Digital PCR device, digital PCR apparatus, and digital PCR conducting method

Abstract

To provide a digital PCR device technology which enables solution introduction without leaving air bubbles in a fraction by introducing a sample solution into a fraction by utilizing the air permeability of a constituent material of a digital PCR device, the digital PCR device technology having high thermal efficiency that enables the conduction of digital PCR.SOLUTION: A digital PCR device for conducting digital PCR comprises: an air-permeable chip having a plurality of fractions on the surface; an inlet for introducing a solution into the fractions on the chip; and an outlet for discharging the solution. The digital PCR device has: a lid that covers the chip via the solution chamber; and a support that is fixed to the surface on the side opposite to the lid with respect to the chip and has a breathable structure. The support has high thermal conductivity.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a digital PCR device, a digital PCR apparatus and a method for performing digital PCR. [Background technology]

[0002] Digital PCR (Polymerase Chain Reaction) is a technology that detects nucleic acids with high sensitivity, and compared to conventional real-time quantitative PCR, it can detect low-frequency gene mutations. The measurement flow in digital PCR includes a process in which a limit-diluted nucleic acid solution is introduced into nanoscale to microscale microareas, and a PCR process in which the temperature of the solution separated into each area is controlled.

[0003] Patent Document 1 discloses a nucleic acid capture device for performing gene expression analysis on a single cell basis, and describes the method of introducing a sample suspension into the sample capture section as follows: "After the cell suspension is dripped, a suction operation is performed by pulling the syringe from the suction port, and the cell suspension is sucked into the single-cell nucleic acid capture chip."

[0004] Patent document 2 discloses a method and device for simultaneously analyzing the entire gene expression profile of an organism, and with regard to the thermal efficiency of fractions during temperature control, it states that "the substrate of the microplate assembly includes a through-hole plate and a backing sheet, where the holes in the plate form the sides of the wells and the backing sheet forms the bottom of the wells. The backing sheet includes a thermally conductive material capable of contacting the thermal cycler and transferring heat to the contents of the reaction chamber." [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US Patent No. 11299727 [Patent Document 2] International Publication No. 2005 / 028629 Summary of the Invention [Problem to be solved by the invention]

[0006] Patent Document 1 describes a method for introducing a sample suspension into a minute fraction in a nucleic acid capture device for performing gene expression analysis for each cell by applying negative pressure from the lower part of the minute fraction in the device into which the sample suspension has been introduced. However, in the device described in Patent Document 1, when performing temperature control required for a reaction such as PCR, the temperature is controlled through the material on the lower surface of the device, the space for applying negative pressure, and the filter, which causes a problem of reduced thermal efficiency. For example, when the device described in Patent Document 1 is used for digital PCR, sufficient nucleic acid amplification does not occur due to the above-mentioned thermal efficiency, and it is possible that nucleic acid cannot be quantified.

[0007] Patent Document 2 describes a device for performing gene expression profile analysis in which a highly thermally conductive material is used for the sheet constituting the bottom surface of the well for holding the sample solution. However, in the device described in Patent Document 2, it is difficult to introduce the sample solution without leaving air bubbles in the well, which can impair solution division and reduce quantitative accuracy when performing digital PCR.

[0008] One object of the present invention is to provide a digital PCR technology that can introduce a sample solution into a fraction by utilizing the breathability of the material that constitutes the device, thereby enabling the introduction of the solution without leaving any air bubbles in the fraction, and has high thermal efficiency that enables digital PCR to be performed. [Means for solving the problem]

[0009] An example of a digital PCR device according to the present invention is A digital PCR device for performing digital PCR, comprising: A breathable tip having a plurality of compartments on its surface; a lid covering the chip through a solution chamber, the lid having an inlet for introducing a solution into the chip and an outlet for discharging the solution; a support fixed to a surface of the chip opposite to the lid and having a structure that allows ventilation; having The support has high thermal conductivity.

[0010] An example of a digital PCR device according to the present invention is A digital PCR apparatus for performing digital PCR using the digital PCR device described above, A vacuum chamber that fixes the digital PCR device by supporting the support and creates a negative pressure in a space facing the support side of the digital PCR device; A temperature controller that heats the digital PCR device from the support side; Equipped with.

[0011] An example of a digital PCR device according to the present invention is A digital PCR apparatus for performing digital PCR using a digital PCR device, comprising: The digital PCR device comprises: A breathable tip having a plurality of compartments on its surface; a lid covering the chip through a solution chamber, the lid having an inlet for introducing a solution into the chip and an outlet for discharging the solution; having The digital PCR device comprises a vacuum chamber, The vacuum chamber has a support having a breathable structure fixed to one surface, and the one surface is made negative pressure; The digital PCR apparatus includes a temperature regulator that heats the digital PCR device disposed relative to the vacuum chamber from the support side.

[0012] An example of a method for performing digital PCR according to the present invention includes: A method for performing digital PCR using the digital PCR device described above, comprising: Introducing a sample solution into the inlet of the digital PCR device; A step of creating a negative pressure in a space facing the support side of the digital PCR device; separating the sample solution by introducing a separation solution through the inlet; A step of controlling the temperature of the digital PCR device via the support; Equipped with. Effect of the Invention

[0013] According to the present invention, high thermal efficiency can be achieved in a device that utilizes the breathability of the device to introduce a sample solution into a fraction. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a side view showing the configuration of a digital PCR device to which a breathable support is fixed. [Diagram 2] FIG. 2 is a top view showing the configuration of the digital PCR device shown in FIG. 1. [Diagram 3] FIG. 2 is a diagram illustrating the configuration of an apparatus in which the digital PCR device shown in FIG. 1 is placed on a vacuum chamber. [Figure 4] FIG. 2 is a diagram illustrating the configuration of an apparatus in which the digital PCR device shown in FIG. 1 is placed on a temperature regulator. [Diagram 5] A diagram showing the introduction of a sample solution into the solution chamber of the digital PCR device shown in Figure 1. [Figure 6] This figure shows how the sample solution is introduced into the solution chamber of the digital PCR device shown in Figure 1, and then the solution is introduced into the fractions by depressurizing the vacuum chamber. [Figure 7] 6 shows the results of three-dimensional fluorescence observation after a fluorescent solution was introduced into the solution chamber of the digital PCR device shown in FIG. 1 by the method shown in FIG. 5. [Figure 8]7 shows the results of three-dimensional fluorescence observation after a fluorescent solution was introduced into a compartment of the digital PCR device shown in FIG. 1 by the method shown in FIG. 6. [Figure 9] 7A and 7B show the process of introducing a sample solution into the solution chamber and fractions of the digital PCR device shown in FIG. 1 by the method shown in FIGS. 5 and 6, and then introducing a separation solution into the solution chamber to divide the sample solution into fractions. [Figure 10] FIG. 11 is a diagram illustrating how a sample solution is introduced into the solution chamber and fractions of the digital PCR device shown in FIG. 1 by the methods shown in FIGS. 5, 6, and 9, and then the sample solution is further divided into fractions, after which the digital PCR device is placed in a temperature controller and temperature-regulated. [Figure 11] Fluorescence images showing the results of digital PCR in Example 1 of the present invention. [Figure 12] FIG. 11 is a side view showing the configuration of a digital PCR device and an apparatus according to Example 2 of the present invention. [Figure 13] FIG. 11 is a side view showing the configuration of a digital PCR device according to Example 3 of the present invention. [Figure 14] FIG. 14 shows the results of fluorescence measurements after introducing and dividing a fluorescent solution into compartments of a digital PCR device configured as shown in FIG. 13. [Figure 15] FIG. 14 shows the results of fluorescence measurements after introducing and dividing a fluorescent solution into compartments of a digital PCR device configured as shown in FIG. 13. [Figure 16] FIG. 11 is a side view showing an outline of the configuration of a digital PCR device and an apparatus according to Example 4 of the present invention. [Figure 17] FIG. 11 is a conceptual diagram for illustrating the results of measurements that can be performed according to Example 4 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0016] [Example 1] Example 1 will be described below with reference to Figures 1 to 11. This example is a method for implementing digital PCR that can suppress deformation of a digital PCR device during the steps of introducing and dividing a solution using the digital PCR device, and can control the temperature with sufficient thermal efficiency during the PCR step.

[0017] 1 is a side view showing an outline of the configuration of a digital PCR device 100 in this embodiment. The digital PCR device 100 is used to perform digital PCR according to the digital PCR performing method described herein. The digital PCR device 100 includes a breathable chip 101 having a plurality of compartments 102 on its surface.

[0018] For example, PDMS (Polydimethylsiloxane) is a suitable material for the chip 101 because it has good breathability. The thickness of the chip will be described later.

[0019] The number of fractions 102 can be 1,000 to 100,000,000. The shape of the fractions 102 in a horizontal plane (hereinafter, fraction shape) can be various shapes such as a circle, a polygon (a rectangle, a pentagon, a hexagon, etc.), and a combination thereof, and is preferably a circle or a hexagon in order to ensure the planar arrangement density of the fractions.

[0020] The width or diameter of the shape of the fraction 102 in a horizontal plane (hereinafter, fraction width) can be 1 μm to 1000 μm, and is preferably 10 μm to 100 μm from the viewpoint of the resolution in microscopic observation and the planar array density of the fraction. The depth of the fraction 102 is determined relative to the fraction width in order to estimate the volume per fraction according to the application of the digital PCR device 100, and can be, for example, 1 μm to 1000 μm.

[0021] The planar arrangement period of the fractions 102 (hereinafter, fraction period) is a value that determines the planar arrangement density of the fractions, and is determined relatively to the fraction width, and can be, for example, 1.01 to 100 times the fraction width.

[0022] The digital PCR device 100 has a solution chamber 103 on the surface of the chip 101 where the fractions 102 are formed. This solution chamber 103 is used to transport the sample solution and the separation liquid. The depth of the solution chamber may be 100 nm to 10 mm, as long as the length allows liquid to be introduced by a syringe, air pressure pump, or pipette operation.

[0023] At least a portion of the internal surface of the digital PCR device 100, which is composed of the surfaces of the solution chamber 103, the compartment 102, and the chip 101, may be subjected to surface treatment such as hydrophilization, hydrophobization, chemical modification, etc., or may not be subjected to any treatment at all.

[0024] The digital PCR device 100 has a lid 104 that covers the chip 101 via the solution chamber 103 (for example, sandwiching the solution chamber 103). The material of this lid 104 is preferably made of a material that is transparent in the visible light range or a material that has high transmittance to visible light in order to detect fluorescence through the lid. Also, it is preferable that the material be made of a material with low autofluorescence. The thickness of the lid can be 100 μm to 10 mm in order to suppress deformation of the chip.

[0025] The material of the lid 104 may be, for example, a resin (acrylic resin, cycloolefin resin, polycarbonate, etc.), a visible light transparent inorganic material (quartz, sapphire, various glasses, ITO, etc.), etc. In particular, when the material of the lid 104 is made of resin or glass, it is easy to make the lid 104 transparent.

[0026] The lid 104 has an inlet 105 for introducing a solution into the compartment 102 of the chip 101, and an outlet 106 for discharging the solution. The inlet 105 and the outlet 106 can be formed by injection molding when molding the lid, drilling holes in a plate material, or the like.

[0027] Here, the thickness of the chip is defined as the sum of the depth of the compartment and the distance from the bottom of the compartment to the surface of the chip opposite the lid (chip bottom thickness). If the chip bottom thickness (i.e., the thickness of the chip excluding the depth of the compartment 102) is thinner than 50 μm, manufacturing becomes difficult, and if it is thicker than 2000 μm, thermal conductivity and air permeability decrease, so it is preferable to set the chip bottom thickness to 50 μm to 2000 μm. In addition, considering the thermal conductivity of the chip, it is more preferable that the chip bottom thickness is 1000 μm or less.

[0028] The digital PCR device 100 has a support 107 fixed to the surface of the chip 101 opposite to the lid 104. The support 107 has a breathable structure.

[0029] The support 107 has high thermal conductivity. The thermal conductivity of the support 107 is preferably higher than that of the chip material, and can be set to 0.2 W / m·K or more. Such a value can improve the thermal efficiency of the digital PCR device 100. Furthermore, the thermal conductivity of the support 107 is preferably 1 W / m·K or more, and more preferably 100 W / m·K or more.

[0030] In addition, since the thermal resistance of the support increases in direct proportion to its thickness, the thinner the support is, the better from the viewpoint of thermal conductivity, but a certain thickness is required from the viewpoint of suppressing deformation of the chip. The thickness of the support is, for example, 10 μm to 5000 μm, and preferably 50 μm to 500 μm.

[0031] The breathable structure has at least one physically continuous through hole. The material of the support 107 is preferably made of an inorganic material in order to provide the above-mentioned thermal conductivity. For example, gold, silver, copper, platinum, aluminum, tungsten, molybdenum, chromium, titanium, nickel, alloys containing these in their composition, silicon, silicon carbide, silicon nitride, glass, etc. can be used. By constructing the support 107 from an inorganic material, strength can be easily ensured.

[0032] The holes (openings) that the through holes make in the surface of the support can have any shape, such as a circle, a polygon (rectangle, pentagon, hexagon, etc.), or a combination thereof.

[0033] The width or diameter of the pore in the horizontal plane (hereinafter referred to as pore width) can be 0.1 μm to 1000 μm, and from the viewpoint of supporting the chip and suppressing deformation, it is preferably 0.1 μm to 100 μm. In addition, when a porous body is used as the support, it is preferable that the average porosity thereof is 90% or less.

[0034] In order to reduce the variation in the area of ​​each fraction overlapping the support surface in the thickness direction, the planar arrangement period of the holes (hereinafter, hole period) is preferably equal to or less than the fraction period, and is most preferably equal to the fraction period. In addition, if the hole period exceeds the fraction period, there is a high possibility that the thermal efficiency will vary depending on the relative position of the fraction with respect to the holes of the support during temperature control, so it is preferable to use a separate measure. Specifically, the temperature is controlled with sufficient thermal efficiency in the fractions overlapping the support surface in the thickness direction, but the thermal efficiency is insufficient in the fractions overlapping the holes in the thickness direction, so there is a possibility that a sufficient temperature control effect cannot be obtained, so measures against these are preferable.

[0035] 2 is a top view showing the outline of the configuration of the digital PCR device 100. Due to the constraint that the components of the digital PCR device 100 are stacked vertically, the illustration of the lid 104 and the support 107 is omitted. The inlet 105 and the outlet 106 formed in the lid 104 may be directly connected to the solution chamber 103 as shown in FIG. 2(a) or indirectly connected to the solution chamber 103 via a channel 200 as shown in FIG. 2(b).

[0036] The shape of the solution chamber 103 in the horizontal plane can be various shapes, such as a circle, a polygon (square, pentagon, hexagon, etc.), and combinations thereof.

[0037] The above is the configuration of the digital PCR device 100 in this embodiment. The configuration of the digital PCR device in this embodiment will be described below.

[0038] 3 is a schematic diagram showing an outline of the configuration of a digital PCR device in the steps of solution introduction and solution division using the digital PCR device 100. This digital PCR device uses the digital PCR device 100 to carry out digital PCR.

[0039] The digital PCR apparatus includes a vacuum chamber 300. The vacuum chamber 300 fixes the digital PCR device 100 by supporting the support 107, and creates a negative pressure in the space facing the support 107 side of the digital PCR device 100.

[0040] The support 107 in the digital PCR device 100 is placed so as to cover an open space 301 of a vacuum chamber 300 connected to a pressure reducer 303. This causes at least a part of the surface of the chip 101 to face the open space 301 across a through hole in the support 107. Therefore, pressure changes in the open space 301 are directly transmitted to the surface of the chip 101.

[0041] For example, when the open space 301 is decompressed using the pressure reducer 303 connected via the connection hole 302, the air pressure on the surface of the chip 101 is also reduced, and a pressure difference occurs between the open space 301 side and the solution chamber 103 and the compartment 102 side across the chip 101 and the support 107. Here, if the support 107 has a structure with through holes as described above, the pressure difference can be appropriately utilized.

[0042] Since the chip 101 is made of a breathable material, air moves from the relatively high-pressure solution chamber 103 and fraction 102 side to the relatively low-pressure open space 301 side across the chip 101. In other words, it is possible to release the air in fraction 102 to the outside of the digital PCR device 100 and introduce the sample solution into fraction 102 instead.

[0043] 4 is a schematic diagram showing an overview of the device configuration in the PCR process using the digital PCR device 100. The digital PCR device is equipped with a temperature regulator 400. The temperature regulator 400 heats the digital PCR device 100 from the support 107 side. In the PCR process, after the solution division process is completed, the digital PCR device 100 is removed from the open space 301 of the vacuum chamber 300 and moved onto the temperature regulator 400.

[0044] The above is the configuration of the digital PCR device in this embodiment. This embodiment will be described below along with the flow of operation of this device.

[0045] 5 is a diagram illustrating the step of introducing a sample solution into the solution chamber 103. As shown in Fig. 5(a), the sample solution 500 is connected to the inlet 105, and pressure is applied to the sample solution 500 or air, thereby introducing the sample solution 500 into the solution chamber 103. Pressure can be applied by pipetting or various pumps such as a syringe pump or a peristaltic pump, and the pressure may be applied from the inlet 105 or reduced pressure from the outlet 106.

[0046] Alternatively, the sample solution 500 may be introduced by utilizing the capillary force due to the surface properties of the inner walls of the solution chamber 103 and the flow channel 200 (see FIG. 2) connecting the solution chamber 103 to the inlet 105 and outlet 106. By the above-mentioned method, the sample solution 500 fills the solution chamber 103 and reaches the outlet 106. If the surface properties of the surfaces of the chip 101, the fraction 102, and the lid 104 facing the solution chamber 103 are not controlled, the air inside the fraction will generally remain without being replaced by the sample solution, as shown in FIG. 5(b).

[0047] 6 is a diagram illustrating the step of introducing a sample solution into the fraction 102. In the method shown in FIG. 6(a), the sample solution is introduced into the solution chamber 103 from the inlet 105 of the digital PCR device 100. After the sample solution is introduced, a pressure reducer 303 is connected via a connection hole 302 of the vacuum chamber 300, and the open space 301 is depressurized as shown in FIG. 6(b). That is, the space facing the support 107 side of the digital PCR device 100 is made negative pressure.

[0048] The point in time when pressure reducer 303 is connected to vacuum chamber 300 may be either before or after the step of introducing the solution into solution chamber 103. This operation completes the step of introducing the sample solution into solution chamber 103 and fraction 102.

[0049] Fig. 7 shows the results of three-dimensional fluorescence imaging performed after introducing a fluorescent solution into a solution chamber of a digital PCR device 100 having a circular compartment with no surface treatment applied to the inner surface, using the method described in Fig. 5. Fig. 7(a) is a fluorescent image in an XY cross section 704 (shown in Fig. 7(b)) passing through the central depth of the compartment, and Fig. 7(b) is a fluorescent image in an XZ cross section 701 (shown in Fig. 7(a)) passing through the center of the compartment. The compartment wall surface 700 is indicated by a dashed line.

[0050] In Fig. 7(a), no area of ​​high fluorescence intensity is observed. This indicates that the fluorescent solution has not been introduced into the fraction. In Fig. 7(b), fluorescence intensity is observed in the solution chamber 702 area, but not in the fraction interior 703 area. This indicates that the fluorescent solution has been introduced into the solution chamber, but not into the fraction.

[0051] Figure 8 shows the results of three-dimensional fluorescence photography of a digital PCR device 100 having circular compartments with no surface treatment on the inner surface, after introducing a fluorescent solution into the solution chamber by the method shown in Figure 5 and then into the compartment by the method shown in Figure 6. Figure 8(a) is a fluorescent image in an XY cross section 704 (shown in Figure 8(b)) passing through the central depth of the compartment, and Figure 8(b) is a fluorescent image in an XZ cross section 701 (shown in Figure 8(a)) passing through the center of the compartment. The compartment wall surface 700 is indicated by a dashed line.

[0052] In Fig. 8(a), high fluorescence intensity is observed inside the compartment wall surface 700. This indicates that the fluorescent solution has been introduced into the compartment up to at least the central height. In Fig. 8(b), high fluorescence intensity is observed not only in the solution chamber 702 but also in the compartment interior 703 region. Furthermore, fluorescence intensity is observed down to the bottom of the compartment. This indicates that the fluorescent solution has been introduced into the solution chamber and the compartment.

[0053] The experimental results in FIGS. 7 and 8 show that the solution introduction method of this example makes it possible to introduce a solution into a compartment without performing surface treatment on the inner surface of the digital PCR device 100.

[0054] Fig. 9 is a diagram illustrating the process of dividing the sample solution into fractions 102. After the process of introducing the sample solution into solution chamber 103 and fractions 102 is completed by the method shown in Fig. 5 and Fig. 6, separation liquid 900 is connected to inlet 105 as shown in Fig. 9(a), and pressure is applied to the separation liquid or air, thereby introducing the separation liquid into solution chamber 103 as shown in Fig. 9(b).

[0055] Here, the separation liquid is a liquid that is immiscible with water, and examples of the method that can be used include mineral oil, silicone oil, fluorine-based oil, etc. Pressure can be applied to the separation liquid by pipetting, or by using various pumps such as a syringe pump or a peristaltic pump. Pressurization can be performed from inlet 105, or depressurization can be performed from outlet 106.

[0056] By this operation, the separation liquid 900 fills the solution chamber 103 and reaches the outlet 106. At this time, it is expected that the introduction of the separation liquid into the solution chamber 103 will remove the sample solution from the surface of the chip 101. This causes the sample solution to be divided into individual fractions and lose continuity as a fluid. In other words, the sample solution can be separated by introducing the separation liquid from the inlet 105.

[0057] FIG. 10 is a diagram illustrating the PCR process. After the solution division process is completed, as shown in FIG. 4, the digital PCR device 100 is removed from the open space 301 of the vacuum chamber 300 and moved onto the temperature regulator 400. In this configuration, the temperature regulator is used to regulate the temperature of the digital PCR device 100 via the support 107, and PCR is performed on the sample solution. At this time, the sample solution divided into the fractions 102 of the digital PCR device 100 is heated and cooled by the temperature regulator via the chip 101 and the support. Although no nucleic acid amplification reaction occurs in the digital PCR negative fraction 1000, nucleic acid is amplified in the digital PCR positive fraction 1001.

[0058] The above is the operation of the digital PCR device in this embodiment. After the PCR process, the digital PCR device 100 detects fluorescence through the lid 104. The method of detecting fluorescence can be designed by a person skilled in the art based on known techniques, and therefore will not be described here.

[0059] Figure 11 shows the results of fluorescence observation after digital PCR according to this embodiment. A clear difference can be seen in the fluorescence intensity between the digital PCR negative fraction 1100 and the digital PCR positive fraction 1101. Furthermore, no areas of clearly high fluorescence intensity can be seen in the non-fractionated portion 1102. This indicates that the solution was stably maintained in a state divided into fractions during the PCR process. These results demonstrate that digital PCR can be performed normally according to this embodiment.

[0060] The digital PCR device 100 of this embodiment comprises a chip 101 having a plurality of compartments 102 on its surface and made of a breathable material, a solution chamber 103, a lid having an inlet 105 and an outlet 106 and covering the solution chamber 103, and a support 107 having high thermal conductivity and a breathable structure.

[0061] Chip deformation causes the divided solutions to jump out into the solution chamber or combine with the solutions in the adjacent compartments, which reduces the discrimination accuracy of digital PCR. In this example, the support is sufficiently hard against the chip, so that deformation of the chip can be suppressed throughout the steps of introducing the solution, dividing the solution, and PCR, even when a thin chip is used.

[0062] There is a concern that inserting a support between the chip and the temperature regulator may reduce the efficiency of temperature control of the solution in the fraction, but by using a material with high thermal conductivity for the support, it is possible to perform digital PCR, which requires high thermal efficiency.

[0063] By utilizing the breathability of the chip material for introducing the solution into the fractions, it becomes unnecessary to perform surface treatment such as hydrophilization, hydrophobization, or chemical modification on the inner surface of the digital PCR device 100, thereby reducing costs. In addition, the solution can be introduced into the fractions regardless of the conditions of the inflow speed, solution viscosity, and wettability of the solution to the inner surface of the digital PCR device 100, so that the percentage of fractions in which the solution introduction can be completed normally increases.

[0064] [Example 2] Next, Example 2 will be described with reference to Fig. 12. Example 2 simplifies the implementation process and reduces the device cost by fixing the support 107 in Example 1 to the open space 301 of the vacuum chamber, rather than to the digital PCR device. In Fig. 12, the same reference numerals as in Figs. 1 to 11 denote the same parts, and therefore repeated explanations will be omitted.

[0065] 12(a) is a schematic diagram of the configuration of a digital PCR apparatus of Example 2. The configuration of the digital PCR device of Example 2 differs from that of Example 1 only in that it does not have a support. In Example 2, a support 1200 having a breathable structure is directly embedded in the opening of a vacuum chamber 1201. The support 1200 is fixed to one surface of the vacuum chamber 1201, and the vacuum chamber 1201 can create a negative pressure on this one surface. Also, in Example 2, the digital PCR device is fixed to the surface of the vacuum chamber 1201 having the support 1200.

[0066] The digital PCR apparatus includes a temperature regulator 1203. The temperature regulator 1203 heats the digital PCR device disposed relative to the vacuum chamber 1201 from the support 1200 side. The vacuum chamber 1201 is fixed to the temperature regulator 1203.

[0067] In the PCR process of Example 2, the sample solution in the fraction is heated and cooled by the temperature regulator 1203 via the chip 101, the support 1200, and the vacuum chamber 1201. Therefore, in Example 2, it is preferable that not only the support 1200 but also the vacuum chamber 1201 are made of a material with high thermal conductivity. The material of the vacuum chamber can be, for example, a metal (gold, silver, copper, platinum, aluminum, tungsten, molybdenum, chromium, titanium, nickel, and alloys containing these in their composition, etc.) or a semiconductor (silicon, silicon carbide, silicon nitride, etc.).

[0068] As shown in FIG. 12(b), in the PCR process of Example 2, the inside of the vacuum chamber and the ventilation structure of the support may be filled with liquid 1204. This improves the thermal efficiency of the entire device, making it easier for heat from the temperature regulator to be transferred to the compartments inside the digital PCR device. The liquid used for filling may be, for example, water, silicone oil, mineral oil, liquid paraffin, or alcohol, but is not limited to these. Furthermore, a filler may be dispersed in the liquid used for filling to improve its thermal conductivity.

[0069] The above is the configuration of the digital PCR device in this embodiment. Note that the flow of operation of this device is basically the same as in embodiment 1, so a description thereof will be omitted.

[0070] The device configuration in Example 2 differs from Example 1 in that the support 1200 is fixed to a vacuum chamber 1201 rather than to a digital PCR device, and that high thermal conductivity is preferable in the vacuum chamber 1201 .

[0071] Since there is no need to move the digital PCR device throughout the entire workflow of digital PCR, including solution introduction, solution division, and PCR, it is possible to achieve stable solution division, simplify the implementation process, and reduce device costs. Furthermore, by incorporating an optical system for detecting fluorescence in digital PCR into the device configuration of this embodiment, there is no need to move the digital PCR device between installation and digital PCR detection. This is an advantageous feature for automating the workflow of digital PCR.

[0072] [Example 3] Next, Example 3 will be described with reference to Figures 13 to 15. Example 3 has a configuration in which the support in Example 1 is provided with an antireflection mechanism, which makes it possible to reduce background light intensity and variations in background light intensity in fluorescence detection.

[0073] 13 is a schematic diagram illustrating the configuration of a digital PCR device in Example 3. The configuration of the digital PCR device in Example 3 differs from that in Example 1 only in that the support 1300 has an antireflection mechanism.

[0074] For the support 1300, a material having a breathable structure and low visible light reflectance can be used. For example, a resin breathable body (made of one or more of polyester, polyethylene, polypropylene, polystyrene, nylon, polyurethane, cellulose, cycloolefin, epoxy, vinyl chloride), a metal breathable body (made of gold, silver, copper, platinum, aluminum, tungsten, molybdenum, chromium, titanium, nickel, and alloys containing these in their composition, etc.), a non-metal breathable body (made of one or more of silicon, silicon carbide, silicon nitride, alumina, glass, and carbon), and a composite of these can be used. In addition, these anti-reflection layers may be subjected to secondary processing such as surface treatment or coating, or may be filled with a filler in order to reduce reflected light.

[0075] In particular, when the reflectance of the support 1300 in the visible light region is low, it is possible to prevent light reflection upward from the digital PCR device during fluorescence detection, and it is possible to reduce background light intensity and variation in background light intensity during fluorescence detection. For example, it is preferable that the reflectance in the visible light region is 20% or less.

[0076] Furthermore, in consideration of the efficiency of temperature control of the sample solution in the compartment, it is preferable that the thermal conductivity of the antireflection layer is 1 W / m·K or more.

[0077] The above is the configuration of the digital PCR device in this embodiment. Note that the flow of operation of this device is basically the same as in embodiment 1, so a description thereof will be omitted.

[0078] Figure 14 shows the results of fluorescence measurements taken after dividing a fluorescent solution in a digital PCR device using a 0.3 mm-thick PDMS chip with circular fractions of 0.1 mm diameter, 0.1 mm depth, and fraction period of 0.15 mm, in which a 0.5 mm-thick aluminum alloy plate with circular through-holes of 0.5 mm diameter and 1 mm pore period was used as the support.

[0079] FIG. 14(a) is a fluorescent image of a digital PCR device in which a fluorescent solution was divided into fractions, and FIG. 14(b) is a histogram of the fluorescence intensity averaged per fraction.

[0080] From FIG. 14(a), it can be seen that the fluorescent molecule aqueous solution is divided, since the intensity is high inside the fraction and low outside. However, there are also areas with high fluorescence intensity according to the shape of the support surface. This is because the excitation light is reflected by the support and is not completely cut by the fluorescence filter for fluorescence measurement, resulting in the fluorescence image. The magnitude relationship between this reflected light intensity and the fluorescence intensity in the well can be seen from FIG. 14(a). That is, the first peak 1400 (intensity: about 7500) in FIG. 14(b) indicates the fluorescence intensity of the fraction contained inside the through-hole of the support, and the second peak 1401 (intensity: about 15000) indicates the sum of the fluorescence intensity of the fraction and the reflected light intensity by the support.

[0081] This result suggests that when an aluminum alloy plate is used as the support, in the fraction of the area overlapping the surface of the support, the reflected light at a level equivalent to the fluorescence intensity within the fraction is superimposed on the fluorescence image, increasing the background intensity and decreasing the measurement accuracy. In addition, when the pore period of the support is larger than the fraction period, a clear difference occurs in the fluorescence intensity between the fraction that overlaps with the pores in the thickness direction and the fraction that does not, which leads to variation in the fluorescence intensity between the fractions, which is thought to interfere with the discrimination of positive and negative results in digital PCR.

[0082] Figure 15 shows the results of fluorescence measurement when a copper fiber-resin fiber composite was used as the antireflection layer inserted between the support and the chip in Example 3. The conditions for the digital PCR device, sample solution introduction, solution separation, and fluorescence measurement differ from those in Figure 14 only in that an antireflection layer was inserted during fluorescence measurement.

[0083] In Figure 15(a), no variation in fluorescence intensity according to the surface shape of the support is observed. Similarly, in Figure 15(b), no variation in fluorescence intensity between fractions is observed. Furthermore, the peak intensity of peak 1500 in Figure 15(b) is approximately 7500, which roughly coincides with the position of the first peak in Figure 14(b), suggesting that the influence of reflected light by the support can be eliminated by inserting a metal fiber / resin fiber composite as an anti-reflection layer.

[0084] The device configuration in Example 3 differs from Example 1 in that an anti-reflection layer is inserted between the chip and the support.

[0085] During fluorescence detection, the antireflection layer prevents excitation light from reaching the support surface and prevents light from being reflected upwards in the digital PCR device, thereby reducing background light intensity and variability in background light intensity during fluorescence detection.

[0086] [Example 4] Next, Example 4 will be described with reference to Figures 16 and 17. Example 4 relates to a method for performing fluorescence measurement while controlling the temperature of a digital PCR device using a temperature regulator in the method or configuration of Examples 1 to 3.

[0087] 16 is a schematic diagram for explaining an outline of an apparatus configuration for measuring fluorescence while controlling the temperature of a digital PCR device in Example 4. In the digital PCR device 100, the fractions are held on the support side with respect to the solution chamber. Therefore, if temperature control is performed from the lid side in the PCR process, heat is transferred to the solution via the lid and the solution chamber filled with a separation solution, resulting in poor thermal efficiency and the temperature of the solution not following the temperature of the temperature controller, which causes no nucleic acid amplification.

[0088] Therefore, in order to simultaneously perform temperature control and fluorescence measurement using the digital PCR device in the first to third embodiments, it is preferable to set the support side in a temperature controller 1601 to control the temperature, and set a fluorescence detection unit 1600 on the lid side to perform fluorescence measurement from the lid side, as shown in Fig. 16. The fluorescence detection unit 1600 includes a light source, an excitation filter, a fluorescence filter, a mirror, a dichroic mirror, a lens, a detector, a spectrometer, etc. By using the device configuration of Fig. 16, it is possible to perform any fluorescence measurement while controlling the temperature using the temperature controller 1601.

[0089] 17 is a schematic diagram showing an example of melting curve analysis that can be performed by Example 4. Melting curve analysis is a technique for identifying the type of a target gene by using a target gene in a sample and a fluorescently labeled probe that hybridizes to the target gene, and measuring and analyzing the melting curve. To measure the melting curve, the temperature dependency of the fluorescence intensity of the fluorescently labeled probe is measured.

[0090] In the device configuration of Example 4, after performing PCR on a sample solution containing a fluorescently labeled probe for melting curve analysis, the digital PCR device is temperature-controlled while performing fluorescence measurement to measure the melting curve of the target gene. By analyzing the inflection point of the melting curve, it is possible to obtain the melting temperature that depends on the target gene. In other words, melting curve analysis can be performed in this embodiment.

[0091] The device configuration in Example 4 is composed of a temperature controller, a digital PCR device, and a fluorescence detection unit, which are arranged in this order in a certain direction. In this device configuration in this Example, fluorescence detection is performed simultaneously with temperature control.

[0092] Melting curve analysis can be performed by measuring fluorescence while controlling the temperature.

[0093] The present invention is not limited to the above-described embodiments, and includes various modified examples. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those including all of the configurations described. It is also possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. It is also possible to add, delete, or replace a part of the configuration of each embodiment with another configuration. [Explanation of symbols]

[0094] 100: Digital PCR device 101: Tip 102: Fraction 103: Solution chamber 104: Lid 105: Inlet 106: Outlet 107:Support 200: Flow path 300: Vacuum chamber 301: Opening space 302: Connection hole 303: Pressure reducer 400: Temperature controller 500: Sample solution 700:Fractional wall 701: XZ section through the center of the fraction 702: Solution chamber 703: Inside the compartment 704: XY cross section through mid-height of fraction 900: Separation liquid 1000: Digital PCR negative fraction 1001: Digital PCR positive fraction 1100: Digital PCR negative fraction 1101: Digital PCR positive fraction 1102: Unfractionated part 1200: Support 1201: Vacuum chamber 1203: Temperature controller 1204:Liquid 1300: Support 1400: First peak 1401: The second peak 1500: Peak 1600: Fluorescence detection unit 1601: Temperature controller

Claims

1. A digital PCR device for performing digital PCR, comprising: A breathable tip having a plurality of compartments on its surface; a lid covering the chip through a solution chamber, the lid having an inlet for introducing a solution into the chip and an outlet for discharging the solution; a support fixed to a surface of the chip opposite to the lid and having a structure that allows ventilation; having The support has high thermal conductivity.

2. The digital PCR device of claim 1 , wherein the material of the chip is PDMS.

3. The digital PCR device of claim 1, wherein the thickness of the chip excluding the depth of the fraction is 50 μm to 2000 μm.

4. The digital PCR device of claim 1 , wherein the lid is made of a material that is transparent in the visible light range.

5. The digital PCR device of claim 4 , wherein the material of the lid is made of resin or glass.

6. The digital PCR device of claim 1 , wherein the support is made of an inorganic material.

7. The digital PCR device of claim 1 , wherein the breathable structure has at least one through-hole.

8. The digital PCR device of claim 1 , wherein the support has low reflectance in the visible light region.

9. The digital PCR device of claim 8 , wherein the reflectance is 20% or less.

10. The digital PCR device according to claim 1 , wherein the support has a thermal conductivity of 0.2 W / (m·K) or more.

11. A digital PCR apparatus for performing digital PCR using the digital PCR device according to claim 1, a vacuum chamber that fixes the digital PCR device by supporting the support and creates a negative pressure in a space facing the support side of the digital PCR device; A temperature controller that heats the digital PCR device from the support side; A digital PCR device equipped with

12. A digital PCR apparatus for performing digital PCR using a digital PCR device, comprising: The digital PCR device comprises: A breathable tip having a plurality of compartments on its surface; a lid covering the chip through a solution chamber, the lid having an inlet for introducing a solution into the chip and an outlet for discharging the solution; having The digital PCR device comprises a vacuum chamber; The vacuum chamber has a support having a breathable structure fixed to one surface, and the one surface is made to have a negative pressure; The digital PCR device includes a temperature controller that heats the digital PCR device disposed in the vacuum chamber from the support side. Digital PCR device.

13. 13. A method for performing digital PCR using the digital PCR device of claim 1, comprising: Introducing a sample solution into the inlet of the digital PCR device; A step of creating a negative pressure in a space facing the support side of the digital PCR device; separating the sample solution by introducing a separation solution through the inlet; Regulating the temperature of the digital PCR device via the support; A method for performing digital PCR comprising:

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