JP5141039B2 - Nucleic acid amplification apparatus and nucleic acid amplification method - Google Patents

Description

  The present invention relates to a nucleic acid amplification apparatus, a nucleic acid amplification container, and a nucleic acid amplification method. More specifically, the present invention relates to a nucleic acid amplification apparatus, a nucleic acid amplification container, and a nucleic acid amplification method that improve the temperature adjustment efficiency with respect to a nucleic acid amplification reaction solution and further facilitate sample injection.

  Nucleic acid amplification reactions such as PCR (Polymerase Chain Reaction) are applied in various fields in biotechnology. For example, in the medical field, diagnosis based on DNA or RNA base sequences is attracting attention, and in the agricultural field, DNA testing is used for determination of genetically modified crops.

  PCR is generally a double-stranded DNA dissociation reaction (hereinafter simply referred to as “dissociation reaction”), an annealing reaction between a single-stranded DNA generated by dissociation and a primer (hereinafter simply referred to as “annealing reaction”). This is carried out by repeating the double-stranded DNA extension reaction (hereinafter simply referred to as “extension reaction”) between the annealed single-stranded DNA and the primer. Specifically, conventionally, a method of adjusting a reaction temperature necessary for each of the above reactions by mainly putting a PCR reaction solution into individual microtubes and repeating heating and cooling has been mainly used.

  However, in order to perform PCR, complicated operations and a lot of time are required due to repeated adjustment of the temperature and handling of a small amount of sample. For example, in the method using the above-described microtube, it takes time to adjust the temperature of the member itself that heats the microtube. Therefore, it takes about 2 to 3 hours to repeat the temperature adjustment.

  Therefore, in recent years, techniques for performing PCR more simply and in a short time have been proposed (Patent Documents 1 to 3).

  Patent Document 1 discloses an apparatus including a plurality of heat blocks that can be adjusted in advance to each reaction temperature such as the above-described dissociation reaction. Patent Document 2 discloses a nucleic acid amplification substrate for use in the apparatus, and the nucleic acid amplification substrate is formed with a reaction liquid reservoir that is filled with a PCR reaction liquid when performing PCR. Patent Documents 1 and 2 disclose a technique in which PCR is performed by sequentially bringing the nucleic acid amplification substrate in which the reaction solution reservoir is filled with a PCR reaction solution into contact with the heat block. Thereby, it is said that the problem that it takes time to adjust the temperature of the member for heating the microtube as described above does not occur.

Patent Document 3 discloses a technique for performing a process from the preparation of a DNA sample to PCR on a single minute system platform. The micro system platform is formed with ports, flow paths, chambers and the like for injecting or mixing a sample and a reagent for extracting DNA. Then, the sample and the reagent move in the flow path and the chamber by the centrifugal force that rotates the micro system platform, so that DNA is extracted. Further, the micro system platform is provided with a heater, and the extracted DNA sample and PCR reagent can be mixed in a chamber in the micro system platform, and PCR can be performed by the heater. Therefore, it is said that operations such as DNA extraction necessary for performing PCR can be simplified, and PCR can be performed in a short time.
JP 2006-115742 A (published May 11, 2006) JP 2006-115741 A (published May 11, 2006) Special table 2003-502656 gazette (published on January 21, 2003)

  However, the devices disclosed in Patent Documents 1 to 3 have a problem that it takes a long time to adjust the temperature of the PCR reaction solution and the operation is not simple.

  In the devices disclosed in Patent Documents 1 and 2, it is necessary to repeat the process of bringing the nucleic acid amplification substrate into contact with only one heat block and adjusting each reaction temperature.

  Thus, a region having the temperature of the heat block in contact with each reaction is widely formed around the reaction liquid reservoir. Therefore, even if the nucleic acid amplification substrate is brought into contact with the next heat block, it takes a long time to adjust the temperature in the reaction liquid reservoir due to the influence of the temperature of the heat block that was in contact with the nucleic acid amplification substrate last time. It will be.

  For example, assume that the nucleic acid amplification substrate is first brought into contact with a heat block at 95 ° C. to perform a dissociation reaction, and then the nucleic acid amplification substrate is brought into contact with a heat block at 65 ° C. for the annealing reaction. At the time of the dissociation reaction, a region having a temperature of 95 ° C. or the vicinity thereof is widely formed not only in the reaction liquid reservoir but also in the vicinity thereof. For this reason, even during the annealing reaction, it takes a long time for the reaction liquid reservoir to reach 65 ° C. due to the influence of this region. That is, it takes a long time to cool the periphery of the reaction liquid reservoir so that the reaction liquid reservoir is not affected by this effect.

  In the micro-system platform disclosed in Patent Document 3, since it takes a long time to adjust the temperature of the heater itself, it takes a long time to adjust the temperature of the PCR reaction solution.

  Thus, when the temperature adjustment is performed by bringing the container used for the nucleic acid amplification reaction into contact with only the heat block adjusted to the respective reaction temperature or changing the temperature of the heater on one heater, It takes a lot of time to adjust the temperature.

  Moreover, in the nucleic acid amplification substrate disclosed in Patent Documents 1 to 3, a very small amount of reaction solution is used. However, precise work is required to inject a small amount of reaction solution. Therefore, work efficiency cannot be improved. Patent Documents 1 to 3 do not describe any such problem, and therefore do not disclose any technique for easily injecting a small amount of reaction solution.

  The present invention has been made in view of the above problems, and an object thereof is to provide a nucleic acid amplification apparatus, a nucleic acid amplification container, and a nucleic acid amplification method for performing a nucleic acid amplification reaction such as PCR in a simple and short time. There is to do.

  In order to solve the above-described problems, the nucleic acid amplification apparatus according to the present invention forms mounting means for mounting a nucleic acid amplification container and a plurality of heating regions adjusted to different temperatures on the mounting means. The first heating means is provided.

  According to said structure, the some heating area | region adjusted to different temperature can each be formed on the said mounting means. For example, a plurality of heating regions adjusted to reaction temperatures for dissociation reaction, annealing reaction, etc. can be formed on one mounting means.

  The mounting means can be mounted with the nucleic acid amplification container in a state where the entire nucleic acid amplification container is simultaneously heated in a plurality of heating regions. Thereby, the site | part which stored the nucleic acid amplification reaction liquid in a nucleic acid amplification container can be arrange | positioned sequentially in each said heating area | region, maintaining the said state. In other words, while maintaining the state where the entire nucleic acid amplification container is heated by at least two heating regions adjusted to different temperatures, the nucleic acid amplification reaction solution is sequentially heated in each of the above heating regions, thereby allowing the dissociation reaction. The reaction can be transferred sequentially such as from annealing to annealing.

  Thereby, the heating efficiency of the nucleic acid amplification reaction solution can be improved.

  The reason for this is that after the dissociation reaction at 95 ° C., the annealing reaction and the extension reaction are performed by cooling to 65 ° C., and the entire nucleic acid amplification vessel is heated simultaneously by the 95 ° C. heating region and the 65 ° C. heating region. Will be described as an example.

  First, during the dissociation reaction, the site where the nucleic acid amplification reaction solution is stored and its surroundings are heated to 95 ° C. According to said structure, the area | region heated at 65 degreeC also exists in the nucleic acid amplification container. Therefore, compared with the case where the nucleic acid amplification container is heated only at 95 ° C., the region at 95 ° C. formed around the site where the nucleic acid amplification reaction solution is stored is reduced. That is, the region of 65 ° C. or a temperature close to this increases on the nucleic acid amplification container. Thereby, at the time of annealing reaction, the area | region which should be cooled is narrow and the amount of heat which should be absorbed for cooling can also be reduced. Therefore, the nucleic acid amplification reaction solution can be quickly adjusted to 65 ° C.

  In this way, the entire nucleic acid amplification container is maintained in a state of being heated in a plurality of heating regions, so that when the nucleic acid amplification reaction solution is heated for the next reaction, the previous nucleic acid amplification reaction solution is heated. The influence of the applied temperature can be suppressed. As a result, the amount of heat necessary for adjusting the temperature of the nucleic acid amplification reaction solution can be reduced, and the temperature can be adjusted quickly.

  Therefore, it is possible to provide a nucleic acid amplification apparatus that can rapidly perform nucleic acid amplification.

  The nucleic acid amplification device according to the present invention includes a moving means for moving the nucleic acid amplification container, wherein the moving means is configured to adjust the entire nucleic acid amplification container to at least different temperatures among the plurality of heating regions. It is preferable to move in a state of being heated simultaneously by two heating regions.

  According to the above configuration, the nucleic acid amplification reaction solution is sequentially heated in each of the heating regions while the entire nucleic acid amplification container is heated by the above-described at least two heating regions adjusted to different temperatures. The operation of moving the nucleic acid amplification container can be performed by the moving means, not manually.

  That is, by providing the moving means, it is possible to provide a nucleic acid amplification apparatus that can perform a nucleic acid amplification reaction more easily and efficiently.

  In the nucleic acid amplification apparatus according to the present invention, it is more preferable that the moving means rotate the nucleic acid amplification container.

  The mechanism for rotating the nucleic acid amplification container can reduce the size and simplification of the nucleic acid amplification apparatus as compared with the mechanism that moves other than the rotation. For example, when a member that moves linearly is provided, in addition to the components that support the nucleic acid amplification container and the components that are to be driven, a rail that determines the direction of movement is further required. However, since the member for rotating the nucleic acid amplification container only needs to support and rotate the nucleic acid amplification container, it can be realized by simple parts or the like. Therefore, a nucleic acid amplification device having a simpler structure can be provided.

  In the nucleic acid amplification apparatus according to the present invention, it is more preferable that the rotation axis of the rotation intersects the nucleic acid amplification container.

  By using the straight line passing through the nucleic acid amplification container as the rotation axis, the space required for rotation can be saved compared to the case where the straight line away from the nucleic acid amplification container is used as the rotation axis. Therefore, the apparatus can be reduced in size. In addition, when the through-hole is formed in the nucleic acid amplification container, the case where the rotating shaft intersects the opening surface of the through-hole is also included in the present invention.

  In the nucleic acid amplification apparatus according to the present invention, it is more preferable that the rotation axis intersects the center of the nucleic acid amplification container.

  By passing the rotation shaft through the center of the nucleic acid amplification container, the space required for rotating the nucleic acid amplification container can be further reduced.

  In the nucleic acid amplification apparatus according to the present invention, it is more preferable that the moving means moves the nucleic acid amplification container in a constant direction at a constant speed.

  When changing the speed and direction of movement of the nucleic acid amplification container, a complicated mechanism and means for controlling the movement are required. However, a mechanism for moving the nucleic acid amplification container in a constant direction at a constant speed is simple. It's okay. Therefore, a nucleic acid amplification device having a simpler structure can be provided.

  In the nucleic acid amplification device according to the present invention, the first heating means is preset with a time when an arbitrary point of the nucleic acid amplification container passes through each of the heating regions when the nucleic acid amplification container moves. In addition, it is more preferable that each of the heating regions is formed so that the time for heating by the heating region matches.

  The nucleic acid amplification container moves at a constant speed and in a constant direction. Therefore, by forming the heating region as described above by the first heating means, an arbitrary point of the nucleic acid amplification container, for example, a site filled with the nucleic acid amplification reaction solution can be obtained in advance by a desired nucleic acid amplification reaction. It passes through the said heating area | region with the time heated in each heating area | region set based on conditions. Thereby, a desired temperature can be applied to the nucleic acid amplification reaction solution for a desired time. Therefore, it is possible to provide a nucleic acid amplification apparatus capable of performing a nucleic acid amplification reaction under more various conditions.

  In the nucleic acid amplification device according to the present invention, the moving means rotates the nucleic acid amplification container, and the first heating means spreads radially from the point where the rotation axis of rotation and the mounting means intersect. More preferably, the heating region is formed so as to have a shape.

  The shape of the heating region is defined by two line segments centered on the point where the rotation axis of rotation intersects the mounting means. The angle formed by the two line segments is proportional to the time for heating in each heating region. Therefore, it is possible to apply a desired temperature to the nucleic acid amplification reaction solution for a desired time by simply rotating the nucleic acid amplification container around the rotation axis at a constant rotation speed in a constant rotation direction. Therefore, the nucleic acid amplification reaction can be performed under various conditions with a more compact apparatus.

  In the nucleic acid amplification device according to the present invention, when the nucleic acid amplification container is mounted on the mounting means, the second heating means is disposed above the mounting means so that the nucleic acid amplification container is sandwiched between the two heating means. It is more preferable to comprise.

  Since the nucleic acid amplification container can be heated from two directions by the second heating means, the time required for adjusting the temperature of the nucleic acid amplification container can be shortened. Therefore, the nucleic acid amplification reaction can be performed more rapidly.

  In the nucleic acid amplification device according to the present invention, the first heating means and the second heating means are each configured to form a heating area in which the overlapping areas are adjusted to the same temperature. preferable.

  The nucleic acid amplification container is heated at the same temperature from two directions by forming a heating region adjusted to the same temperature in a region where the first heating unit and the second heating unit overlap each other. be able to. Thereby, the time required for temperature adjustment of the nucleic acid amplification reaction solution can be further shortened. Therefore, the nucleic acid amplification reaction can be performed more rapidly.

  In the nucleic acid amplification device according to the present invention, the nucleic acid amplification container is provided with an opening, a channel, and a tank in the substrate, the opening and the tank are connected by the channel, and the opening is In the case of a cylindrical structure protruding from the substrate, it is further provided with external force applying means for applying an external force for moving the nucleic acid amplification reaction solution injected from the opening into the tank through the flow path. preferable.

  The opening has a shape in which a nucleic acid amplification reaction solution can be easily injected. That is, since the opening is cylindrical, the inner wall can prevent the nucleic acid amplification reaction liquid from leaking and the like, and the aim can be easily determined even using a fine pipe such as a pipette.

  Then, by applying an external force to the nucleic acid amplification reaction solution injected into the opening by the external force adding means, the nucleic acid amplification reaction solution can be easily filled into the tank. For example, in order to improve the thermal conductivity with respect to the nucleic acid amplification reaction solution, the tank portion filled with the nucleic acid amplification reaction solution is formed into a flat shape or the like so that the filled nucleic acid amplification reaction solution and the heating region overlap. It is preferable to increase the area. And according to said structure, even such a flat tank part can be filled easily. Therefore, it is possible to provide a nucleic acid amplification apparatus capable of performing a nucleic acid amplification reaction simply and efficiently.

  In the nucleic acid amplification apparatus according to the present invention, the external force applying means is such that the nucleic acid amplification container is rotated about a straight line intersecting the substrate as a rotation axis at a point closer to the opening than the tank. preferable.

  When the nucleic acid amplification container is rotated by the rotation shaft, an external force is generated from the opening toward the tank by centrifugal force. Therefore, by simply rotating the nucleic acid amplification container, the external force can be applied to the nucleic acid amplification reaction solution injected into the opening, and the tank portion can be filled with the nucleic acid amplification reaction solution. Therefore, it is possible to provide a nucleic acid amplification apparatus that can perform a nucleic acid amplification reaction more easily and efficiently.

  The nucleic acid amplification apparatus according to the present invention includes a moving means for moving the nucleic acid amplification container, and the moving means is configured to adjust the entire nucleic acid amplification container to different temperatures in the plurality of heating regions. More preferably, the nucleic acid amplification container is rotated while being heated simultaneously by at least two heating regions, and the moving means and the external force applying means are formed of the same member.

  By configuring the moving means and the external force applying means with the same member, the nucleic acid amplification reaction liquid is filled into the tank section and the nucleic acid amplification reaction liquid is heated by the heating region, using the same straight line as the rotation axis. Temperature adjustment can be performed. Thereby, after performing the temperature adjustment of the nucleic acid amplification reaction liquid after filling the tank portion with the nucleic acid amplification reaction liquid, there is no need to switch between the moving means and the external force applying means, so that A nucleic acid amplification reaction can be performed rapidly. In addition, the number of parts of the nucleic acid amplification device can be reduced. Therefore, it is possible to perform a nucleic acid amplification reaction more quickly and easily, and a nucleic acid amplification apparatus with a simplified structure can be provided.

  In the nucleic acid amplification device according to the present invention, it is more preferable that a window portion is provided in the mounting region, and further provided with a light detection means for detecting light emitted from the window portion.

  When using a nucleic acid amplification reaction solution that changes light such as fluorescence emitted in response to nucleic acid amplification, the light detection means detects and analyzes the light through the window, The progress of the nucleic acid amplification reaction can be determined in real time. Therefore, it is possible to provide a nucleic acid amplification apparatus that can easily perform the operation of confirming the result of the nucleic acid amplification reaction.

  In order to solve the above problems, the nucleic acid amplification container according to the present invention is provided with an opening, a channel, and a tank in a substrate, and the opening and the tank are connected by the channel, and the opening The part is characterized by a cylindrical structure protruding from the substrate.

  According to said structure, the said opening part has a shape which is easy to inject | pour a nucleic acid amplification reaction liquid. That is, since the opening is cylindrical, it is possible to prevent the nucleic acid amplification reaction solution from leaking due to its inner wall.

  Furthermore, by applying an external force such as centrifugal force or pressure to the nucleic acid amplification reaction solution injected into the opening, the tank portion can be easily filled with the nucleic acid amplification reaction solution. In general, in order to improve the thermal conductivity with respect to the nucleic acid amplification reaction solution, the tank part filled with the nucleic acid amplification reaction solution has a flat structure or the like, and the filled nucleic acid amplification reaction solution and the heating region overlap. It is preferable to increase the area. And according to said structure, even if it is a tank part of such a flat structure, it can be filled easily.

  In other words, a tank portion that is difficult to inject a nucleic acid amplification reaction liquid by simply injecting the nucleic acid amplification reaction liquid into an opening of a shape that is easy to inject the nucleic acid amplification reaction liquid and applying an external force to the nucleic acid amplification reaction liquid. Even so, the nucleic acid amplification reaction solution can be easily filled.

  Therefore, a nucleic acid amplification container capable of performing a nucleic acid amplification reaction simply and with high efficiency can be provided.

  In the nucleic acid amplification container according to the present invention, it is more preferable that the substrate has a disk shape, and the opening is provided closer to the center of the substrate than the tank.

  Centrifugal force toward the tank is applied to the nucleic acid amplification reaction solution injected into the opening by rotating the straight line that passes through the center of the substrate and intersects the substrate as a rotation axis. Further, since the substrate is disk-shaped, the position of the entire nucleic acid amplification container does not move even if the substrate is rotated by the rotation shaft. Therefore, when manufacturing a nucleic acid amplification device for using the nucleic acid amplification container having the above-described configuration, the nucleic acid amplification device can be reduced in size.

  Further, in the case of a disk shape, the flow path is formed radially from the center of the disk such that the opening is closer to the center of the substrate than the tank. Thus, more can be formed. Therefore, a nucleic acid amplification container capable of performing a large amount of nucleic acid amplification at a time can be provided.

  In the nucleic acid amplification container according to the present invention, it is more preferable that the substrate includes a light transmission part that transmits light emitted from the tank part.

  When using a nucleic acid amplification reaction solution that changes light such as fluorescence emitted in response to nucleic acid amplification, the light detection means detects and analyzes the light through the window, The progress of the nucleic acid amplification reaction can be determined in real time. Therefore, it is possible to provide a nucleic acid amplification container capable of simply performing the operation of confirming the result of the nucleic acid amplification reaction.

  In order to solve the above problems, the nucleic acid amplification method according to the present invention includes a filling step of filling a nucleic acid amplification container with a nucleic acid amplification reaction solution, and a temperature adjustment step of adjusting the temperature of the filled nucleic acid amplification reaction solution. The temperature adjusting step includes a step of moving the nucleic acid amplification container in a state where the entire nucleic acid amplification container is simultaneously heated by a plurality of heating regions adjusted to different temperatures. .

  According to the above configuration, while maintaining the state in which the entire nucleic acid amplification container is simultaneously heated in a plurality of heating regions, the portions where the nucleic acid amplification reaction liquid in the nucleic acid amplification container is stored are sequentially added to the respective heating regions. Can be placed inside. In other words, while maintaining the state where the entire nucleic acid amplification container is heated by at least two heating regions adjusted to different temperatures, the nucleic acid amplification reaction solution is sequentially heated in each of the above heating regions, so that the dissociation reaction can be performed. The annealing reaction can be transferred to the next reaction.

  Therefore, when the nucleic acid amplification reaction solution is heated for the next reaction, it is possible to suppress the influence of the temperature previously heated on the nucleic acid amplification reaction solution. As a result, the amount of heat necessary for adjusting the temperature of the nucleic acid amplification reaction solution can be reduced, and the temperature can be adjusted quickly.

  Therefore, nucleic acid amplification can be performed rapidly.

  In the nucleic acid amplification method according to the present invention, as the nucleic acid amplification container, an opening, a channel, and a tank are provided in a substrate, the opening and the tank are connected by the channel, and the opening is The nucleic acid amplification container having a cylindrical structure protruding from the substrate is used, and the filling step includes a step of applying an external force to the nucleic acid amplification reaction solution injected from the opening and moving it into the tank portion. More preferably.

  According to said structure, the said opening part has a shape which is easy to inject | pour a nucleic acid amplification reaction liquid. That is, since the opening is cylindrical, the inner wall can prevent the nucleic acid amplification reaction liquid from leaking and the like, and the aim can be easily determined even using a fine pipe such as a pipette.

  Furthermore, by applying an external force such as centrifugal force or pressure to the nucleic acid amplification reaction solution injected into the opening, the tank portion can be easily filled with the nucleic acid amplification reaction solution.

  In other words, a tank portion that is difficult to inject a nucleic acid amplification reaction liquid by simply injecting the nucleic acid amplification reaction liquid into an opening of a shape that is easy to inject the nucleic acid amplification reaction liquid and applying an external force to the nucleic acid amplification reaction liquid. Even so, the nucleic acid amplification reaction solution can be easily filled.

  Therefore, nucleic acid amplification can be performed more simply.

  As described above, the nucleic acid amplification apparatus according to the present invention includes a mounting means for mounting a nucleic acid amplification container and a plurality of heating regions adjusted to different temperatures on the mounting means. One heating means. For this reason, when the nucleic acid amplification reaction solution is heated, it is possible to suppress the influence of the previously heated temperature. As a result, the amount of heat necessary for adjusting the temperature of the nucleic acid amplification reaction solution can be reduced, and the temperature can be adjusted quickly. Therefore, there is an effect that it is possible to provide a nucleic acid amplification apparatus capable of performing nucleic acid amplification quickly.

  Further, as described above, the nucleic acid amplification container according to the present invention is provided with the opening, the channel, and the tank in the substrate, and the opening and the tank are connected by the channel. Is a cylindrical structure protruding from the substrate. Therefore, it is difficult to inject the nucleic acid amplification reaction liquid simply by injecting the nucleic acid amplification reaction liquid into a cylindrical opening having a shape that is easy to inject the nucleic acid amplification reaction liquid and applying an external force to the nucleic acid amplification reaction liquid. Even a tank portion having a shape can be easily filled with a nucleic acid amplification reaction solution. Therefore, it is possible to provide a nucleic acid amplification container capable of performing a nucleic acid amplification reaction easily and efficiently.

[Embodiment 1]
An embodiment of the nucleic acid amplification device according to the present invention will be described below with reference to FIGS.

(Configuration of PCR device 1)
First, the schematic configuration of the PCR device 1 (nucleic acid amplification device) according to the present embodiment will be described with reference to FIG. FIG. 1 is a diagram showing a schematic configuration of a PCR device 1 according to the present embodiment, and also shows a PCR disk 10 (nucleic acid amplification container) mounted on the PCR device 1.

  As shown in FIG. 1, the nucleic acid amplification apparatus 1 includes a heat block 2 (mounting means, first heating means), a heat block 3 (second heating means), a disk drive unit 4 (moving means), and fluorescence detection. A unit 6 (light detection means), a dispensing arm 7 and a sample container 15 are provided.

  The heat block 2 is provided on the disk drive unit 4. On the heat block 2, a PCR disk 10 to be described later can be mounted. That is, in the present embodiment, the case where the mounting means and the first heating means included in the nucleic acid amplification device according to the present invention are configured by the heat block 2 that is the same member will be described, but the present invention is not limited thereto. It is not a thing. For example, a heating region may be formed on the substrate by a structure in which a substrate formed of a material having high thermal conductivity is used as the mounting unit, and a first heating unit configured by a heating wire or the like is disposed below the substrate. Can be formed.

  The heat block 3 is provided together with the heat block 2 so that the PCR disk 10 can be sandwiched from above and below.

  The heat blocks 2 and 3 are formed with fluorescent transmission portions 9a and 9b. A more detailed structure of the heat blocks 2 and 3 will be described later.

  A rotating unit 5 is provided on the disk drive unit 4. The disk drive unit 4 can rotate the PCR disk 10 at a constant rotational speed in the direction of arrow a with the straight line A shown in FIG. Further, the rotating part 5 can be fitted into a hole provided in the center of the PCR disk 10. As a result, the PCR disk 10 can be rotated.

  The fluorescence detection unit 6 is provided so as to sandwich the fluorescence transmission units 9a and 9b. The fluorescence detection unit 6 can receive fluorescence emitted from the reaction solution storage unit 13 via the fluorescence transmission units 9a and 9b, and calculate the illuminance and the like.

The dispensing arm 7 includes a dispensing tube 8. The dispensing arm 7 is rotatable in the arrow B ₂ direction, the dispensing tube 8 is stretchable in the arrow B ₁ direction shown in FIG. The rotation of the dispensing arm 7 and the operation of extending the dispensing tube 8 can be set in advance, and a predetermined reagent and / or sample stored in advance in the sample container 15 can be automatically set according to the setting. It can be injected into the reaction liquid injection part 11 on the disk 10 for use. The nucleic acid amplification device according to the present invention is not limited to the configuration including the dispensing arm 7 and the sample container 15. For example, a desired reagent, sample, or the like may be separately manually injected into the PCR disk 10 or a conventionally known autosampler or the like may be externally attached.

(Configuration of heat blocks 2 and 3)
Next, the configuration of the heat blocks 2 and 3 will be described in more detail with reference to FIGS. FIG. 2 is a diagram schematically showing the configuration of the heat blocks 2 and 3, and also shows the PCR disk 10 to be mounted.

  As shown in FIG. 2, the heat block 2 is formed with heating portions 2 a and 2 b, and the heat block 3 is formed with heating portions 3 a and 3 b. The heating units 2a, 2b, 3a, and 3b can be independently adjusted in temperature. And a heating area | region is formed by each heating part each temperature-controlled. In FIG. 2, the temperature of the heating parts 2a and 3a is set to 65 ° C., and the temperature of the heating parts 2b and 3b is set to 95 ° C. This is set on the assumption that shuttle PCR is performed by the PCR device 1. That is, the temperature of the double-stranded DNA dissociation reaction is set to 95 ° C., the temperature of the annealing reaction and the temperature of the extension reaction are set to 65 ° C. In addition, the temperature of heating part 2a, 2b, 3a, 3b can be changed according to the conditions of desired nucleic acid amplification reaction, etc. For example, when hot start PCR is performed, the temperature of the heating units 2a and 3a is first set at 95 ° C., and after starting PCR, a desired time has passed and then adjusted to 65 ° C.

  The heating unit 2a and the heating unit 3a are formed so as to overlap each other. Similarly, the heating unit 2b and the heating unit 3b are formed so as to overlap each other. This is because the overlapping heating parts are adjusted to the same temperature, and the PCR disk 10 is sandwiched between the heating parts from above and below, so that a predetermined reaction can be performed more accurately and quickly with respect to the PCR reaction solution. This is to give temperature. Thereby, a 65 degreeC heating area | region is formed by the heating parts 2a and 3a, and a 95 degreeC heating area | region is formed by the heating parts 2b and 3b.

  The heating parts 2a and 2b have a shape that spreads radially from the point a 'shown in FIG. Point a 'is a point where the straight line A and the surface on the heat block 2 on which the PCR disk 10 is mounted intersect.

  Each of the heating units 2a and 2b can be changed in shape. The heating parts 3a and 3b are formed so as to overlap the heating parts 2a and 2b, respectively.

  Specifically, depending on the time required for an arbitrary point of the PCR disk 10 moving at a constant rotational speed to pass through the heating area formed by each heating unit, and the preset heating area. The heating portions 2a and 2b are formed so that the time for heating matches. The preset time for heating in the heating region is a time for heating the nucleic acid amplification reaction solution in each heating region, and is a desired reaction time for performing a dissociation reaction, an annealing reaction, an extension reaction, and the like. is there.

The thus formed heating unit 2a, 2b is the shape of, defined by line segments L ₁ and L ₂ extending toward the outer edge of the heat block 2 from the point a ', a line segment L _1, L ₂ and heat block Each shape is formed by two outer edges. In other words, the positions of the line segments L ₁ and L ₂ are calculated so that the time required for the above-described arbitrary point to pass through the heating region matches the desired reaction time, and the heating units 2a and 2b are calculated. Form. Further, the positions of the line segments L ₃ and L ₄ on the heat block 3 parallel to the line segments L ₁ and L ₂ are calculated to form 3a and 3b. Thereby, a heating region capable of heating the nucleic acid amplification reaction solution for a desired time can be formed.

The method for calculating the line segments L _{1 to} L ₄ is not particularly limited. For example, the ratio between θ1 and 360−θ1, which is the angle formed by the line segment L ₁ and the line segment L ₂ , is calculated to be the ratio of the dissociation reaction time, the annealing reaction time, and the extension reaction time. That's fine. Moreover, the nucleic acid amplification device according to the present invention may further include a control means capable of such calculation. In addition, as said arbitrary point, the said calculation should just be performed on the basis of the site | part by which the PCR reaction liquid of PCR disc 10 was stored, for example. In the PCR apparatus 1 according to the present embodiment, θ1 is formed to be 90 degrees for use in the examples described later.

  On the heat blocks 2 and 3, a desired number of heating parts can be formed in a desired shape according to desired PCR conditions. For example, each of the heat blocks 2 and 3 is divided into four by four line segments extending radially from the point a ′, and two heating regions of 95 ° C. and 65 ° C. are formed. You may make it the shape heated alternately at 95 degreeC, 65 degreeC, 95 degreeC, and 65 degreeC, while the disk for 1 round is carried out. A configuration in which a plurality of sets of a plurality of heating regions adjusted to different temperatures as described above are formed is also included in the scope of the nucleic acid amplification device according to the present invention. In addition, when the annealing reaction and the extension reaction are performed at different temperatures, three heating portions may be formed on each of the heat blocks 2 and 3.

  A hole is formed in the center of the heat blocks 2 and 3. This is for penetrating the rotating part 5. Further, the holes formed in the heat block 3 are for causing the reaction liquid injection part 11 to protrude above the heat block 3 as shown in FIG. 1 when the PCR device 1 is loaded with the PCR disk 10. .

  The heat blocks 2 and 3 are formed with fluorescence transmitting portions 9a and 9b (window portions) for detecting fluorescence emitted from the reaction solution storage portion 13 formed on the PCR disk 10, respectively.

(Nucleic acid amplification container according to the present invention)
Next, an embodiment of the nucleic acid amplification container according to the present invention will be described with reference to FIG. FIG. 3 is a diagram schematically showing the structure of the PCR disk 10 according to the present embodiment, where (a) is a plan view and (b) is a perspective view.

  As shown in FIGS. 3 (a) and 3 (b), the PCR disk 10 is formed of a reaction liquid injection section 11 (opening section), a flow path 12, a reaction liquid storage section 13 (tank section), and a substrate 14. Yes.

  Further, as shown in FIG. 3A, a plurality of reaction liquid injection sections 11, flow paths 12, and reaction liquid storage sections 13 are formed on the substrate 14 one by one. For convenience of explanation, FIG. 3B shows the reaction liquid injection section 11, the flow path 12, and the reaction liquid storage section 13 one by one.

  As shown in FIG. 3B, the reaction liquid injection part 11 has a cylindrical structure protruding from the substrate 14. Thereby, when the PCR reaction liquid is injected, the PCR reaction liquid can be prevented from leaking from the reaction liquid injection portion 11 by the inner wall portion of the cylindrical structure, and a fine pipe such as a pipette can be provided. Since it is easy to determine the aim even if it is used, the PCR reaction solution can be easily injected.

  The flow path 12 is connected to the reaction liquid injection part 11 at one end and is connected to the reaction liquid storage part 13 at the other end. The flow path 12 extends radially from the center of the substrate 14. The flow path 12 communicates only with the reaction liquid injection section 11 and the reaction liquid storage section 13. Thereby, the movement of the PCR reaction solution from the reaction solution injection unit 11 to the reaction solution storage unit 13 can be performed quickly and accurately.

  The reaction liquid storage unit 13 is formed inside the substrate 14. Moreover, the reaction liquid storage part 13 is formed in a flat shape. Thereby, since the surface area of the PCR reaction liquid in the reaction liquid storage part 13 can be enlarged, the thermal conductivity with respect to the PCR reaction liquid can be increased. Therefore, the temperature of the PCR reaction solution can be adjusted more rapidly.

  The substrate 14 has a disk-like structure. A hole is formed in the center of the substrate 14. The PCR disk 10 and the rotating part 5 can be fitted through the hole. Thus, by adopting a disk-shaped substrate as the substrate of the nucleic acid amplification container according to the present invention, it can be suitably used for the nucleic acid amplification device according to the present invention, and has many openings, flow paths, A tank part can be formed.

  Here, as shown in FIG. 2, all the reaction liquid injection part 11, the flow path 12, and the reaction liquid storage part 13 are formed in the range which can be accommodated in the area | region which overlaps with the heating parts 2a and 3a. Yes. This is because when the PCR disk 10 is rotated, each reaction solution storage unit 13 first equalizes the time for passing through the region where the heating units 2b and 3b overlap.

  That is, first, for the reaction liquid storage unit 13 arranged in the heating region by the heating units 2a and 3a, the time for passing through the heating region by the heating units 2b and 3b depends on the heating units 2b and 3b. This is the time from entering the warming area until leaving. On the other hand, first, for the reaction liquid storage unit 13 arranged in the heating region by the heating units 2b and 3b, the time for passing through the heating region by the heating units 2b and 3b is the time when the reaction is started. To the time from the second heating area. As described above, there may be a case where the time for first passing through the heating region by the heating units 2b and 3b differs depending on the position where the reaction liquid storage unit 13 is initially arranged. Therefore, if all the reaction liquid storage units 13 can be mounted so as to be within the region overlapping the heating units 2a and 3a at the beginning, this time lag can be eliminated.

  The material of the PCR disk 10 may be any conventionally known material for a container for nucleic acid amplification reaction, and is not limited. For example, polycarbonate or polypropylene may be used. The PCR disk 10 according to the present embodiment is formed using polycarbonate. Since polycarbonate is highly transparent, it is easy to detect fluorescence when using a PCR reaction solution in which fluorescence emitted by DNA amplification such as real-time PCR (RT-PCR) is changed. That is, the light transmission part provided in the nucleic acid amplification container according to the present invention does not need to be separately formed around the tank part using a highly transparent material, and the light transmission part for transmitting fluorescence emitted from the tank part is provided. Can be formed.

(Nucleic acid amplification reaction using PCR apparatus 1 and PCR disk 10)
Next, a method for performing a nucleic acid amplification reaction using the PCR device 1 and the PCR disk 10 will be described with reference to FIGS.

  First, the PCR disk 10 is mounted on the PCR device 1. As shown in FIGS. 1 and 2, the PCR disk 10 is mounted on the heat block 2. Next, in order to heat from the upper surface of the PCR disk 10, the heat block 3 is arranged so as to sandwich the PCR disk 10 together with the heat block 2.

  At this time, it is good to mount so that all the reaction liquid storage parts 13 may be settled in the area | region which overlaps with the heating parts 2a and 3a. Thereby, as above-mentioned, the time for all the reaction liquid storage parts 13 to pass through the heating area | region formed of the heating parts 2b and 3b can be made to correspond.

  Next, as shown in FIG. 1, reagents and samples necessary for PCR are injected into the reaction liquid injection unit 11 by the dispensing arm 7. Specifically, first, the dispensing tube 8 expands and sucks a predetermined amount of reagent and / or sample from the sample container 15 and then contracts. Next, the dispensing arm 7 is rotated, and the dispensing tube 8 is disposed on the reaction liquid injection part 11. Next, the dispensing tube 8 extends to the vicinity of the reaction liquid injection unit 11 and then injects the aspirated reagent and / or sample into the reaction liquid injection unit 11.

  After injecting the PCR reaction solution into the reaction solution injecting unit 11, the disc driving unit 4 rotates the PCR disk 10 at a high speed. Thereby, the PCR reaction solution is filled in the reaction solution storage unit 13. Specifically, as shown in FIG. 3B, the PCR disk 10 is rotated in the direction of the arrow a with the straight line A as the rotation axis. Centrifugal force acts in the direction of arrow C on the PCR reaction solution injected into the reaction solution injection unit 11 by the rotation. Then, due to the centrifugal force, the PCR reaction solution passes through the flow path 12 and fills the reaction solution storage unit 13.

  By rotating the PCR disk 10 in this way, the reaction solution reservoir 13 can be filled with the PCR reaction solution. Thereby, even if it is a reaction liquid storage part of the shape where it is difficult to inject a PCR reaction liquid directly, a PCR reaction liquid can be easily inject | poured.

  That is, as described above, the reaction solution storage unit 13 has a flat structure in order to improve the thermal conductivity with respect to the PCR reaction solution. Here, in general, it is difficult to fill a tank portion having a flat structure with a reagent or the like. For example, when the reaction solution is poured into a thinly dished dish, it is difficult to aim the tip of the pipette, and it is easy to leak outside. Furthermore, when covering from the top of the plate, the reaction solution may leak out of the plate and enter another plate by pressing it from above. Further, in a configuration in which a flat tank is formed in a container, a minute hole is formed in the tank, and the reaction liquid is injected from the hole, a precise operation is required for the injection. In this case, it is difficult to perform the operation manually, and also when automating using an autosampler or the like, a precise structure is required for the autosampler, and the cost is increased. Therefore, the present inventors provide an opening having a cylindrical structure that is easy to inject even a small amount of a nucleic acid amplification reaction solution, and separately form a flat tank portion in the container. It was found that if the tank part is connected with a flow path, the PCR reaction liquid can be easily filled into the flat tank part simply by injecting the PCR reaction liquid into the opening and rotating it. The present invention has been completed on the basis of this completely new technical idea that even those skilled in the art cannot conceive.

  The straight line A passes through a point a ″ which is the center of the PCR disk 10. Thus, by rotating the PCR disk 10 about a straight line that passes through the center and is perpendicular to the surface, the position of the entire PCR disk 10 does not move during rotation. For this reason, a PCR apparatus can be reduced in size. In addition, the said rotating shaft is not restricted to the form which cross | intersects the center of a nucleic acid amplification container. That is, if the rotation axis is a straight line that passes through a point closer to the opening than the tank, the nucleic acid amplification reaction solution filled in the opening by centrifugal force receives force in the direction toward the tank, The tank can be easily filled.

  The number of rotations of the PCR disk 10 when filling the reaction solution reservoir 13 with the PCR reaction solution can cause the reaction solution reservoir 13 to be filled with the PCR reaction solution injected into the reaction solution injector 11. What is necessary is just to rotate the disk 10 for PCR by 1000 rpm-6000 rpm, for example, if a centrifugal force is obtained. The inventors rotated at 3000 rpm in the examples described later.

  In the present embodiment, the case where the PCR disk 10 is rotated as means for moving the PCR reaction solution injected into the reaction solution injection unit 11 to the reaction solution storage unit 13 has been described. However, the present invention is not limited to this. Absent. That is, it is only necessary that an external force for moving the PCR reaction solution injected into the reaction solution injection unit 11 to the reaction solution storage unit 13 can be added to the PCR reaction solution. For example, after injecting the PCR reaction solution into the reaction solution injection unit 11, air may be sent into the reaction solution injection unit 11, and the PCR reaction solution may be sent into the reaction solution storage unit 13 by pressure.

  After filling the reaction solution reservoir 13 with the PCR reaction solution, the PCR is performed by repeatedly adding temperatures necessary for the dissociation reaction, annealing reaction, and extension reaction to the PCR reaction solution. In this embodiment, the case where the dissociation reaction is performed at 95 ° C. and the annealing reaction and the extension reaction are performed at 65 ° C. will be described.

  First, the heating parts 2a and 3a are adjusted to 65 ° C, and the heating parts 2b and 3b are adjusted to 95 ° C. Thus, a 95 ° C. heating region and a 65 ° C. heating region are formed between the heat blocks 2 and 3. The shapes of the heating parts 2a, 2b, 3a, 3b are formed according to the desired reaction time and the like as described above. The warming region may be formed before or after the step of filling the reaction solution reservoir 13 with the PCR reaction solution, or may be performed in parallel. For example, even if the above-described PCR reaction solution is filled after a warming region is formed in advance, the rotational speed for the filling is high as described above, and therefore the influence on the reaction is very small.

  Next, the PCR disk 10 is rotated by the disk drive unit 4. What is necessary is just to set the rotation speed of the said rotation suitably according to time, such as desired dissociation reaction and annealing reaction. For example, the present inventors rotated the PCR disk 10 at 7 rpm in Examples described later.

  The manner in which PCR is performed by this rotation will be described with reference to FIG. FIG. 4 is a diagram schematically showing how PCR is performed by rotating the PCR disk 10, and (a) schematically shows how the PCR disk 10 as viewed from the heat block 3 is rotated. It is a figure and (b) is a perspective view which shows typically a mode that the disk 10 for PCR rotates.

  As shown in FIGS. 4A and 4B, the PCR disk rotates in the direction of arrow a. Thereby, as shown to Fig.4 (a), each reaction liquid storage part 13 is first heated to 95 degreeC. Then, as the rotation further proceeds, each reaction liquid storage unit 13 is heated to 65 ° C. This is repeated, and the PCR reaction solution in the reaction solution storage unit 13 is repeatedly adjusted to 95 ° C. and 65 ° C. to perform PCR.

  At this time, the entire PCR disk 10 is simultaneously heated to 95 ° C. and 65 ° C. Therefore, on the PCR disk 10, there is a region having a temperature higher than 65 ° C. and lower than 95 ° C., and there are few regions where either temperature is reached. Thereby, the calorie | heat amount required in order to adjust the reaction liquid storage part 13 to 95 degreeC or 65 degreeC can be reduced. Therefore, the time for adjusting each reaction liquid storage part 13 to 65 degreeC or 95 degreeC can be shortened.

  Conventionally, a PCR container is brought into contact with each temperature necessary for a dissociation reaction or the like, or a PCR container is mounted on one heater, and the entire temperature of the heater is repeatedly adjusted. . However, in this case, there are many regions where the reaction temperature is reached in addition to the location where the reaction solution is stored, and even in the next temperature adjustment, the amount of heat required for temperature increase or cooling is reduced due to the effect of the region having the previous reaction temperature. It was a lot. Therefore, the efficiency of temperature adjustment is reduced due to loss of heat, and the temperature adjustment has a long time. The present inventors have found that the efficiency of the temperature adjustment described above is improved by simultaneously heating the entire container in a plurality of heating regions while heating the reaction liquid reservoir to a desired temperature. . The present invention has been completed on the basis of this completely new technical idea that even those skilled in the art cannot conceive.

  In the present embodiment, the rotation axis of the PCR disk 10 rotated when the PCR reaction solution is heated is a straight line A as shown in FIG. As described above, the straight line A passes through the point a ″, which is the center of the PCR disk 10, and is a straight line perpendicular to the surface direction of the PCR disk 10. As described above, the rotation axis that is rotated when the PCR disk 10 is heated preferably passes through the center of the PCR disk 10, but is not limited thereto. That is, the moving means provided in the nucleic acid amplification device according to the present invention can simplify the mechanism of the moving means and reduce the number of parts, as long as the nucleic acid amplification container is configured to rotate. The effect that the apparatus can be reduced in size is obtained. In other words, in the case of only rotation, a mechanism for supporting the nucleic acid amplification container and a motor for rotating the nucleic acid amplification container may be used. Further parts such as rails are required. However, if the rotation axis is a straight line that intersects with the PCR disk 10, the range in which the PCR disk 10 moves can be further saved and the apparatus can be further miniaturized. Further, as in the present embodiment, the rotation axis is a straight line passing through the center of the PCR disk 10, so that only the position of the reaction solution storage unit 13 is moved while the mounting position of the PCR disk 10 is fixed. Further, the apparatus can be miniaturized.

  In the present embodiment, the rotation for filling the reaction solution reservoir 13 with the PCR reaction solution and the rotation for heating the reaction solution reservoir 13 are performed on the same straight line A on the rotation axis. However, the present invention is not limited to this. In order to perform each rotation, a plurality of rotation means may be provided, and each rotation may be performed by separate rotation shafts. However, as described in the present embodiment, the rotation for filling the reaction solution storage unit 13 with the PCR reaction solution and the rotation for heating the reaction solution storage unit 13 are the same rotation axis. Thus, both rotations can be performed using one member (disk drive unit 4). Therefore, the number of parts can be reduced and the apparatus can be downsized.

  Further, the moving means provided in the nucleic acid amplification device according to the present invention is not limited to only the configuration for rotating the nucleic acid amplification container. What is necessary is just to move the whole nucleic acid amplification container in the state heated simultaneously by the at least two heating area | regions each adjusted to different temperature among several heating area | regions. For example, a 95 ° C. heating region and a 65 ° C. heating region are alternately formed in a strip shape on a belt-like plate (mounting means), and one of the 95 ° C. heating region and the 65 ° C. heating region is formed. Place the nucleic acid amplification container so as to straddle at the same time. When this is moved so as to reciprocate in the length direction of the plate, the entire nucleic acid amplification container is simultaneously heated at 95 ° C. and 65 ° C., while the nucleic acid amplification reaction solution in the nucleic acid amplification container is filled Can be warmed at 95 ° C or 65 ° C. However, the configuration in which the nucleic acid amplification container is rotated as in the present embodiment is preferable in that the effects of reducing the number of parts and reducing the size of the apparatus can be obtained.

  Further, the PCR disk 10 by the disk drive unit 4 is driven in a constant direction at a constant rotational speed, but the present invention is not limited to this. For example, when the region where the heating region can be formed is limited, the moving speed and the moving direction may be appropriately changed according to the heating region, desired reaction conditions, or the like. However, as in this embodiment, by moving the nucleic acid amplification container at a constant speed in a constant direction, it is not necessary to control the direction and speed, and the line segment that defines the shape of the heating region described above is used. Can be easily calculated. Therefore, it becomes easy to control the operation of the nucleic acid amplification device.

  Confirmation of DNA amplification by PCR may be performed by a conventionally known electrophoresis method and the like, and is not limited. However, if a PCR reaction solution in which fluorescence emitted by DNA amplification changes is used, fluorescence detection is performed. Amplification can be confirmed by just doing. That is, by detecting the fluorescence emitted from the reaction liquid storage unit 13 by the fluorescence detection unit 6 via the fluorescence transmission units 9a and 9b, it is possible to confirm amplification of DNA by PCR in real time.

[Embodiment 2]
Another embodiment of the nucleic acid amplification container according to the present invention will be described with reference to FIG. For convenience of explanation, description of constituent elements having functions similar to those of the constituent elements according to Embodiment 1 is omitted. In the present embodiment, differences from the first embodiment will be mainly described.

  FIG. 5 is a perspective view schematically showing the structure of the PCR chip 20 according to the present embodiment. FIG. 5A shows the reaction solution reservoir 23 filled with the PCR reaction solution injected into the reaction solution injector 21. (B) shows another example of filling the reaction solution storage unit 23 with the PCR reaction solution injected into the reaction solution injection unit 21.

  As shown in FIGS. 5 (a) and 5 (b), the PCR chip 20 is formed from a reaction liquid injection section 21 (opening section), a flow path 22, a reaction liquid storage section 23 (tank section), and a substrate 24. Yes.

  The reaction liquid injection part 21 has a cylindrical structure protruding from the substrate 24. Thereby, the PCR reaction liquid can be prevented from leaking from the reaction liquid injection section 21 by the inner wall portion of the reaction liquid injection section 21, and the aim is determined even by using a fine dispensing tube such as a pipette. For reasons such as ease, the PCR reaction solution can be easily injected.

  The flow path 22 is connected to the reaction liquid injection part 21 at one end and is connected to the reaction liquid storage part 23 at the other end. Further, as described later, the substrate 24 has a rectangular shape, and the flow path 22 is formed in parallel to the length direction of the substrate 24.

  The reaction liquid reservoir 23 is formed inside the substrate 24. The reaction liquid reservoir 23 is formed in a flat shape. Thereby, since the surface area of the PCR reaction liquid when it fills in the reaction liquid storage part 23 can be enlarged, the thermal conductivity with respect to the said PCR reaction liquid can be raised. Therefore, the temperature of the PCR reaction solution can be adjusted more rapidly.

  The substrate 24 has a rectangular shape. If it is rectangular, the substrate 24 can be cut out in large quantities from a single sheet formed of the material of the substrate 24, and less material is discarded than when a disk-shaped substrate is used. This is advantageous in that the manufacturing cost can be reduced.

  When filling the reaction solution reservoir 23 with the PCR reaction solution injected into the reaction solution injector 21, it passes through a point on the substrate 24 that is closer to the reaction solution injector 21 than the reaction solution reservoir 23. What is necessary is just to rotate a straight line as a rotating shaft.

  For example, as shown in FIG. 5A, it is preferable to rotate a straight line D perpendicular to the surface direction of the substrate 24 as a rotation axis. As a result, the injected PCR reaction solution receives a centrifugal force in the direction of arrow E and is filled in the reaction solution reservoir 23.

  Further, for example, as shown in FIG. 5B, the PCR chip 20 may be rotated with the straight line D as the rotation axis while the PCR chip 20 is inclined from the horizontal as compared with the case of FIG. Thereby, the injected PCR reaction solution receives a force in the direction of arrow E due to the centrifugal force and gravity due to the rotation, and is filled in the reaction solution reservoir 23.

  When performing PCR using the PCR chip 20, a conventionally known PCR device may be used, but if the nucleic acid amplification device according to the present invention is used, PCR can be performed simply and rapidly as described above.

  For example, when the PCR chip 20 is used in the PCR apparatus 1, an arm structure member or the like that can grip and rotate the PCR chip 20 may be provided instead of the rotating unit 5. And while maintaining the state heated simultaneously by the heating area | region formed by the heating parts 2a and 3a and the heating area | region formed by the heating parts 2b and 3b, the reaction liquid storage part 23 is respectively What is necessary is just to hold | grip and rotate the PCR chip | tip 20 with the member of the said arm structure so that it may heat alternately to this heating area | region. For example, the PCR reaction solution 20 is first filled in the reaction solution reservoir 23 by gripping and rotating the PCR chip 20 with a member of the arm structure so that the rotation axis is a straight line D1, and the PCR reaction solution is heated. In some cases, a part to be gripped by a member of the arm structure is changed so that a straight line that passes through the center of the PCR chip 20 and is perpendicular to the surface of the PCR chip 20 is used as a rotation axis, or a member that is rotated separately It may be provided and rotated.

[Example: Detection of Cytochrome P450 2C19 (CYP2C19) gene]
(Synthesis of primer for detecting CYP2C19 gene)
First, a normal primer and a reverse primer were synthesized. The base sequence of the normal primer is shown in SEQ ID NO: 1, and the base sequence of the reverse primer is shown in SEQ ID NO: 2.

  The normal primer and the reverse primer were synthesized by a phosphoramidite method using a DNA synthesizer (Perkin Elmer, Inc .; type 392) according to the attached manual. The synthesized forward primer and reverse strand primer were deprotected by dissolving in aqueous ammonia and allowing to stand overnight in an environment at 55 ° C. Then, it refine | purified in the OPC column (made by Perkin Elmer). The primer synthesis may be requested from a DNA synthesis contract company (Japan Bioservice Co., Ltd., Sigma Aldrich Japan Co., Ltd., Operon Biotechnology Co., Ltd., etc.).

(Analysis of CYP2C19 gene by PCR)
As a sample to be subjected to PCR, a DNA solution obtained from human leukocytes by the phenol / chloroform method was used. Two types of human leukocytes were obtained from two types of individuals, and two types of DNA solutions were prepared (hereinafter referred to as “sample A” and “sample B”).

Next, a PCR reaction solution was prepared. The total amount of PCR reaction solution is 25 μl, 20 pmol of normal primer, 20 pmol of reverse primer, KOD plus DNA polymerase (manufactured by Toyobo Co., Ltd., product number KOD-201), 2.5 μl of x10 buffer attached to KOD plus DNA polymerase, 2 mM dNTPs 2.5 [mu] l, a 25 mM MgCl ₂ 2 [mu] l and the sample a or B, DNA which is dissolved is mixed so that the 100 ng, the balance was water.

  The PCR apparatus 1 and the PCR disk 10 described in the first embodiment were used for PCR.

  First, 20 μl of the prepared PCR reaction solution was injected into the reaction solution injection unit 11. Next, the PCR disk 10 was rotated at 3000 rpm for 5 seconds, and the PCR reaction solution in the reaction solution injection unit 11 was moved to the reaction solution storage unit 13.

  The temperature conditions for PCR were such that the heating parts 2a and 3a were 95 ° C. and allowed to stand for 5 minutes, then the heating parts 2a and 3a were adjusted to 65 ° C., and the PCR disk was rotated at 7 rpm for 5 minutes. As described above, since the angle θ1 is 90 degrees, the rotation of the PCR reaction solution is performed for 35 cycles at 95 ° C. for 2.1 seconds and at 65 ° C. for 6.4 seconds.

(Confirmation of nucleic acid amplification by agarose gel electrophoresis)
After the PCR reaction, 5 μl of the PCR reaction solution was recovered from the reaction solution reservoir 13. The collected PCR reaction solution was subjected to electrophoresis using a 3% agarose gel. Electrophoresis was performed for 60 minutes by applying a voltage of 100V. The result is shown in FIG. FIG. 6 is a diagram showing the results of electrophoresis in this example. Lanes 1 and 2 show the results of PCR using samples A and B, respectively. Lane 3 shows the results of samples A and B for comparison. Instead, water is mixed and the results of PCR performed by the method described in this example are shown. Lane M shows the result of electrophoresis using a molecular weight marker.

  As shown in FIG. 6, the CYP2C19 gene could be detected satisfactorily in PCR using any of the samples A and B. This indicates that PCR was performed well in a short time of 5 minutes.

  The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments can be appropriately combined. Such embodiments are also included in the technical scope of the present invention. Moreover, all the patent documents described in this specification are used as reference in this specification.

  The nucleic acid amplification apparatus and the nucleic acid amplification container according to the present invention can perform a nucleic acid amplification reaction extremely quickly and easily. It can be used in all industries that use nucleic acid amplification reactions such as PCR, such as gene analysis and bioassays performed by monitoring genes.

It is a figure which shows schematic structure of the PCR apparatus which concerns on embodiment of this invention. It is a figure which shows typically the structure of the heat block with which the PCR apparatus which concerns on embodiment of this invention is provided. It is a figure which shows typically the structure of the disk for PCR which concerns on embodiment of this invention. It is a figure which shows typically a mode that PCR is rotated by rotating the disc for PCR which concerns on embodiment of this invention. It is a perspective view which shows typically the structure of the chip | tip for PCR which concerns on other embodiment of this invention. It is a figure which shows the result of the electrophoresis in the Example of this invention.

Explanation of symbols

1 PCR equipment (nucleic acid amplification equipment)
2 Heat block (mounting means, first heating means)
2a, 2b, 3a, 3b Heating part 3 Heat block (second heating means)
4 Disk drive (moving means)
5 Rotating part 6 Fluorescence detecting part (light detecting means)
9a, 9b Fluorescent transmission part (window part)
10 PCR disc (nucleic acid amplification container)
11, 21 Reaction liquid injection part (opening)
12, 22 Flow path 13, 23 Reaction liquid storage part (tank part)
14, 24 Substrate 20 PCR chip (nucleic acid amplification container)
A, D Straight line (rotary axis)

Claims (13)

Mounting means for mounting the nucleic acid amplification container;
A first heating means for forming a plurality of heating regions adjusted to different temperatures on the mounting means ,
The nucleic acid amplification container is provided with an opening, a channel, and a tank in a substrate, the opening and the tank are connected by the channel, and the opening protrudes from the substrate. And
The apparatus further comprises an external force applying means for applying an external force for moving the nucleic acid amplification reaction solution injected from the opening through the flow path into the tank.
The nucleic acid amplification apparatus , wherein the external force applying means rotates the nucleic acid amplification container about a straight line intersecting the substrate at a point closer to the opening than the tank . A moving means for moving the nucleic acid amplification container;
The moving means moves the entire nucleic acid amplification container in a state of being simultaneously heated by at least two heating regions adjusted to different temperatures among the plurality of heating regions. The nucleic acid amplification device according to claim 1.   The nucleic acid amplification apparatus according to claim 2, wherein the moving means rotates a nucleic acid amplification container.   4. The nucleic acid amplification apparatus according to claim 3, wherein the rotation axis of the rotation intersects the nucleic acid amplification container.   The nucleic acid amplification apparatus according to claim 4, wherein the rotation shaft intersects the center of the nucleic acid amplification container.   The nucleic acid amplification apparatus according to claim 2, wherein the moving means moves the nucleic acid amplification container in a constant direction at a constant speed.   When the nucleic acid amplification container is moved, the first warming means is configured to warm the predetermined time by which an arbitrary point of the nucleic acid amplification container passes through each of the warming areas. The nucleic acid amplification device according to claim 6, wherein each of the heating regions is formed so as to coincide with a time to be performed. The moving means rotates the nucleic acid amplification container,
The said 1st heating means forms the said heating area | region so that it may become a shape which spreads radially from the point where the rotating shaft of the said rotation and the said mounting means cross. The nucleic acid amplification device according to 1.   The second heating means is provided above the mounting means so that the nucleic acid amplification container is sandwiched by two heating means when the nucleic acid amplification container is mounted on the mounting means. 1. The nucleic acid amplification apparatus according to 1.   The nucleic acid according to claim 9, wherein each of the first warming means and the second warming means forms a warming region in which regions overlapping each other are adjusted to the same temperature. Amplification equipment. Moving means for moving the nucleic acid amplification container, wherein the moving means simultaneously heats the entire nucleic acid amplification container with at least two heating regions adjusted to different temperatures among the plurality of heating regions. The nucleic acid amplification container is rotated while being heated,
2. The nucleic acid amplification apparatus according to claim 1 , wherein the moving means and the external force applying means are composed of the same member. A window is provided in the mounting means ,
The nucleic acid amplification apparatus according to claim 1, further comprising a light detection unit configured to detect light emitted from the window portion. A filling step of filling the nucleic acid amplification container with the nucleic acid amplification reaction solution; and
Including a temperature adjustment step of adjusting the temperature of the nucleic acid amplification reaction solution filled,
The temperature adjustment step, the entire above-mentioned nucleic acid amplification container, while being simultaneously warmed by a plurality of heating regions which are adjusted to different temperatures, comprising the step of moving the nucleic acid amplification container,
As the nucleic acid amplification container, the substrate is provided with an opening, a channel, and a tank, the opening and the tank are connected by the channel, and the opening is a cylindrical structure protruding from the substrate Using a nucleic acid amplification container
The filling step includes a step of applying an external force to the nucleic acid amplification reaction solution injected from the opening and moving the solution into the tank.
The nucleic acid amplification method according to claim 1, wherein the external force is applied by rotating the nucleic acid amplification container about a straight line intersecting the substrate at a point closer to the opening than the tank portion .