without gel film-forming material) prepared in the same manner as in Comparative Example 1 was placed in the microtube, and then 30 μL of the fluorine-based oil to which barium chloride had been added, prepared in the same manner as in Example 2, was placed in the microtube to prepare a non-dispersible sample in which only the top of the aqueous sample was coated with the fluorine-based oil.

[0070] The microtubes containing the non-dispersive sample were subjected to a thermal cycle of PCR using the PCR analyzer under the same conditions as in Example 1. Furthermore, the fluorescence intensity for each cycle was measured using the PCR measuring instrument, and the Cq value was determined. Based on the above, the nucleic acid detection method according to Comparative Example 2 was carried out. The nucleic acid detection method according to Comparative Example 2 corresponds to the measurement of the Cq value in the conventional qPCR method.

[0071] Figure 6 shows the results of applying statistical analysis using ANOVA (analysis of variance) and Tukey's HSD test to the Cq value data measured by each nucleic acid detection method related to Example 2 (gddPCR method), Comparative Example 1 (vddPCR method), and Comparative Example 2 (qPCR method). As shown in Figure 6, it was confirmed that the nucleic acid detection methods in Example 2 (gddPCR method) and Comparative Example 1 (vddPCR method), in which the reaction system is accumulated in a minute droplet or hydrogel capsule, yield significantly lower Cq values ​​compared to the nucleic acid detection method in Comparative Example 2 (qPCR method), which does not involve this accumulating system. Furthermore, the slightly higher Cq value of the nucleic acid detection method in Example 2 (gddPCR method) compared to the nucleic acid detection method in Comparative Example 1 (vddPCR method) is thought to be due to a decrease in the diffusion rate of substances within the hydrogel capsule, which in turn reduced the reaction rates.

[0072] Next, the reaction solutions from Example 2 (gddPCR method), Comparative Example 1 (vddPCR method), and Comparative Example 2 (qPCR method) were transferred to 96-well plates (STEM, P96F25S), and the fluorescence intensity was measured using a plate reader (ThermoScientific, VarioSkan).

[0073] Figure 7 shows the results of applying ANOVA and Tukey's HSD test to the fluorescence intensity measurement data for each reaction solution in Example 2 (gddPCR method), Comparative Example 1 (vddPCR method), and Comparative Example 2 (qPCR method). As shown in Figure 7, it was confirmed that there was no significant difference in the fluorescence intensity measurement results between the nucleic acid detection method according to Example 2 (gddPCR method) and the nucleic acid detection method according to Comparative Example 1 (vddPCR method). In other words, although the nucleic acid detection method according to Example 2 (gddPCR method) was slightly slower in reaction rate compared to the nucleic acid detection method according to Comparative Example 1 (vddPCR method) (see Figure 6), it was confirmed that the reaction itself proceeded to almost the same level (see Figure 7). Therefore, the gddPCR method does not have disadvantages such as being a PCR inhibitor, and compared to the vddPCR method, it only has the advantage of improving the reliability of quantitative analysis by eliminating problems such as the fusion of droplets. On the other hand, in the nucleic acid detection method according to Comparative Example 2 (qPCR method), the fluorescence intensity was significantly lower, confirming that the reaction itself had not progressed (see Figure 7). In other words, under the conditions of the thermal cycle, a large amount of unreacted material remained after the thermal cycle.

[0074] (Example 3) The diffusion detection method according to Example 3 was carried out in the same manner as in Example 2, except that 30 μL of soybean oil to which barium chloride had been added to achieve an oil concentration of 20 mM was used instead of 30 μL of the fluorine-based oil to which barium chloride had been added to achieve an oil concentration of 20 mM.

[0075] Next, the reaction solutions from Example 2 (the fluorine-based oil) and Example 3 (the soybean oil) after PCR were transferred to the 96-well plate, and the fluorescence intensity was measured using the plate reader.

[0076] Figure 8 shows the results of applying statistical analysis using ANOVA and Tukey's HSD test to the fluorescence intensity measurement data for each reaction solution in Example 2 (the fluorine-based oil) and Example 3 (the soybean oil). As shown in Figure 8, the reaction solution in Example 3 (the soybean oil) exhibits a higher fluorescence intensity compared to the reaction solution in Example 2 (the fluorine-based oil).

[0077] Here, photographs of the states of the reaction solutions of Example 2 (the fluorinated oil) and Example 3 (the soybean oil) stored in the microtube are shown in Fig. 9. As shown in Fig. 9, in the reaction solution of Example 2 (the fluorinated oil), an oil phase is confirmed to separate below the phase in which the hydrogel capsule and the oil are suspended, whereas in the reaction solution of Example 3 (the soybean oil), the whole is a phase in which the hydrogel capsule and the oil are suspended and can be homogeneously suspended.

[0078] In addition, image images of the fluorescence states in the reaction solutions of Example 2 (the fluorinated oil) and Example 3 (the soybean oil) are shown in Fig. 10. Note that the left side (Fluorescence) of Fig. 10 shows a fluorescence image, and the right side (Merge) is an image obtained by superimposing a bright-field image on the fluorescence image. As shown in Fig. 10, it is confirmed that the hydrogel capsules are densely dispersed in the reaction solution of Example 2 (the fluorinated oil) compared to the reaction solution of Example 3 (the soybean oil).

[0079] The analysis results of the fluorescence intensity shown in Fig. 8 are, firstly, regarding the specific gravity of the fluorinated oil (about 1.4 to 1.6) and the specific gravity of the soybean oil (about 0.9), the specific gravity of the fluorinated oil is a value far from the specific gravity of water (1.0), which is the main component of the hydrogel capsule, and it is considered that this is due to the fact that the fluorinated oil is more likely to separate from the hydrogel capsule than the soybean oil. Secondly, the thermal conductivity of the soybean oil (0.18 Wm -1 K -1 about) is higher than the thermal conductivity of the fluorinated oil (0.06 Wm -1 K -1 ~0.08 Wm -1 K -1 about), and it is considered that this is due to the fact that the heat exchange between the hydrogel capsule and the oil is made more efficient. In other words, when using the soybean oil, the homogeneous suspension of the hydrogel capsules and the oil results in a sparse dispersion of the hydrogel capsules within a unit area, rather than dense clustering. Furthermore, the high thermal conductivity allows for efficient heat exchange between the hydrogel capsules and the oil, which is thought to enable the reaction associated with the thermal cycle to proceed efficiently.

[0080] Figure 11 shows the relationship between specific gravity and thermal conductivity for typical oils and heat transfer fluids. As shown in Figure 11, fluorine oils are grouped in the lower right region of the figure, which has a high specific gravity (large difference in specific gravity from water) and low thermal conductivity. Therefore, groups such as vegetable oils, mineral oils, and silicone oils, which have a small difference in specific gravity from water and high thermal conductivity, can be expected to perform more efficient heat exchange between the hydrogel capsules and the oils. This has significant technical importance because it allows the PCR reaction associated with the thermal cycle to be achieved without using the aforementioned fluorine oil, whose manufacture is being restricted due to its harmful effects, and in fact, the reactivity is better with oils other than the aforementioned fluorine oil. [Explanation of Symbols]

[0081] 1 Hydrogel Capsule 2 Core section 3. Heat-resistant gel film 4 Dispersion medium 10 Dispersion

Claims

1. A dispersion formation step in which a core-shell type hydrogel capsule is dispersed in a dispersion medium, the core portion of which contains at least an aqueous reaction solution comprising nucleic acid, an amplification component necessary for amplifying the nucleic acid, and a detection component that emits a detection signal in response to the amplification of the nucleic acid, and the outer circumference of the core portion is covered with a heat-resistant gel film, thereby forming a dispersion; A nucleic acid amplification step is performed by subjecting the dispersion to a PCR thermal cycle to obtain the nucleic acid amplification product in the hydrogel capsule, A detection step for detecting the aforementioned detection signal, A nucleic acid detection method characterized by including the following.

2. The nucleic acid detection method according to claim 1, wherein the volume of the hydrogel capsule is 0.5 fL to 20 μL.

3. The nucleic acid detection method according to claim 1 or 2, wherein the hydrogel capsule has mechanical strength that prevents it from rupturing even when pressed from one direction and its diameter is increased by up to two times.

4. The nucleic acid detection method according to claim 1 or 2, wherein the heat-resistant gel film can be sol-formed while maintaining the nucleic acid sequence of the amplification product amplified within the core.

5. The nucleic acid detection method according to claim 1 or 2, wherein the heat-resistant gel film forming material is selected from sodium alginate, poly(N-isopropylacrylamide-co-acrylic acid), polylactic acid, and photocurable chitosan.

6. The nucleic acid detection method according to claim 1 or 2, wherein the specific gravity of the dispersion medium is 0.6 to 1.4, and the thermal conductivity of the dispersion medium is 0.05 W / m·K to 0.23 W / m·K.

7. The nucleic acid detection method according to claim 1 or 2, wherein the viscosity of the dispersion medium is 0.5 mPa·s to 500 mPa·s.

8. The nucleic acid detection method according to claim 1 or 2, wherein the thermal cycle in the nucleic acid amplification step is provided by replacing the dispersion medium in the dispersion with at least a high-temperature component of the dispersion medium that is temperature-controlled to the high-temperature side and a low-temperature component of the dispersion medium that is temperature-controlled to the low-temperature side.

9. A hydrogel capsule characterized by having a core-shell structure in which at least an aqueous reaction solution containing nucleic acid, an amplification component necessary for amplifying the nucleic acid, and a detection component that emits a detection signal in response to the amplification of the nucleic acid is disposed in the core portion, and the outer circumference of the core portion is covered with a heat-resistant gel film.

10. A dispersion of hydrogel capsules, characterized in that a core-shell type hydrogel capsule is dispersed in a dispersion medium, the core portion of which contains at least an aqueous reaction solution comprising a nucleic acid, an amplification component necessary for amplifying the nucleic acid, and a detection component that emits a detection signal in response to the amplification of the nucleic acid, and the outer circumference of the core portion is covered with a heat-resistant gel film.

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