JP2018042482A - Nucleic acid amplification reaction container, nucleic acid amplification reaction device and nucleic acid amplification reaction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid amplification reaction container that satisfies the moving speed of reaction liquid and improvement of thermal conductivity to reaction liquid.SOLUTION: A nucleic acid amplification reaction container 100 has: a first region 101 in which reaction liquid 140 is brought to a first temperature; a second region 102 in which the reaction liquid 140 is brought to a second temperature; and first and second flow channels 103,104 which communicate the first region 101 and the second region 102, and move the reaction liquid 140, in which the first region 101 and the second region 102 are formed in a space sandwiched between a first inner wall face 111 and a second inner wall face 112, and a distance between the first inner wall face 111 and the second inner wall face 112 is a distance at which reaction liquid 140 contacts both of the first inner wall face 111 and the second inner wall face 112.SELECTED DRAWING: Figure 6

Description

  The present invention relates to a nucleic acid amplification reaction vessel, a nucleic acid amplification reaction apparatus, and a nucleic acid amplification reaction method.

  Conventionally, as a technique for growing nucleic acids at high speed, PCR (Polymerase Chain) in which a nucleic acid amplification reaction solution filled in a nucleic acid amplification reaction vessel is alternately moved to a first region and a second region having different temperatures and subjected to thermal cycling. Reaction) and other technologies are widely used. In the PCR method, it is required to shorten the heat cycle time by improving the moving speed for moving the nucleic acid amplification reaction solution and the heat transfer property to the nucleic acid amplification reaction solution. For example, Patent Document 1 discloses a nucleic acid amplification reaction vessel in which a nucleic acid amplification reaction solution contacts both the inner surface of a first side wall and the inner surface of a second side wall. Thereby, it is described that the heat transfer property to the nucleic acid amplification reaction solution can be improved.

JP-A-2015-154722

  The nucleic acid amplification reaction vessel is filled with a reaction solution (nucleic acid amplification reaction solution) and a gas, and the reaction solution is moved from the first region to the second region or from the second region to the first region. It is necessary to exchange the reaction liquid and gas using gravity. However, the nucleic acid amplification reaction vessel described in Patent Document 1 has only one flow path for moving the reaction solution between the first region and the second region. Accordingly, depending on the amount of the reaction liquid, the cross-sectional area of the flow path, and the like, the exchange of the reaction liquid and the gas is prevented, and the reaction liquid moves from the first area to the second area or from the second area to the first area. There was a possibility that the moving speed of moving to would decrease.

  SUMMARY An advantage of some aspects of the invention is to solve at least a part of the problems described above, and the invention can be implemented as the following forms or application examples.

  [Application Example 1] A nucleic acid amplification reaction container according to this application example includes a first region for bringing a reaction solution to a first temperature, a second region for bringing the reaction solution to a second temperature different from the first temperature, A plurality of flow paths for communicating the first region and the second region to move the reaction solution, wherein the first region and the second region include a first inner wall surface, and the first inner A distance between the first inner wall surface and the second inner wall surface is formed in a space between the second inner wall surface facing the wall surface, and the distance between the first inner wall surface and the second inner wall surface is the first region or the second through the flow path. When the reaction solution is introduced into the region, the reaction solution is in contact with both the first inner wall surface and the second inner wall surface.

  According to this application example, the nucleic acid amplification reaction vessel includes a plurality of flow paths that move the reaction solution by communicating the first region and the second region, so that the reaction solution is transferred from the first region to the second region. When moving from the second region to the first region, the reaction solution and the gas can be efficiently exchanged by different flow paths. Thereby, the moving speed which moves a reaction liquid can be improved. The first region and the second region are heated from the outside of the nucleic acid amplification reaction vessel. Since the reaction solution is in contact with both the first inner wall surface and the second inner wall surface where the first region and the second region are formed, the thermal conductivity for bringing the reaction solution to the first temperature or the second temperature. Is improved. Therefore, it is possible to provide a nucleic acid amplification reaction vessel that achieves both improvements in the moving speed of the reaction solution and the thermal conductivity to the reaction solution.

  Application Example 2 In the nucleic acid amplification reaction container described in the above application example, it is preferable that the flow path is formed in a space sandwiched between the first inner wall surface and the second inner wall surface.

  According to this application example, the flow path of the nucleic acid amplification reaction vessel is formed in the same space between the first inner wall surface and the second inner wall surface as the first region and the second region. The second region and the flow path can be integrally formed.

  Application Example 3 In the nucleic acid amplification reaction container described in the above application example, oil is disposed in the first region, the second region, and the flow path.

  According to this application example, since the oil is disposed in the first region, the second region, and the flow path of the nucleic acid amplification reaction container, the reaction solution is transferred from the first region to the second region or from the second region. It is possible to suitably perform the movement to the first region and the temperature change from the first temperature to the second temperature or from the second temperature to the first temperature.

  Application Example 4 In the nucleic acid amplification reaction container according to the application example described above, it is preferable that 50% or more of the volume formed by the first region, the second region, and the flow path is filled with gas. .

  According to this application example, the nucleic acid amplification reaction container is filled with 50% or more of the volume formed by the first region, the second region, and the flow path. In other words, since the reaction solution is less than 50% of the volume, the reaction solution can be accommodated in the first region or the second region of the nucleic acid amplification reaction vessel. Thereby, the temperature of the reaction solution can be efficiently changed from the first temperature to the second temperature or from the second temperature to the first temperature.

  Application Example 5 In the nucleic acid amplification reaction container described in the above application example, the distance is preferably 0.1 mm or more and 3.0 mm or less.

  According to this application example, the nucleic acid amplification reaction container is formed such that the distance between the first inner wall surface and the second inner wall surface is 0.1 mm or more and 3.0 mm or less. As a result, the thermal conductivity to the reaction solution is improved, so that a high-temperature thermal cycle of, for example, 40 cycles / 3 minutes or less can be realized.

  Application Example 6 In the nucleic acid amplification reaction container according to the application example described above, the wall thickness of at least one of the first wall constituting the first inner wall surface and the second wall constituting the second inner wall surface is: It is preferable that it is 0.01 mm or more and 0.5 mm or less.

  According to this application example, the nucleic acid amplification reaction container has a wall thickness of at least one of the first wall constituting the first inner wall surface and the second wall constituting the second inner wall surface of 0.01 mm or more and 0.5 mm. It is formed as follows. As a result, the thermal conductivity to the reaction solution is improved, so that a high-temperature thermal cycle of, for example, 40 cycles / 3 minutes or less can be realized.

  Application Example 7 In the nucleic acid amplification reaction container according to the application example described above, at least one of the first wall and the second wall has an increased internal pressure in the first region, the second region, and the flow path. It is preferable to be deformed in such a case.

  According to this application example, at least one of the first wall and the second wall of the nucleic acid amplification reaction container is formed of a material that deforms when the internal pressure of the first region, the second region, and the flow path rises. Therefore, it is possible to prevent the nucleic acid amplification reaction vessel from being damaged due to an increase in internal pressure.

  Application Example 8 In the nucleic acid amplification reaction container according to the application example described above, it is preferable that at least one of the first wall and the second wall has light transmittance.

  According to this application example, since the nucleic acid amplification reaction vessel has light permeability on at least one of the first wall and the second wall, the side wall of the nucleic acid amplification reaction vessel is irradiated with excitation light to fluoresce. The amount can be measured. Thereby, the amount of nucleic acids in the reaction solution can be detected.

  Application Example 9 In the nucleic acid amplification reaction container according to the application example, it is preferable that the first inner wall surface and the second inner wall surface are hydrophobic.

  According to this application example, since the first inner wall surface and the second inner wall surface of the nucleic acid amplification reaction vessel are hydrophobic, the first region formed between the first inner wall surface and the second inner wall surface, The flow resistance of the reaction solution flowing in the second region and the flow path is reduced. Thereby, the moving speed when moving the reaction solution from the first region to the second region or from the second region to the first region can be improved.

  [Application Example 10] A nucleic acid amplification reaction apparatus according to this application example is equipped with the nucleic acid amplification reaction container according to any one of Application Examples 1 to 9 in which at least a reaction solution and a gas are arranged. A first heating part that heats the first region of the nucleic acid amplification reaction vessel to a first temperature in a state where the nucleic acid amplification reaction vessel is attached to the attachment unit, and a second region of the nucleic acid amplification reaction vessel A second heating unit that heats the second heating unit to a second temperature different from the first temperature, and a drive that switches the mounting unit, the first heating unit, and the second heating unit between the first arrangement and the second arrangement And the first arrangement is an arrangement in which the first region is located below the second region in the direction of gravity, and the second arrangement has the second region in the direction of gravity. It is characterized by being arranged below the first region. .

  According to this application example, the nucleic acid amplification reaction apparatus includes a mounting unit to which the nucleic acid amplification reaction vessel is mounted, a first heating unit that heats the first region of the nucleic acid amplification reaction vessel to the first temperature, and a first of the nucleic acid amplification reaction vessel. A drive mechanism for switching the second heating unit that heats the two regions to the second temperature between the first arrangement and the second arrangement is provided. In the first arrangement, the first area is located below the second area in the direction of gravity, and in the second arrangement, the second area is located below the first area in the direction of gravity. That is, the reaction solution filled in the nucleic acid amplification reaction vessel is held in the first region in the first arrangement and in the second region in the second arrangement by the action of gravity. The first region is heated to the first temperature by the first heating unit, and the second region is heated to the second temperature different from the first temperature by the second heating unit. The nucleic acid amplification reaction apparatus can bring the reaction solution to the first temperature by holding the mounting unit, the first heating unit, and the second heating unit in the first arrangement, and the mounting unit, the first heating unit, and the second heating unit. By holding the heating part in the second arrangement, the reaction solution can be brought to the second temperature. Therefore, it is possible to provide a nucleic acid amplification reaction apparatus that can easily apply a thermal cycle to a reaction solution.

  Application Example 11 In the nucleic acid amplification reaction method according to this application example, the nucleic acid amplification reaction container according to any one of Application Example 1 to Application Example 9 in which at least a reaction solution and a gas are arranged is applied to Application Example 10. A mounting step of mounting on the mounting portion of the nucleic acid amplification reaction apparatus described above; a first heating step of heating the first region of the nucleic acid amplification reaction vessel to a first temperature; and a second region of the nucleic acid amplification reaction vessel of the first region. A second heating step for heating to a second temperature different from the first temperature; and a first arrangement in which the first region is located below the second region in the direction of gravity, and the second region is below the first region. And a second driving step for switching from the second arrangement to the first arrangement. In the first driving step, the nucleic acid amplification reaction vessel comprises: Forming a first region and a second region; The nucleic acid amplification reaction vessel is rotated in a first direction around a rotation axis that intersects the inner wall surface and has a horizontal component, and in the second driving step, the nucleic acid amplification reaction container is in a second direction opposite to the first direction. It is characterized by being rotated.

  According to this application example, in the nucleic acid amplification reaction method, in the gravity direction, the first region is located below the second region, and the second region is located below the first region. And a second driving step for switching from the second arrangement to the first arrangement. That is, by switching from the first arrangement to the second arrangement in the first driving process, the reaction liquid held in the first area is moved and held in the second area by the action of gravity. By switching from the second arrangement to the first arrangement in the second driving process, the reaction liquid held in the second area is moved and held in the first area by the action of gravity. The first region is heated to the first temperature by the first heating step, and the second region is heated to the second temperature different from the first temperature by the second heating step. In the nucleic acid amplification reaction method, the reaction solution can be changed from the first temperature to the second temperature by the first driving step, and the reaction solution can be changed from the second temperature to the first temperature by the second driving step. Thereby, a heat cycle is added to the reaction solution. In the first driving step, the nucleic acid amplification reaction vessel is rotated in a first direction around a rotation axis that intersects the first inner wall surface and has a horizontal component. In the second driving step, the nucleic acid amplification reaction is performed. The container is rotated in a second direction opposite to the first direction. Thereby, since the twist of the conducting wire that supplies electric power for heating the first and second heating units is reduced, the reliability of the nucleic acid amplification reaction apparatus is improved. Therefore, it is possible to provide a nucleic acid amplification reaction method that can improve the reliability of the nucleic acid amplification reaction apparatus and can easily apply a thermal cycle to the reaction solution.

1 is a perspective view showing an appearance of a nucleic acid amplification reaction device according to Embodiment 1. FIG. The perspective view which shows the structure of the state with which the nucleic acid amplification reaction container was mounted | worn with the nucleic acid amplification reaction apparatus. The disassembled perspective view of a nucleic acid amplification reaction apparatus. Sectional drawing in the AA of FIG. Sectional drawing in the AA line of FIG. 1 at the time of rotating the main body of a nucleic acid amplification reaction apparatus. The front view which shows schematic structure of a nucleic acid amplification reaction container. Sectional drawing in the BB line of FIG. Sectional drawing in the CC line | wire of FIG. The flowchart figure explaining a nucleic acid amplification reaction method. The figure which shows a mode that oil and a reaction liquid are sequentially switched from a 1st area | region to a 2nd area | region by a 1st drive process. The figure which shows a mode that oil and a reaction liquid are sequentially switched from a 1st area | region to a 2nd area | region by a 1st drive process. The figure which shows a mode that oil and a reaction liquid are sequentially switched from a 1st area | region to a 2nd area | region by a 1st drive process. The figure which shows a mode that oil and a reaction liquid are sequentially switched from a 1st area | region to a 2nd area | region by a 1st drive process. The figure which shows a mode that oil and a reaction liquid are sequentially switched from a 1st area | region to a 2nd area | region by a 1st drive process. The figure which shows a mode that oil and a reaction liquid are sequentially switched from a 2nd area | region to a 1st area | region by a 2nd drive process. The figure which shows a mode that oil and a reaction liquid are sequentially switched from a 2nd area | region to a 1st area | region by a 2nd drive process. The figure which shows a mode that oil and a reaction liquid are sequentially switched from a 2nd area | region to a 1st area | region by a 2nd drive process. The figure which shows a mode that oil and a reaction liquid are sequentially switched from a 2nd area | region to a 1st area | region by a 2nd drive process. The figure which shows a mode that oil and a reaction liquid are sequentially switched from a 2nd area | region to a 1st area | region by a 2nd drive process. FIG. 3 is a perspective view illustrating a schematic configuration of a nucleic acid amplification reaction device according to a second embodiment. The perspective view which shows the structure of the state with which the nucleic acid amplification reaction container was mounted | worn with the nucleic acid amplification reaction apparatus. Sectional drawing in the DD line | wire of FIG.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following drawings, the scale of each member or the like is shown differently from the actual scale so as to make each member or the like recognizable. 1 to 7 and 9 to 14, for convenience of explanation, the X axis, the Y axis, and the Z axis are illustrated as three axes orthogonal to each other, and the tip end side of the arrow illustrating the axial direction is shown. The “+ side” and the base end side are “− side”. The direction parallel to the X axis is referred to as “X axis direction”, the direction parallel to the Y axis is referred to as “Y axis direction”, and the direction parallel to the Z axis is referred to as “Z axis direction”.

(Embodiment 1)
<Configuration of nucleic acid amplification reaction apparatus>
FIG. 1 is a perspective view showing an appearance of a nucleic acid amplification reaction apparatus according to Embodiment 1. FIG. FIG. 2 is a perspective view showing a configuration in a state where a nucleic acid amplification reaction vessel is attached to the nucleic acid amplification reaction device. FIG. 3 is an exploded perspective view of the nucleic acid amplification reaction apparatus. 4 is a cross-sectional view taken along line AA in FIG. FIG. 5 is a cross-sectional view taken along line AA in FIG. 1 when the main body of the nucleic acid amplification reaction apparatus is rotated. 1 to 4 show a state where the nucleic acid amplification reaction apparatus 1 is located in a first arrangement described later, and FIG. 5 shows a state where the nucleic acid amplification reaction apparatus 1 is located in a second arrangement. . The configuration of the nucleic acid amplification reaction apparatus 1 will be described with reference to FIGS.

As shown in FIGS. 1 and 2, the nucleic acid amplification reaction apparatus 1 includes a main body 10 and a drive mechanism 20 that are substantially rectangular in a plan view. In the present embodiment, the vertical direction along the direction of gravity is the Z axis, and the + Z axis side is “up”. The longitudinal direction of the main body 10 that intersects the Z axis is taken as the Y axis. A direction intersecting both the Z axis and the Y axis is taken as an X axis.
As shown in FIG. 3, the main body 10 includes a mounting part 11, a first heating part 12, and a second heating part 13. A spacer 14 is provided between the first heating unit 12 and the second heating unit 13. The 1st heating part 12, the 2nd heating part 13, and the spacer 14 comprise rectangular plate shape, and are laminated | stacked along the Z-axis direction. In the first arrangement described later, the main body 10 of the present embodiment is arranged such that the first heating unit 12 is arranged on the side of the bottom plate 17 positioned in the −Z-axis direction, and the lid 50 is positioned on the side of the + Z-axis direction. Two heating units 13 are arranged. In the main body 10 of the present embodiment, the first heating unit 12, the second heating unit 13 and the spacer 14 are fixed to the bottom plate 17, the fixing plate 19 facing the ± X axis direction, and the flange 16 facing the ± Y axis direction. Has been.

  The mounting unit 11 is mounted with a nucleic acid amplification reaction vessel 100 described later. As shown in FIGS. 2 and 3, the mounting unit 11 of the present embodiment has a slot structure in which the nucleic acid amplification reaction vessel 100 is inserted and mounted. The first heat block 12 b of the first heating unit 12, the spacer 14, and The nucleic acid amplification reaction vessel 100 is inserted into a hole that penetrates the second heat block 13b of the second heating unit 13 in the Z-axis direction. There may be a plurality of mounting parts 11, and in the present embodiment, eight mounting parts 11 are provided in the main body 10.

  The nucleic acid amplification reaction apparatus 1 preferably includes a structure that holds the nucleic acid amplification reaction vessel 100 at a predetermined position with respect to the first heating unit 12 and the second heating unit 13. Thereby, the predetermined region of the nucleic acid amplification reaction vessel 100 can be heated by the first heating unit 12 and the second heating unit 13. More specifically, as shown in FIG. 4, the first region 101 constituting the nucleic acid amplification reaction vessel 100 described later can be heated by the first heating unit 12, and the second region 102 can be heated by the second heating unit 13. In the present embodiment, the structure that determines the position of the nucleic acid amplification reaction vessel 100 is the bottom plate 17, and as shown in FIG. 4, the first heating unit 12 is inserted by inserting the nucleic acid amplification reaction vessel 100 to a position in contact with the bottom plate 17. In addition, the nucleic acid amplification reaction vessel 100 can be held at a predetermined position with respect to the second heating unit 13.

  The first heating unit 12 heats a first region 101 of the nucleic acid amplification reaction container 100 described later to a first temperature in a state where the nucleic acid amplification reaction container 100 is mounted on the mounting unit 11. As shown in FIG. 4, the first heating unit 12 is disposed in the main body 10 at a position for heating the first region 101 of the nucleic acid amplification reaction vessel 100.

  The first heating unit 12 may include a mechanism that generates heat and a member that transmits the generated heat to the nucleic acid amplification reaction vessel 100. As shown in FIG. 3, the first heating unit 12 includes a first heater 12a and a first heat block 12b. In the present embodiment, the first heater 12a is a cartridge heater, and is connected to an external power source (not shown) by a conducting wire 15 that passes through the flange 16 on the + Y axis side. The first heater 12a is inserted in the first heat block 12b along the Y-axis direction, and the first heat block 12b is heated when the first heater 12a generates heat. The first heat block 12 b is a member that transfers heat generated from the first heater 12 a to the nucleic acid amplification reaction vessel 100. In this embodiment, it is an aluminum block.

  Since the temperature control of the cartridge heater is easy, the temperature of the first heating unit 12 can be easily stabilized by using the first heater 12a as a cartridge heater. Therefore, a more accurate thermal cycle can be realized. Since aluminum has high thermal conductivity, the nucleic acid amplification reaction vessel 100 can be efficiently heated by making the first heat block 12b made of aluminum. Moreover, since the heating unevenness hardly occurs in the first heat block 12b, a highly accurate thermal cycle can be realized. Further, since the processing is easy, the first heat block 12b can be accurately molded, and the heating accuracy can be improved. Therefore, a more accurate thermal cycle can be realized.

  The first heating unit 12 is preferably in contact with the nucleic acid amplification reaction vessel 100 when the nucleic acid amplification reaction vessel 100 is attached to the attachment unit 11. Thereby, when the nucleic acid amplification reaction vessel 100 is heated by the first heating unit 12, the heat of the first heating unit 12 can be stably transmitted to the nucleic acid amplification reaction vessel 100. Can be stabilized. When the mounting part 11 is formed as a part of the first heating unit 12 as in this embodiment, it is preferable that the mounting part 11 contacts the nucleic acid amplification reaction vessel 100. Thereby, since the heat of the 1st heating part 12 can be stably transmitted to the nucleic acid amplification reaction container 100, the nucleic acid amplification reaction container 100 can be heated efficiently.

  The second heating unit 13 heats the second region 102 of the nucleic acid amplification reaction container 100 to a second temperature different from the first temperature in a state where the nucleic acid amplification reaction container 100 is mounted on the mounting unit 11. In the example shown in FIG. 4, the second heating unit 13 is disposed in the main body 10 at a position where the second region 102 of the nucleic acid amplification reaction vessel 100 is heated. As shown in FIG. 3, the second heating unit 13 includes a second heater 13a and a second heat block 13b. The second heating unit 13 is the same as the first heating unit 12 except that the region of the nucleic acid amplification reaction vessel 100 to be heated and the heating temperature are different from those of the first heating unit 12.

  In addition, although the material of the 1st, 2nd heat block 12b, 13b was demonstrated that it was aluminum, it is not limited to this. The material of the heat block can be selected in consideration of conditions such as thermal conductivity, heat retention and ease of processing. For example, a copper alloy may be used and a plurality of materials may be combined. Further, the first heat block 12b and the second heat block 13b may be made of different materials.

  Moreover, although the 1st heating part 12 and the 2nd heating part 13 demonstrated that it was a cartridge heater, it is not limited to this. The 1st heating part 12 should just heat the 1st field 101 to the 1st temperature, and the 2nd heating part 13 should just be able to heat the 2nd field 102 to the 2nd temperature. As the 1st heating part 12 and the 2nd heating part 13, a carbon heater, a sheet heater, IH (electromagnetic induction heating), a Peltier device, heating liquid, heating gas, etc. can be used, for example. In addition, different heating mechanisms may be employed for the first heating unit 12 and the second heating unit 13.

  In this embodiment, the temperature of the 1st heating part 12 and the 2nd heating part 13 is controlled by the temperature sensor which is not shown in figure and the control part mentioned later. The temperatures of the first heating unit 12 and the second heating unit 13 are preferably set so that the nucleic acid amplification reaction vessel 100 is heated to a desired temperature. In the present embodiment, by controlling the first heating unit 12 to the first temperature and the second heating unit 13 to the second temperature, the first region 101 of the nucleic acid amplification reaction vessel 100 is set to the first temperature, and the second temperature is set to the second temperature. Region 102 can be heated to a second temperature. As the temperature sensor, a thermocouple, a resistance temperature detector, a thermistor, or the like can be used.

  The drive mechanism 20 is a mechanism that switches the mounting unit 11, the first heating unit 12, and the second heating unit 13 between the first arrangement and the second arrangement. As shown in FIG. 4, the first arrangement is such that the first region 101 of the nucleic acid amplification reaction vessel 100 is below the second region 102 in the direction of gravity when the nucleic acid amplification reaction vessel 100 is attached to the attachment unit 11. It is a positioned arrangement. As shown in FIG. 5, the second arrangement is such that the second region 102 of the nucleic acid amplification reaction vessel 100 is below the first region 101 in the direction of gravity when the nucleic acid amplification reaction vessel 100 is attached to the attachment unit 11. It is a positioned arrangement. In the present embodiment, the drive mechanism 20 is provided on the −Y axis side of the main body 10 and includes a motor and a drive shaft (not shown). The drive shaft extends from the drive mechanism 20 along the + Y-axis direction, and the drive shaft and the flange 16 of the main body 10 are connected. In the present embodiment, when the motor is operated, the main body 10 rotates about the Y axis with the drive shaft as the axis of rotation.

  The nucleic acid amplification reaction apparatus 1 of this embodiment includes a control unit (not shown). The control unit controls at least one of a first temperature, a second temperature, a first time, a second time, and the number of cycles of the thermal cycle, which will be described later. When the control unit controls the first time or the second time, the control unit controls the operation of the drive mechanism 20 so that the mounting unit 11, the first heating unit 12, and the second heating unit 13 are predetermined. Control the time kept in the placement. The control unit may provide a different mechanism for each item to be controlled, or may control all items at once.

  The control part in the nucleic acid amplification reaction apparatus 1 of this embodiment is electronic control, and controls all the above items. The control unit of the present embodiment includes a processor such as a CPU (not shown) and a storage device such as a ROM (Read Only Memory) and a RAM (Random Access Memory). The storage device stores various programs and data for controlling the above items. The storage device also has a work area for temporarily storing in-process data and processing results of various processes.

  As shown in FIGS. 3 and 4, the main body 10 of this embodiment is provided with a spacer 14 between the first heating unit 12 and the second heating unit 13. The spacer 14 of this embodiment is a member that holds the first heating unit 12 or the second heating unit 13. By providing the spacer 14, the distance between the first heating unit 12 and the second heating unit 13 can be determined more accurately. That is, the position of the 1st heating part 12 and the 2nd heating part 13 with respect to the 1st field 101 and the 2nd field 102 of nucleic acid amplification reaction container 100 mentioned below can be defined more correctly.

  The material of the spacer 14 can be appropriately selected as necessary, but is preferably a heat insulating material. Thereby, since the influence which the heat of the 1st heating part 12 and the 2nd heating part 13 mutually has can be decreased, temperature control of the 1st heating part 12 and the 2nd heating part 13 becomes easy. When the spacer 14 is a heat insulating material, when the nucleic acid amplification reaction vessel 100 is attached to the attachment portion 11, the nucleic acid amplification reaction vessel 100 is surrounded in the region between the first heating portion 12 and the second heating portion 13. Thus, it is preferable that the spacer 14 is arranged. Thereby, since the heat radiation from the region between the first heating unit 12 and the second heating unit 13 of the nucleic acid amplification reaction vessel 100 can be suppressed, the temperature of the nucleic acid amplification reaction vessel 100 becomes more stable. In the present embodiment, the spacer 14 is a heat insulating material, and the mounting portion 11 penetrates the spacer 14 in the example of FIG. Thereby, when the nucleic acid amplification reaction vessel 100 is heated by the first heating unit 12 and the second heating unit 13, the heat of the nucleic acid amplification reaction vessel 100 is difficult to escape, so the temperature of the first region 101 and the second region 102 Can be made more stable.

  The main body 10 of the present embodiment includes a fixing plate 19. The fixed plate 19 is a member that holds the mounting unit 11, the first heating unit 12, and the second heating unit 13 from the ± X axis directions. In the example shown in FIGS. 2 and 3, two fixing plates 19 are fitted to the flange 16, and the first heating unit 12, the second heating unit 13, and the bottom plate 17 are fixed. Since the structure of the main body 10 is further strengthened by the fixing plate 19, the main body 10 is hardly damaged.

  The main body 10 of this embodiment includes a lid 50. In the example of FIGS. 1 and 4, the mounting portion 11 is covered with a lid 50. By covering the mounting portion 11 with the lid 50, when the first and second heating portions 12 and 13 are heated, heat radiation from the main body 10 to the outside can be suppressed, so that the temperature inside the main body 10 is stabilized. Can do. The lid body 50 may be fixed to the main body 10 by the fixing portion 51. In the present embodiment, the fixing part 51 is a magnet. As shown in the example of FIGS. 2 and 3, a magnet is provided on the surface of the main body 10 that contacts the lid 50. Although not shown in FIG. 2 and FIG. 3, the lid 50 is also provided with a magnet at a position where the magnet of the main body 10 comes into contact. 50 is fixed to the main body 10. Thereby, when the main body 10 is driven by the drive mechanism 20, it is possible to prevent the lid 50 from being detached or moved. Accordingly, it is possible to prevent the temperature in the nucleic acid amplification reaction apparatus 1 from changing due to the removal of the lid 50, so that a more accurate thermal cycle can be applied to the reaction solution 140 described later. In addition, although the fixing | fixed part 51 was demonstrated that it was a magnet, the fixing | fixed part should just be what can fix the cover body 50 and the main body 10. FIG.

  The main body 10 preferably has a highly airtight structure. If the main body 10 has a highly airtight structure, the air inside the main body 10 is difficult to escape to the outside of the main body 10, so that the temperature inside the main body 10 becomes more stable. In the present embodiment, as shown in FIG. 3, the space inside the main body 10 is sealed by the two flanges 16, the bottom plate 17, the two fixing plates 19, and the lid 50. The fixed plate 19, the bottom plate 17, the lid 50, and the flange 16 are preferably formed using a heat insulating material. Thereby, since the heat radiation from the main body 10 to the outside can be further suppressed, the temperature in the main body 10 can be further stabilized.

FIG. 6 is a front view showing a schematic configuration of the nucleic acid amplification reaction vessel. 7 is a cross-sectional view taken along line BB in FIG. 8 is a cross-sectional view taken along the line CC of FIG. 6 to 8 show a state in which the nucleic acid amplification reaction vessel 100 is held in the first arrangement state.
Next, the configuration of the nucleic acid amplification reaction vessel 100 will be described with reference to FIGS.

  The nucleic acid amplification reaction container 100 includes a first region 101 for bringing the reaction solution 140 to a first temperature, a second region 102 for bringing the reaction solution 140 to a second temperature different from the first temperature, a first region 101, and a second region. 102 and a plurality of flow paths (first flow path 103 and second flow path 104) for moving the reaction solution 140 in communication with 102.

  Specifically, the nucleic acid amplification reaction vessel 100 has a flat rectangular shape, in which a first region 101 is formed on one short side of the rectangle, and a second region 102 is formed on the other short side. In addition, a first flow path 103 that connects the first region 101 and the second region 102 is formed along one long side of the rectangle, and the first region 101 and the second region are formed along the other long side of the rectangle. A second flow path 104 communicating with 102 is formed. Thereby, the 1st field 101, the 1st channel 103, the 2nd field 102, and the 2nd channel 104 are connected annularly.

  The first area 101, the second area 102, the first flow path 103, and the second flow path 104 are in a space sandwiched between the first inner wall surface 111 and the second inner wall surface 112 that faces the first inner wall surface 111. Is formed. As a material of the first wall 111a constituting the first inner wall surface 111 and the second wall 112a constituting the second inner wall surface 112, for example, a plastic resin in which polypropylene (polypropylene) or the like is formed in a film shape is used. . The first region 101, the second region 102, the outer periphery of the first and second flow paths 103, 104, and the region surrounded by the first area 101, the second region 102, the first and second flow paths 103, 104 are The first wall 111a and the second wall 112a are in close contact by heat welding or the like. Thereby, in the nucleic acid amplification reaction vessel 100, a space communicated by the first region 101, the second region 102, the first channel 103, and the second channel 104 is integrally formed.

  In the first region 101, the second region 102, the first channel 103, and the second channel 104, at least the reaction solution 140 and the gas 150 are disposed. Since the nucleic acid amplification reaction vessel 100 has a plurality of channels, the first channel 103 and the second channel 104, the reaction solution 140 is transferred from the first region 101 to the second region 102 or the second region 102. When moving from the first region 101 to the first region 101 by the action of gravity, the moving speed of moving the reaction solution 140 can be improved by replacing the reaction solution 140 and the gas 150 with different first and second flow paths 103 and 104. it can. As described above, when the nucleic acid amplification reaction vessel 100 attached to the nucleic acid amplification reaction apparatus 1 is in the first arrangement state, the first region 101 brings the reaction solution 140 to the first temperature. When the nucleic acid amplification reaction vessel 100 attached to the nucleic acid amplification reaction apparatus 1 is in the second arrangement state, the second region 102 brings the reaction solution 140 to the second temperature.

  Oil 130 is further disposed in the first region 101, the second region 102, the first flow path 103, and the second flow path 104 of the present embodiment. Since the oil 130 has a specific gravity smaller than that of the reaction liquid 140 and does not mix with the reaction liquid 140, that is, does not mix, the reaction liquid 140 is held in the oil 130 in a droplet state. Since the reaction liquid 140 has a specific gravity greater than that of the oil 130, the reaction liquid 140 is positioned at the lowermost portion of the first region 101 in the first arrangement state, and is positioned at the lowermost portion of the second region 102 in the second state. As the oil 130, for example, dimethyl silicone oil or paraffin oil can be used. By disposing oil 130 in the first region 101, the second region 102, the first channel 103, and the second channel 104, the reaction solution 140 is transferred from the first region 101 to the second region 102 or second. The movement from the region 102 to the first region 101 and the temperature change from the first temperature to the second temperature or from the second temperature to the first temperature can be suitably performed.

  The reaction liquid 140 is a liquid containing components necessary for the reaction. When the reaction is PCR, DNA (target nucleic acid) amplified by PCR, DNA polymerase necessary for amplifying DAN, primers, and the like are included. For example, when PCR is performed using the oil 130, the reaction solution 140 is preferably an aqueous solution containing the above components.

  50% or more of the volume formed by the first region 101, the second region 102, the first flow path 103, and the second flow path 104 is filled with the gas 150. In other words, since the reaction solution 140 and the oil 130 are less than 50% of the volume, the nucleic acid amplification reaction vessel 100 that can store the reaction solution 140 and the oil 130 in the first region 101 or the second region 102 is formed. be able to. Thereby, the temperature of the reaction solution 140 can be efficiently changed from the first temperature to the second temperature or from the second temperature to the first temperature.

  The first inner wall surface 111 and the second inner wall surface 112 are preferably hydrophobic. By making the first inner wall surface 111 and the second inner wall surface 112 of the nucleic acid amplification reaction vessel 100 hydrophobic, the first region 101 formed between the first inner wall surface 111 and the second inner wall surface 112, the first The flow resistance of the reaction solution 140 flowing through the two regions 102 and the first and second flow paths 103 and 104 is reduced. Thereby, the moving speed when moving the reaction solution 140 from the first region 101 to the second region 102 or from the second region 102 to the first region 101 can be improved.

  At least one of the first wall 111a constituting the first inner wall surface 111 and the second wall 112a constituting the second inner wall surface 112 has the first region 101, the second region 102, and the first and second flow paths 103. , 104 is preferably deformed when the internal pressure rises. In the nucleic acid amplification reaction vessel 100 of the present embodiment, the first wall 111a and the second wall 112a are made of plastic resin whose shape is deformed, so that the internal pressure increases by heating the first region 101 and the second region 102. In this case, the nucleic acid amplification reaction vessel can be prevented from being damaged.

  The distance L between the first inner wall surface 111 and the second inner wall surface 112 is such that the reaction liquid 140 is introduced into the first region 101 or the second region 102 via the first channel 103 or the second channel 104. In this case, the reaction liquid 140 is in contact with both the first inner wall surface 111 and the second inner wall surface 112. Since the reaction liquid 140 is in contact with both the first inner wall surface 111 and the second inner wall surface 112, the first heating unit 12 or the second heating unit 13 described above via the first wall 111a and the second wall 112a. Heated directly from. Thereby, the heat conductivity for making the reaction liquid 140 1st temperature or 2nd temperature improves.

  Furthermore, the distance L between the first inner wall surface 111 and the second inner wall surface 112 is preferably 0.1 mm or greater and 3.0 mm or less. The smaller the distance L between the first inner wall surface 111 and the second inner wall surface 112, the wider the contact area between the reaction liquid 140 and the first and second inner wall surfaces 111, 112. Conductivity is improved. By attaching the nucleic acid amplification reaction vessel 100 having the distance L of 3 mm or less to the nucleic acid amplification reaction apparatus 1 described above, it is possible to enable thermal cycling at a high speed of, for example, 40 cycles / 3 minutes or less. When the distance L between the first inner wall surface 111 and the second inner wall surface 112 is 0.1 mm or less, the effect of shortening the heat cycle time due to the improvement in thermal conductivity is reduced, and conversely the movement of the reaction solution 140 is reduced. There is a risk of being disturbed.

  The wall thickness of at least one of the first wall 111a and the second wall 112a is preferably 0.01 mm or more and 0.5 mm or less. As the wall thickness of the first wall 111a and the second wall 112a is thinner, the thermal conductivity to the reaction liquid 140 is improved. By attaching the nucleic acid amplification reaction vessel 100 having a wall thickness of 0.5 mm or less to the above-described nucleic acid amplification reaction apparatus 1, it is possible to enable thermal cycling at a high speed of, for example, 40 cycles / 3 minutes or less. When the wall thickness is 0.01 mm or less, it becomes difficult to manufacture the nucleic acid amplification reaction vessel 100, and the manufacturing cost increases.

  The volume of the reaction solution 140 introduced into the first region 101, the second region 102, and the first and second flow paths 103, 104 is not particularly limited. The volume of the reaction solution 140 introduced into the first region 101, the second region 102, and the first and second flow paths 103, 104 is, for example, 1 μl or more and 100 μl or less, preferably 5 μl or more and 50 μl or less, more preferably 10 μl or more. It is 40 μl or less, more preferably 20 μl or more and 30 μl or less. If it is the volume of this range, it will be easy to amplify the nucleic acid in the reaction liquid 140. FIG.

Although the nucleic acid amplification reaction vessel 100 has been described as having a rectangular shape, the nucleic acid amplification reaction vessel 100 may have a rounded rectangular shape, a circular shape, or an elliptical shape. The first region 101, the second region 102, What is necessary is just the shape in which the space connected so that the 1st flow path 103 and the 2nd flow path 104 may circulate was formed.
The first region 101, the second region 102, and the first and second flow paths 103, 104 have been described as being formed between the first inner wall surface 111 and the second inner wall surface 112. An inner wall surface (side wall surface) that connects the first inner wall surface 111 and the second inner wall surface 112 may be provided.
In addition, the first region 101 and the second region 102 have been described as being connected by the two first and second flow paths 103 and 104, but may be connected by three or more flow paths. .

<Nucleic acid amplification reaction method>
FIG. 9 is a flowchart for explaining the nucleic acid amplification reaction method.
Next, a nucleic acid amplification reaction method (thermal cycle process) using the nucleic acid amplification reaction vessel 100 and the nucleic acid amplification reaction apparatus 1 will be described with reference to FIGS. 4, 5, and 9. In addition, the direction of the arrow g in FIG.4 and FIG.5 has shown the direction where gravity acts. In the present embodiment, a case where shuttle PCR (two-stage temperature PCR) is performed will be described as an example of thermal cycle processing for performing nucleic acid amplification. In addition, each process demonstrated below shows an example of a heat cycle process. If necessary, the order of processes may be changed, two or more processes may be performed continuously or in parallel, or processes may be added.

  Shuttle PCR is a technique for amplifying nucleic acids in the reaction solution 140 by repeatedly applying a high-temperature and low-temperature treatment to the reaction solution 140. Dissociation (denaturation reaction) of double-stranded DNA occurs in high-temperature treatment, and annealing (reaction in which a primer binds to single-stranded DNA) and extension reaction (primer starts as a complementary strand of DNA) are formed in low-temperature treatment. Reaction) is performed.

  Generally, the high temperature in shuttle PCR is a temperature between 80 ° C. and 100 ° C., and the low temperature is a temperature between 50 ° C. and 70 ° C. The treatment at each temperature is performed for a predetermined time, and the time for keeping at a high temperature is generally shorter than the time for keeping at a low temperature. For example, the high temperature may be about 0.2 to 10 seconds, and the low temperature may be about 0.5 to 60 seconds. The time required for transferring heat from the heating units 12 and 13 to the reaction solution 140, the reaction (denaturation reaction, The optimum time is selected in consideration of the time required for the annealing and elongation reaction) to occur.

  In addition, since the appropriate time, temperature, and number of cycles (the number of repetitions of high temperature and low temperature) differ depending on the type and amount of reagent used, an appropriate protocol was determined in consideration of the type of reagent and the amount of reaction solution 140. It is preferred to carry out the reaction above.

  Step S <b> 1 is a mounting step for mounting the nucleic acid amplification reaction vessel 100 in which at least the reaction solution 140 and the gas 150 are disposed on the mounting portion 11 of the nucleic acid amplification reaction apparatus 1. In the present embodiment, the reaction solution 140 is introduced into the first region 101, the second region 102, and the first and second flow paths 103 and 104 filled with the oil 130, and then the sealed nucleic acid amplification reaction vessel 100 is mounted. 11 is attached. The volume of the reaction liquid 140 added to the oil 130 is substantially equal to or slightly smaller than the volume of the first region 101 and the second region 102, and the first region 101, the second region 102, and the first and second flow paths 103, 104 are. Is filled with oil 130, reaction liquid 140 and gas 150.

  The reaction solution 140 can be introduced into the nucleic acid amplification reaction vessel 100 using a micropipette, an inkjet type dispensing device, or the like. In a state where the nucleic acid amplification reaction vessel 100 is attached to the attachment unit 11, the first heating unit 12 is placed in the nucleic acid amplification reaction vessel 100 at a position including the first region 101 and the second heating unit 13 includes the second region 102. Touching. In this embodiment, as shown in FIG. 4, the flat rectangular nucleic acid amplification reaction vessel 100 has a plane formed by a short side and a long side and an XZ plane formed by an X axis and a Z axis. It is inserted into the mounting portion 11 from the first region 101 side so as to be parallel, and is mounted so that the short side on the first region 101 side contacts the bottom plate 17. Thereby, the nucleic acid amplification reaction vessel 100 can be held in a predetermined direction and position with respect to the first heating unit 12 and the second heating unit 13.

  In this embodiment, arrangement | positioning of the mounting part 11, the 1st heating part 12, and the 2nd heating part 13 in step S1 is a 1st arrangement | positioning. As shown in FIG. 4, the first arrangement is an arrangement in which the first region 101 is located below the second region 102 in the direction of gravity. In this embodiment, the nucleic acid amplification reaction is performed in the direction in which gravity acts. In this arrangement, the first region 101 of the container 100 is positioned at the bottom. In the first arrangement, the oil 130 and the reaction liquid 140 are accommodated in the first region 101, and the reaction liquid 140 having a specific gravity greater than that of the oil 130 is located at the lowermost part of the first region 101. After mounting the nucleic acid amplification reaction vessel 100 on the mounting part 11, the mounting part 11 is covered with the lid 50.

  Step S2 includes a first heating step of heating the first region 101 of the nucleic acid amplification reaction vessel 100 to a first temperature, and a second heating step of heating the second region 102 of the nucleic acid amplification reaction vessel 100 to a second temperature different from the first temperature. 2 heating process. By the first and second heating steps, a temperature gradient in which the temperature gradually changes between the first temperature and the second temperature is formed between the first region 101 and the second region 102. In the present embodiment, the first temperature is a relatively high temperature among the temperatures suitable for the target reaction in the thermal cycle process, and the second temperature is a temperature suitable for the target reaction in the thermal cycle process. Of these, the temperature is relatively low. Therefore, in step S <b> 2 of the present embodiment, a temperature gradient is formed in which the temperature decreases from the first region 101 toward the second region 102. Since the thermal cycle treatment of this embodiment is shuttle PCR, it is preferable that the first temperature is a temperature suitable for dissociation of double-stranded DNA, and the second temperature is a temperature suitable for annealing and extension reaction.

  Since the arrangement of the mounting part 11, the first heating part 12 and the second heating part 13 in step S2 is the first arrangement, when the nucleic acid amplification reaction vessel 100 is heated in step S2, the reaction solution 140 is brought to the first temperature. Heated. Accordingly, in step S2, the reaction at the first temperature is performed on the reaction liquid 140.

  In step S3, it is determined whether or not the first time has elapsed in the first arrangement. In this embodiment, the determination is performed by a control unit (not shown). The first time is a time during which the mounting unit 11, the first heating unit 12, and the second heating unit 13 are held in the first arrangement state. In the present embodiment, when step S3 is performed following the mounting in step S1, that is, when the first step S3 is performed, the time after the nucleic acid amplification reaction apparatus 1 is operated is the first time. It is determined whether or not In the first arrangement, since the reaction solution 140 is heated to the first temperature, the first time is preferably set to a time for causing the reaction solution 140 to react at the first temperature in the target reaction. In this embodiment, it is preferable to set the time required for dissociation of double-stranded DNA. When it determines with 1st time having passed (step S3: Yes), it progresses to step S4. If it is determined that the first time has not elapsed (step S3: No), step S3 is repeated.

  Step S4 is a first driving process for switching from the first arrangement to the second arrangement. The second arrangement is an arrangement in which the second region 102 is vertically lower than the first region 101. In this embodiment, the second region 102 of the nucleic acid amplification reaction vessel 100 is placed at the maximum in the direction in which gravity acts. It is an arrangement located at the bottom. In the second arrangement, the oil 130 and the reaction liquid 140 are accommodated in the second region 102, and the reaction liquid 140 having a specific gravity greater than that of the oil 130 is located at the lowermost part of the second region 102. In the first driving step, the arrangement of the mounting unit 11, the first heating unit 12, and the second heating unit 13 is switched from the state of the first arrangement shown in FIG. 4 to the state of the second arrangement shown in FIG. In the nucleic acid amplification reaction apparatus 1 of the present embodiment, the switching is performed by the drive mechanism 20 rotating the main body 10 connected to the drive shaft under the control of the control unit.

  The nucleic acid amplification reaction vessel 100 has a rotation axis having a horizontal component that intersects the first inner wall surface 111 forming the first region 101 and the second region 102, that is, the plane formed by the long side and the short side. It is rotated in the first direction around. Specifically, the rotation axis is a drive axis extending in the + Y-axis direction (horizontal direction) from the drive mechanism 20, and in this embodiment, the plane formed by the long side and the short side is a drive axis as the rotation axis. It is almost orthogonal. In the first driving process, the main body 10 is rotated 180 ° counterclockwise (first direction) toward the + Y axis. As a result, the positional relationship in the direction in which gravity acts between the first region 101 and the second region 102 is opposite to that in the first arrangement, so that the oil 130 and the reaction liquid 140 are separated from the first region 101 by the action of gravity. The second area 102 is switched. When the control unit stops the operation of the driving mechanism 20 when the placement of the mounting unit 11, the first heating unit 12, and the second heating unit 13 reaches the second configuration, the mounting unit 11, the first heating unit 12, and the second heating unit 13 are stopped. The arrangement of the two heating units 13 is held in the second arrangement.

FIG. 10A to FIG. 10E are views showing a state in which the oil and the reaction liquid are sequentially switched from the first region to the second region by the first driving process. Here, the movement of the oil 130 and the reaction liquid 140 will be described.
FIG. 10A shows the state of the nucleic acid amplification reaction vessel 100 when the mounting unit 11, the first heating unit 12, and the second heating unit 13 are located in the first arrangement. Since the first region 101 is located at the lowermost part in the direction of gravity, the oil 130 and the reaction liquid 140 are accommodated in the first region 101, and the first flow path 103, the second area 102, and the second flow path. 104 is filled with a gas 150. By the first driving process, the nucleic acid amplification reaction vessel 100 is rotated counterclockwise toward the + Y axis (clockwise toward the paper surface of FIG. 10A).

  FIG. 10B shows a state in which the nucleic acid amplification reaction vessel 100 is rotated 45 ° from the state of FIG. 10A. In this state, the tip formed by the short side on the first region 101 side and the long side on the first flow path 103 side is located at the lowest position in the direction of gravity, so that part of the oil 130 is moved by gravity. It enters the first flow path 103 from one area 101. At the same time, a gas 150 having the same volume as the oil 130 that has entered the first flow path 103 flows from the first flow path 103 into the first area 101 via the second area 102 and the second flow path 104. At this time, the reaction solution 140 is located at the lowermost part.

  FIG. 10C shows a state in which the nucleic acid amplification reaction vessel 100 is further rotated by 45 ° from the state of FIG. 10B, that is, a state in which the nucleic acid amplification reaction vessel 100 is rotated by 90 ° from the state of FIG. In this state, the long side on the first flow path 103 side is located at the lowermost part in the direction of gravity, so that the oil 130 and the reaction liquid 140 remaining in the first region 101 in the state of FIG. It moves into one flow path 103. At the same time, the gas 150 having the same volume as the volume of the oil 130 and the reaction solution 140 moved to the first flow path 103 flows into the first area 101 from the first flow path 103 through the second area 102 and the second flow path 104. To do. Thereby, the inside of the first region 101 is filled with the gas 150.

  FIG. 10D shows a state in which the nucleic acid amplification reaction vessel 100 is further rotated by 45 ° from the state in FIG. 10C, that is, a state in which the nucleic acid amplification reaction vessel 100 is rotated by 135 ° from the state in FIG. 10A. In this state, in the gravity direction, the tip formed by the long side on the first flow path 103 side and the short side on the second region 102 side is located at the bottom, so that part of the oil 130 is moved by gravity. The reaction liquid 140 moves from the first flow path 103 to the second area 102 and moves from the first flow path 103 to the second area 102. At the same time, after the oil 130 that has entered the second region 102 and the gas 150 having the same volume as the volume of the reaction solution 140 that has moved to the second region 102 pass from the second region 102 through the second flow path 104 and the first region 101. It flows into the first flow path 103. At this time, the reaction solution 140 is located at the lowermost part.

  FIG. 10E shows a state in which the nucleic acid amplification reaction vessel 100 is further rotated by 45 ° from the state of FIG. 10D, that is, a state in which the nucleic acid amplification reaction vessel 100 is rotated by 180 ° from the state of FIG. In this state, the short side on the second region 102 side is located at the bottom in the direction of gravity, so that the oil 130 remaining in the first flow path 103 in the state of FIG. Move to. At the same time, a gas 150 having the same volume as the oil 130 moved to the second region 102 flows from the second region 102 into the first channel 103 through the second channel 104 and the first region 101. Thereby, the oil 130 and the reaction liquid 140 are accommodated in the second region 102, and the second channel 104, the first region 101, and the first channel 103 are filled with the gas 150.

  In step S5, it is determined whether the second time has elapsed in the second arrangement. In this embodiment, the determination is performed by a control unit (not shown). The second time is a time for holding the mounting unit 11, the first heating unit 12, and the second heating unit 13 in the second arrangement. In the present embodiment, since the second region 102 is heated to the second temperature in step S2, the placement unit 11, the first heating unit 12, and the second heating unit 13 are arranged in the second configuration in step S5. It is determined whether or not the time after reaching the placement has reached the second time. In the second arrangement, since the reaction solution 140 is held in the second region 102, the reaction solution 140 is heated to the second temperature for the time that the main body 10 is held in the second arrangement. Therefore, the second time is preferably a time for heating the reaction liquid 140 to the second temperature in the target reaction. In the present embodiment, it is preferable to set the time required for annealing and extension reaction. When it determines with 2nd time having passed (step S5: Yes), it progresses to step S6. If it is determined that the second time has not elapsed (step S5: No), step S5 is repeated.

  In step S6, it is determined whether the number of thermal cycles has reached a predetermined number of cycles. The determination is performed by a control unit (not shown). Specifically, it is determined whether or not the procedure from step S3 to step S5 has been completed a predetermined number of times. When Step S3 to Step S5 are performed once, the reaction solution 140 is subjected to one thermal cycle. Therefore, the number of times determined as “Yes” in Step S5 can be set as the number of thermal cycles. . Therefore, it can be determined by step S6 whether or not the number of thermal cycles necessary for the target reaction has been performed. When the thermal cycle is performed for the predetermined number of cycles (step S6: Yes), the process is completed. If the number of thermal cycles has not been performed (step S6: No), the process proceeds to step S7, and steps S3 to S6 are repeated.

  Step S7 is a second driving process for switching from the second arrangement to the first arrangement. The arrangement of the mounting unit 11, the first heating unit 12, and the second heating unit 13 is changed from the second arrangement to the first by rotating the main body 10 connected to the drive shaft by the drive mechanism 20 under the control of the control unit. Switch to placement. In the second driving process of the present embodiment, the main body 10 is rotated 180 ° clockwise (second direction opposite to the first direction) toward the + Y axis. As a result, the positional relationship between the first region 101 and the second region 102 in the direction in which gravity acts is opposite to that in the second arrangement, so that the oil 130 and the reaction liquid 140 are separated from the second region 102 by the action of gravity. The first area 101 is switched. When the control unit stops the operation of the drive mechanism 20 when the placement of the mounting unit 11, the first heating unit 12, and the second heating unit 13 reaches the first configuration, the mounting unit 11, the first heating unit 12, and the second heating unit 13 are stopped. The arrangement of the two heating units 13 is held in the first arrangement. In addition, in step S3 performed following step S7, that is, in step S3 after the second time, the placement of the mounting portion 11, the first heating portion 12, and the second heating portion 13 has reached the first placement. It is determined whether the time has reached the first time.

FIG. 11A to FIG. 11E are diagrams illustrating how oil and a reaction liquid are sequentially switched from the second region to the first region in the second driving process. Here, the movement of the oil 130 and the reaction liquid 140 will be described.
FIG. 11A shows a state of the nucleic acid amplification reaction vessel 100 when the mounting unit 11, the first heating unit 12, and the second heating unit 13 are located in the second arrangement. Since the second region 102 is located at the lowermost part in the direction of gravity, the oil 130 and the reaction liquid 140 are accommodated in the second region 102, and the first channel 103, the first region 101, and the second channel are stored. 104 is filled with a gas 150. By the second driving process, the nucleic acid amplification reaction vessel 100 is rotated clockwise toward the + Y axis (counterclockwise toward the paper surface of FIG. 10A).

  FIG. 11B shows a state in which the nucleic acid amplification reaction vessel 100 is rotated 45 ° from the state of FIG. 11A. In this state, the tip formed by the short side on the second region 102 side and the long side on the first flow path 103 side is located at the bottom in the direction of gravity, so that part of the oil 130 is moved by gravity. 2 enters the first flow path 103 from the region 102. At the same time, a gas 150 having the same volume as the oil 130 that has entered the first flow path 103 flows from the first flow path 103 into the second area 102 via the first area 101 and the second flow path 104. At this time, the reaction solution 140 is located at the lowermost part.

  FIG. 11C shows a state in which the nucleic acid amplification reaction vessel 100 is further rotated by 45 ° from the state of FIG. 11B, that is, a state in which the nucleic acid amplification reaction vessel 100 is rotated by 90 ° from the state of FIG. 11A. In this state, in the direction of gravity, the long side on the first flow path 103 side is located at the bottom, so that the oil 130 and the reaction liquid 140 remaining in the second region 102 in the state of FIG. It moves into one flow path 103. At the same time, the gas 150 having the same volume as the volume of the oil 130 and the reaction solution 140 moved to the first channel 103 flows into the second region 102 from the first channel 103 via the first region 101 and the second channel 104. To do. Thereby, the inside of the second region 102 is filled with the gas 150.

  FIG. 11D shows a state in which the nucleic acid amplification reaction vessel 100 is further rotated by 45 ° from the state of FIG. 11C, that is, a state in which the nucleic acid amplification reaction vessel 100 is rotated by 135 ° from the state of FIG. In this state, the tip formed by the long side on the first flow path 103 side and the short side on the first region 101 side is located at the bottom in the direction of gravity, so that part of the oil 130 is moved by gravity. The reaction liquid 140 moves from the first flow path 103 to the first area 101 through the first flow path 103. At the same time, the oil 130 that has entered the first region 101 and the gas 150 having the same volume as the volume of the reaction solution 140 that has moved to the first region 101 are transferred from the first region 101 through the second channel 104 and the second region 102. It flows into one flow path 103. At this time, the reaction solution 140 is located at the lowermost part.

  FIG. 11E shows a state in which the nucleic acid amplification reaction container 100 is further rotated by 45 ° from the state of FIG. 11D, that is, a state in which the nucleic acid amplification reaction vessel 100 is rotated by 180 ° from the state of FIG. In this state, since the short side on the first region 101 side is located at the bottom in the direction of gravity, the oil 130 remaining in the first flow path 103 in the state of FIG. Move to. At the same time, the gas 150 having the same volume as the oil 130 moved to the first region 101 flows from the first region 101 into the first channel 103 via the second channel 104 and the second region 102. Thereby, the oil 130 and the reaction liquid 140 are accommodated in the first region 101, and the second channel 104, the second region 102, and the first channel 103 are filled with the gas 150.

The nucleic acid amplification reaction container 100 includes a flow path (first flow path 103 in the present embodiment) for moving the oil 130 and the reaction solution 140 between the first area 101 and the second area 102, and a gas 150 for the first area. 101 and the second region 102 (in this embodiment, the second channel 104), the movement speed of moving the oil 130 and the reaction liquid 140 can be improved. it can.
Further, since the main body 10 of the nucleic acid amplification reaction apparatus 1 is rotated in the opposite directions in the first driving process and the second driving process, the conductor 15 for supplying electric power to the first and second heaters 12a and 13a is twisted. Can be reduced.

In the present embodiment, the main body 10 of the nucleic acid amplification reaction apparatus 1 is described as being rotated counterclockwise toward the + Y axis in the first driving step and clockwise toward the + Y axis in the second driving step. However, it may be rotated clockwise toward the + Y axis in the first driving step and counterclockwise toward the + Y axis in the second driving step.
In addition, the main body 10 of the nucleic acid amplification reaction apparatus 1 has been described as rotating in the reverse direction in the first driving process and the second driving process, but after rotating in the same direction a plurality of times, May be rotated the same number of times. Thereby, since the twist which arose in the conducting wire 15 can be eliminated, compared with the case where the rotation to a reverse direction is not performed, deterioration of the conducting wire 15 can be suppressed.

  In the present embodiment, the first inner wall surface 111 (a plane formed by the long side and the short side) and the rotation axis (drive shaft) are described as being substantially orthogonal to each other. The rotation axis only needs to intersect at an angle other than 0 ° or 180 ° (parallel). As a result, when the nucleic acid amplification reaction vessel 100 is rotated about the rotation axis, the first flow path 103 and the second flow path 104 are rotated in a state of being vertically shifted in the direction of gravity. In addition, the reaction solution 140 and the gas 150 can be moved in different flow paths.

  In the present embodiment, the nucleic acid amplification reaction vessel 100 has been described as being hermetically sealed. However, a communication hole that communicates with the outside air is provided in the flow path for moving the gas 150 (the second flow path 104 in the present embodiment). It may be done. As a result, the gas 150 and the outside air in the nucleic acid amplification reaction vessel 100 circulate in accordance with the movement of the oil 130 and the reaction liquid 140, so that the moving speed for moving the oil 130 and the reaction liquid 140 can be further improved.

As described above, according to the nucleic acid amplification reaction vessel 100, the nucleic acid amplification reaction apparatus 1, and the nucleic acid amplification reaction method according to this embodiment, the following effects can be obtained.
The nucleic acid amplification reaction vessel 100 includes first and second flow paths 103 and 104 that connect the first region 101 and the second region 102. For example, the first channel 103 is a channel that moves the oil 130 and the reaction solution 140 between the first region 101 and the second region 102 by the action of gravity, and the second channel 104 is a gas 150 that moves the gas 150 to the first region 103. By using a flow path that moves between the first region 101 and the second region 102, the moving speed of moving the oil 130 and the reaction solution 140 can be improved. The distance L between the first inner wall surface 111 and the second inner wall surface 112 of the nucleic acid amplification reaction vessel 100 is set to a distance at which the reaction solution 140 contacts both the first inner wall surface 111 and the second inner wall surface 112. Has been. Thereby, since the reaction liquid 140 is directly heated from the 1st heating part 12 or the 2nd heating part 13 via the 1st wall 111a and the 2nd wall 112a, the reaction liquid 140 is made into 1st temperature or 2nd temperature. Therefore, the thermal conductivity is improved. Therefore, it is possible to provide the nucleic acid amplification reaction vessel 100 that achieves both improvement in the moving speed for moving the reaction solution 140 and the thermal conductivity to the reaction solution 140.

  The nucleic acid amplification reaction apparatus 1 includes a drive mechanism 20 that switches the mounting unit 11, the first heating unit 12, and the second heating unit 13 between the first arrangement and the second arrangement. The nucleic acid amplification reaction vessel 100 is mounted on the mounting unit 11 such that the first region 101 is heated to the first temperature by the first heating unit 12 and the second region 102 is heated to the second temperature by the second heating unit 13. Is done. The nucleic acid amplification reaction container 100 has the first region 101 located below the second region 102 in the first arrangement, and the second region 102 located below the first region 101 in the second arrangement. That is, the reaction liquid 140 is held in the first region 101 in the first arrangement and in the second region 102 in the second arrangement by the action of gravity. The nucleic acid amplification reaction apparatus 1 can bring the reaction solution 140 to the first temperature by holding the mounting unit 11, the first heating unit 12, and the second heating unit 13 in the first arrangement by the drive mechanism 20, By holding the mounting unit 11, the first heating unit 12, and the second heating unit 13 in the second arrangement, the reaction solution 140 can be brought to the second temperature. Therefore, it is possible to provide the nucleic acid amplification reaction apparatus 1 that can easily apply a heat cycle to the reaction solution 140.

  In the nucleic acid amplification reaction method, the first region 101 of the nucleic acid amplification reaction vessel 100 is positioned below the second region 102 in the direction of gravity, and the second region 102 is positioned below the first region 101 in the first direction. A first driving process for switching to the second arrangement, and a second driving process for switching from the second arrangement to the first arrangement. The nucleic acid amplification reaction vessel 100 is mounted on the mounting unit 11 such that the first region 101 is heated to the first temperature by the first heating unit 12 and the second region 102 is heated to the second temperature by the second heating unit 13. Is done. That is, by switching from the first arrangement to the second arrangement in the first driving process, the reaction liquid 140 held in the first area 101 is moved and held in the second area 102 by the action of gravity. . By switching from the second arrangement to the first arrangement in the second driving process, the reaction liquid 140 held in the second area 102 is moved and held in the first area 101 by the action of gravity. In the nucleic acid amplification reaction method, the reaction solution 140 can be changed from the first temperature to the second temperature by the first driving step, and the reaction solution 140 can be changed from the second temperature to the first temperature by the second driving step. Thereby, a thermal cycle is added to the reaction liquid 140. Moreover, the main body 10 of the nucleic acid amplification reaction apparatus 1 is rotated in the opposite direction between the first driving process and the second driving process. Thereby, since the twist of the conducting wire 15 which supplies electric power to the 1st, 2nd heater 12a, 13a is reduced, the reliability of the nucleic acid amplification reaction apparatus 1 is improved. Therefore, the reliability of the nucleic acid amplification reaction apparatus 1 can be improved, and a nucleic acid amplification reaction method capable of easily applying a heat cycle to the reaction solution 140 can be provided.

(Embodiment 2)
FIG. 12 is a perspective view illustrating a schematic configuration of the nucleic acid amplification reaction device according to the second embodiment. FIG. 13 is a perspective view showing a configuration in a state where a nucleic acid amplification reaction vessel is attached to the nucleic acid amplification reaction device. 14 is a cross-sectional view taken along the line DD in FIG. The configuration of the nucleic acid amplification reaction apparatus 2 will be described with reference to FIGS. In addition, in FIG. 12, the figure which saw through the cover body 50 is shown. Moreover, about the same component as Embodiment 1, the same number is used and the overlapping description is abbreviate | omitted.

  In the first embodiment, an example in which the nucleic acid amplification reaction device 1 does not include a detection device has been shown. However, as shown in FIGS. 12 and 13, the nucleic acid amplification reaction device 2 according to the second embodiment measures the amount of nucleic acid. The fluorescence detector 40 is included. It is preferable that at least one of the first wall 111a and the second wall 112a of the nucleic acid amplification reaction vessel 100 has light transmittance. The first and second walls 111a and 112a of the present embodiment are formed of a plastic resin such as polypropylene that transmits light. In addition, as shown in FIG. 13, the flat rectangular nucleic acid amplification reaction vessel 100 has an acute inner angle formed by the plane formed by the short side and the long side and the XZ plane formed by the X axis and the Z axis. It is attached to the attachment part 11 so as to be. In the present embodiment, the interior angle formed by the plane formed by the short side and the long side and the XZ plane is set to about 45 °. Thereby, it is possible to irradiate the first wall 111a or the second wall 112a with the excitation light from the measurement window 18, and the effects described in the first embodiment can be obtained.

  With such a configuration, the nucleic acid amplification reaction apparatus 2 can be used for applications involving fluorescence detection such as real-time PCR. The number of the fluorescence detectors 40 is arbitrary as long as detection can be performed without any problem. In the present embodiment, the fluorescence detection is performed by moving one fluorescence detector 40 along the slide 22. In order to detect fluorescence, a hole is provided in a side surface of the main body 10a on the second heating unit 13 side, and a measurement window 18 (see FIGS. 13 and 14) is formed. When the reaction solution 140 is located in the second region 102, the fluorescence detector 40 irradiates the side wall of the second region 102 of the nucleic acid amplification reaction vessel 100 through the measurement window 18 and emits the emitted fluorescence. The amount of nucleic acid amplification in the reaction solution 140 can be measured.

  As shown in FIGS. 12, 13, and 14, in the nucleic acid amplification reaction device 2 of the present embodiment, the first heating unit 12 is provided on the side of the lid 50 and the second heating is provided on the side far from the lid 50. A portion 13 is provided. That is, the positional relationship between the first heating unit 12 and the second heating unit 13 and other members included in the main body 10 is different from that of the nucleic acid amplification reaction apparatus 1. The functions of the first heating unit 12 and the second heating unit 13 are the same as those in the first embodiment except that the positional relationship is different. In the present embodiment, as shown in FIG. 14, a measurement window 18 is provided on the side surface of the second heating unit 13. Thereby, appropriate fluorescence measurement can be performed in real-time PCR in which fluorescence measurement is performed on the low temperature side (temperature at which annealing and extension reaction are performed).

  The nucleic acid amplification reaction apparatus 2 includes a setting unit 25. The setting unit 25 is a UI (user interface), and is a device that sets conditions for thermal cycling. By operating the setting unit 25, at least one of the first temperature, the second temperature, the first time, the second time, and the number of thermal cycles can be set. The setting unit 25 is mechanically or electronically linked with the control unit, and the setting in the setting unit 25 is reflected in the control of the control unit. Thereby, since the conditions of reaction can be changed, a desired thermal cycle can be applied to the reaction solution 140. Even if the setting unit 25 can individually set any of the above items, for example, if one is selected from a plurality of reaction conditions registered in advance, the necessary items are automatically set. It may be a thing. In this embodiment, the setting unit 25 is a button type, and reaction conditions can be set by pressing a button for each item.

  The nucleic acid amplification reaction apparatus 2 includes a display unit 24. The display unit 24 is a display device, and the control unit causes the display unit 24 to display various information related to the nucleic acid amplification reaction device 2. The control unit may cause the display unit 24 to display the conditions set by the setting unit 25 and the actual time and temperature during the heat cycle process. For example, when setting is performed, the input conditions are displayed, or during the thermal cycle process, the temperature measured by the temperature sensor, the time elapsed in the first arrangement or the second arrangement, and the thermal cycle are applied. The number of cycles may be displayed. Further, when the heat cycle process is completed or when some abnormality occurs in the apparatus, the fact may be displayed. Furthermore, notification by voice may be performed. By performing notification by display or voice, the user of the apparatus can easily grasp the progress or termination of the thermal cycle process.

As described above, according to the nucleic acid amplification reaction device 2 according to this embodiment, the following effects can be obtained.
The first wall 111a and the second wall 112a of the nucleic acid amplification reaction vessel 100 are light transmissive. Measuring the amount of nucleic acid amplification in the reaction solution 140 by measuring the fluorescence emitted by irradiating the reaction solution 140 with excitation light through the first wall 111a or the second wall 112a of the nucleic acid amplification reaction vessel 100. Can do.

  DESCRIPTION OF SYMBOLS 1, 2 ... Nucleic acid amplification reaction apparatus 10, 10a ... Main body, 11 ... Mounting part, 12 ... 1st heating part, 12a ... 1st heater, 12b ... 1st heat block, 13 ... 2nd heating part, 13a ... 1st 2 heaters, 13b ... second heat block, 14 ... spacer, 15 ... conductor, 16 ... flange, 17 ... bottom plate, 18 ... measurement window, 19 ... fixing plate, 20 ... drive mechanism, 22 ... slide, 24 ... display unit, 25 ... setting unit, 40 ... fluorescence detector, 50 ... lid, 51 ... fixing unit, 100 ... nucleic acid amplification reaction vessel, 101 ... first region, 102 ... second region, 103 ... first channel, 104 ... first 2 flow paths, 111 ... first inner wall surface, 111a ... first wall, 112 ... second inner wall surface, 112a ... second wall, 130 ... oil, 140 ... reaction liquid, 150 ... gas.

Claims (11)

A first region for bringing the reaction liquid to a first temperature;
A second region for bringing the reaction liquid to a second temperature different from the first temperature;
A plurality of flow paths for communicating the first region and the second region to move the reaction solution,
The first region and the second region are formed in a space sandwiched between a first inner wall surface and a second inner wall surface facing the first inner wall surface,
The distance between the first inner wall surface and the second inner wall surface is such that when the reaction solution is introduced into the first region or the second region via the flow path, A nucleic acid amplification reaction vessel characterized in that the reaction solution is in contact with both of the second inner wall surfaces.   The nucleic acid amplification reaction container according to claim 1, wherein the flow path is formed in a space sandwiched between the first inner wall surface and the second inner wall surface.   The nucleic acid amplification reaction container according to claim 1 or 2, wherein oil is disposed in the first region, the second region, and the flow path.   The nucleic acid according to any one of claims 1 to 3, wherein 50% or more of a volume constituted by the first region, the second region, and the flow path is filled with a gas. Amplification reaction vessel.   The nucleic acid amplification reaction container according to any one of claims 1 to 4, wherein the distance is 0.1 mm or more and 3.0 mm or less.   The wall thickness of at least one of the first wall constituting the first inner wall surface and the second wall constituting the second inner wall surface is 0.01 mm or more and 0.5 mm or less. The nucleic acid amplification reaction container according to any one of claims 1 to 5.   The at least one of the first wall and the second wall is deformed when an internal pressure of the first region, the second region, and the flow path is increased. Nucleic acid amplification reaction vessel.   The nucleic acid amplification reaction container according to claim 6 or 7, wherein at least one of the first wall and the second wall has light permeability.   The nucleic acid amplification reaction container according to any one of claims 1 to 8, wherein the first inner wall surface and the second inner wall surface are hydrophobic. A mounting part to which the nucleic acid amplification reaction container according to any one of claims 1 to 9 is mounted, wherein at least a reaction solution and a gas are disposed;
In the state where the nucleic acid amplification reaction vessel is attached to the attachment part,
A first heating unit for heating the first region of the nucleic acid amplification reaction vessel to a first temperature;
A second heating unit for heating the second region of the nucleic acid amplification reaction vessel to a second temperature different from the first temperature;
A drive mechanism for switching the mounting unit, the first heating unit, and the second heating unit between a first arrangement and a second arrangement;
The first arrangement is an arrangement in which the first region is positioned below the second region in the direction of gravity.
The nucleic acid amplification reaction apparatus, wherein the second arrangement is an arrangement in which the second region is located below the first region in the direction of gravity. A mounting step of mounting the nucleic acid amplification reaction vessel according to any one of claims 1 to 9 on the mounting portion of the nucleic acid amplification reaction device according to claim 10, wherein at least a reaction solution and a gas are disposed;
A first heating step of heating the first region of the nucleic acid amplification reaction vessel to a first temperature;
A second heating step of heating the second region of the nucleic acid amplification reaction vessel to a second temperature different from the first temperature;
A first driving step of switching from a first arrangement in which the first area is located below the second area to a second arrangement in which the second area is located below the first area in the direction of gravity;
A second driving step of switching from the second arrangement to the first arrangement;
Including
In the first driving step, the nucleic acid amplification reaction vessel rotates in a first direction around a rotation axis that intersects a first inner wall surface forming the first region and the second region and has a horizontal component. Moved,
In the second driving step, the nucleic acid amplification reaction container is rotated in a second direction opposite to the first direction.

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