JP4147292B2 - Reactor - Google Patents

Description

    The present invention relates to a reaction apparatus using a closed cassette, and more particularly to an improvement in a temperature control apparatus of a reaction apparatus for nucleic acid amplification reaction and nucleic acid detection.

    In recent years, technologies relating to genetic information have been actively developed. Among them, a particularly important technique is a technique for amplifying genes. With the advent of nucleic acid amplification technology for amplifying such genes and the improvement of nucleic acid detection technology, detection of specific DNA strands is increasing. However, in the nucleic acid amplification, the possibility of contamination by the amplified nucleic acid, or complicated operations such as temperature conditions, injection, mixing, etc. are required, so that these techniques are limited to test research.

  Conventionally, in order to solve these problems and enable nucleic acid detection in hospitals, clinical laboratories, quarantine stations, etc., processing from samples containing nucleic acids to detection of target nucleic acids is performed automatically and consistently. A disposable closed type detection container and a detection apparatus using the same are disclosed.

  Regarding a method for amplifying a gene, for example, JP-A-62-2000081 (Patent Document 1) discloses a gene amplification method called a polymerase chain reaction. Japanese Patent Laid-Open No. 06-233670 (Patent Document 2) discloses an apparatus for performing a polymerase chain reaction in a microtiter tray using a sample block. As an unpublished related application related to the prior application of the applicant of the present application, Japanese Patent Application No. 2003-400878 (Patent Document 3) discloses an automatic nucleic acid detection apparatus using a nucleic acid detection cassette, and as a part of the detection process, Implementation of the polymerase chain reaction is also envisioned.

Regarding a disposable closed-type detection container and a detection apparatus using the same, for example, Japanese Patent No. 2536945 (Patent Document 4) discloses a cuvette for amplification and detection of nucleic acid containing all necessary reagents (see FIG. cuvette), Japanese Patent Application Laid-Open No. Hei 8-62225 (Patent Document 5) has a test unit for feeding by utilizing centrifugal force, and Japanese Patent Publication No. Hei 9-511407 (Patent Document 6) has a fine processing technique. Disclosed are microchannels and reaction chambers that are used to form solid substrates.
JP-A 62-000281 Japanese Patent Laid-Open No. 06-233670 Japanese Patent Application No. 2003-400878 Japanese Patent No. 2536945 JP-A-8-62225 JP 9-511407 gazette

  Gene amplification by polymerase chain reaction involves three steps: dissociating double-stranded DNA into single strands (thermal denaturation), binding primers (annealing), and extending DNA with polymerase (extension reaction). The step is performed as one cycle, and the same step is repeated 30 to 40 cycles. Although the temperature and time differ depending on the target to be amplified, heat denaturation (94 ° C. × 30 seconds), annealing (50 ° C. to 65 ° C. × 1 minute) and extension reaction (72 ° C. × 1 to 5 minutes) are usually performed. Is done.

  Thus, in the polymerase chain reaction, the reaction solution is heated to a high temperature close to 100 ° C. in a process called heat denaturation. Therefore, when a cassette-shaped container as disclosed in Patent Document 3, that is, a plate-shaped container is used, and the cassette is sandwiched by heat transfer blocks from both sides and temperature controlled, the following problems occur. May occur.

(1) Liquid Leakage A flat plate-shaped container is usually composed of a fixing member that becomes a substrate and a flexible member that seals the liquid. In the case where the bonding force between the two is weak, there is a possibility that the liquid leaks from the bonding surface when the inside of the container becomes high pressure due to high temperature heating.

(2) Temperature non-uniformity When a flat container is directly sandwiched between heat media and the temperature is controlled, a temperature difference is generated between the central part and the end part of the container due to the influence of outside air. When the set temperature is higher than the outside air, the temperature at the end portion is lowered, and when the set temperature is lower than the outside air, the temperature at the end portion may be increased.

  In particular, when the polymerase chain reaction is performed, the constant temperature time is about 10 seconds to 60 seconds. Therefore, before the temperature of the entire liquid becomes constant, the process proceeds to the next step, which may cause a problem that the reaction efficiency is lowered.

  In the above description, the problem in the polymerase chain reaction has been pointed out. However, the present invention is not limited to this polymerase chain reaction, and there is a similar problem in other reactions that require temperature control.

  The present invention has been made in order to solve the above-described problems, and an object of the present invention is to provide a closed type capable of preventing temperature nonuniformity during temperature control and preventing liquid leakage. It is in providing the reaction container using a cassette.

According to this invention,
A closed-type nucleic acid detection cassette comprising a closed channel formed between a fixed substrate and a flexible sheet and including a holding channel for holding a sample and a reagent;
A first heat transfer block that transfers heat to the sample and reagent in the holding flow path via the flexible sheet ;
A second heat transfer block for sandwiching the nucleic acid detection cassette with the first heat transfer block, wherein the nucleic acid detection cassette is in contact with the fixed substrate and transfers heat to the sample and reagent in the holding channel. Two heat transfer blocks ;
A contact member having a recess and forming a closed space on the flexible sheet for contacting the flexible sheet and transferring heat from the first heat transfer block to the sample and the reagent in the recess. A contact member having a volume that allows the flexible sheet to expand due to thermal expansion of the sample and reagent ;
The reaction apparatus characterized by comprising is provided.

  When the reaction apparatus according to the present invention is used, even if the cassette is sandwiched by the heat transfer block and temperature control including a high temperature state close to 100 ° C. is performed on the sample held in the flow path, the flow path The internal pressure does not increase and no liquid leakage occurs. Further, the temperature difference between the central portion and the end portion of the flow path can be kept within a predetermined range, for example, 2 ° C.

  Hereinafter, a nucleic acid detection device and a nucleic acid detection system using a nucleic acid detection cassette according to an embodiment of the present invention will be described with reference to the drawings as necessary.

  FIG. 1 is a perspective view schematically showing the reaction apparatus according to the first embodiment of the present invention. As shown in FIG. 1, the reaction apparatus includes a closed nucleic acid detection cassette 100 in which a sample and a reagent are housed in a sealed channel, a heat transfer unit 200 for controlling the temperature of the nucleic acid detection cassette 100, and a detailed description later. The auxiliary member 300 is described.

  Although the case where the reaction apparatus shown in FIG. 1 is applied to a nucleic acid amplification reaction will be described below, it is apparent that the reaction apparatus of the present invention can be applied not only to a nucleic acid amplification reaction but also to a reaction that requires temperature control.

  2 and 3 show an automatic control mechanism for automatically executing a nucleic acid amplification reaction. FIG. 2 is a perspective view showing a form when the automatic control mechanism is used, and FIG. 3 is a perspective view showing the automatic control mechanism shown in FIG. 2 in an exploded manner for convenience of explanation.

  As shown in FIG. 2, a cassette holder 721 and a fixed X stage 722 are fixedly disposed on the support base 720. The cassette holder 721 has rails 721a and 721b that support the two sides of the outer frame of the nucleic acid detection cassette 100 and guide the nucleic acid detection cassette 100 to the temperature control operation position in the X-axis direction. A movable X stage 723 is placed on the fixed X stage 722, and the X driving device 714 can position the fixed X stage 722 in the X direction.

  In the operation unit, an electrical connector 703 used for current measurement, two drive units 701 and 702, and two heat transfer units 200 and 210 are provided as objects to be driven. The operation unit can be freely moved in the X, Y, and Z axis directions by the above-described X-direction drive system and the Y-direction and Z-direction drive systems.

  As shown in FIG. 3, the Y-direction drive system includes a fixed Y stage 731, a movable Y stage 713 a that can be driven by a Y drive device 732 so that it can be positioned in the Y direction with respect to the fixed Y stage 731, and this movable Y The movable mounting plate 713b moves together with the stage 713a. The fixed Y stage 731 is fixed to the movable X stage 723. Accordingly, the fixed Y stage 731 moves in the X direction as the movable X stage 723 moves in the X direction.

  The first drive system in the Z direction includes a fixed Z stage 725a and a movable Z stage 726a that can be driven by the first Z drive device 711 so as to be positioned in the Z direction with respect to the fixed Z stage 725a. The fixed Z stage 725a is fixed to a first Z stage mounting plate 724a fixed to the movable mounting plate 713b, and the fixed Z stage 725a also moves in the X and Y directions along with the movement in the X and Y directions.

  The second driving system in the Z direction includes a fixed Z stage 725b and a movable Z stage 726b that can be driven by the second Z driving device 712 so as to be positioned in the Z direction with respect to the fixed Z stage 725b. The fixed Z stage 725b is fixed to a second Z stage mounting plate 724b fixed to the movable mounting plate 713b, and the fixed Z stage 725b also moves in the X and Y directions along with the movement in the X and Y directions.

  The drive units 701 and 702 are fixed to the movable Z stages 726a and 726b via attachment plates 727a and 727b, respectively. As a result, the drive units 701 and 702 freely move in the X, Y, and Z directions as the movable Z stages 726a and 726b move in the X, Y, and Z directions.

  Further, the heat transfer units 200 and 210 are also fixed to the movable Z stages 726a and 726b so as to be selectively movable via mounting bases 741 and 742 or further mounting plates 727a and 727b, and the electric connector 703 is driven in the same manner It is fixed to the movable Z stages 726a and 726b so as to be selectively movable so as to be positioned in the vicinity immediately above the units 701 and 702. As a result, the heat transfer units 200 and 210 and the electrical connector 703 also move freely in the X, Y, and Z directions as the movable Z stages 726a and 726b move in the X, Y, and Z directions. However, the Z direction can be selectively moved, and the heat transfer units 200 and 210 and the electrical connector 703 can be brought close to or in contact with the nucleic acid detection cassette 100 separately at the required timing.

  Next, a nucleic acid detection cassette used for the nucleic acid amplification reaction will be described with reference to FIGS.

  FIG. 4 is a perspective view showing the components of the nucleic acid detection cassette 100 shown in FIG. 1 separately, and FIG. 5 is a perspective view showing an overview of the nucleic acid detection cassette 100 shown in FIGS. The nucleic acid detection cassette 100 is a flow channel closed type cassette that is roughly composed of a fixed substrate 1, a flexible sheet 2 made of silicone rubber, and a cover plate 3.

  The fixed substrate 1 is composed of a fixing member, and a continuous flow path 111 is formed in the fixed substrate 1. The flexible sheet 2 covers the upper surface of the flow path 111 formed by the fixed substrate 1 and is composed of a flexible member. The cover plate 3 includes a pressing block 4 that supports the flexible sheet 2 while sandwiching it between the fixed substrate 1 and presses and deforms the flexible sheet 2 locally.

  The nucleic acid detection cassette 100 is blocked by its function. In the example of FIG. 5, an end block 102, two intermediate blocks 101, a detector block 106, an intermediate block 101, and an end block 103 are formed in this order from the upstream side, that is, the left side of FIG. 5. The detection block 106 is a block for nucleic acid detection, and the other end blocks 102 and 103 and the intermediate block 101 are blocks for various reactions other than nucleic acid detection. These blocks are joined to each other so that the flow paths provided on the surfaces thereof are connected. Each block is joined by a fastening member or a fastening portion (not shown). Alternatively, each block is integrated and formed integrally.

  As shown in FIG. 4, a groove for the flow path 111 is provided on the upper surface of the fixed substrate 1. The lower surface of the flexible sheet 2 is bonded or welded to the upper surface of the fixed substrate 1 so as to cover the groove, thereby functioning as a flow path 111 in which the groove is closed. That is, the bottom surface and the side surface are constituted by the fixed substrate 1, and the upper surface is formed with the holding channel 111 and the retreat channel 131 covered with the flexible sheet 2. In the nucleic acid detection cassette 100 shown in FIGS. 4 and 5, holding channels such as the holding channels 111 and the retreat channels 131 are arranged in series.

  The fixed substrate 1 can be made of a polymer material such as polypropylene, polycarbonate, or POM, silicon, glass, ceramics, or a metal such as stainless steel or aluminum. The flexible sheet 2 can be composed of a polymer elastomer such as polypropylene rubber or urethane rubber. The cover plate 3 is made of a polymer material such as polypropylene, polycarbonate, POM, or PMMA, or a metal such as silicon, stainless steel, or aluminum.

  The thickness of the flexible sheet is 0.2 to 0.5 mm, the rubber hardness is relatively high such as JIS-A 20 ° to 30 °, and the softness is low such as Asker C 20 ° to 40 °. Any of these can be used.

  The nucleic acid detection chip 500 is fixed to the lower surface of the detection unit block 106 with a detection flow path seal 520 provided with a detection flow path 111 interposed therebetween. Further, the detection unit block 106 is provided with two contact portion openings 151 formed so as to penetrate from the front surface to the back surface. By inserting an electrical connector into the contact portion opening 151 and bringing it into contact with the exposed surface of the nucleic acid detection chip 500, an electrical signal can be extracted from the chip surface.

  The flexible sheet 2 can be deformed by the pressing block 4 and the volume of a required portion of the flow path can be varied to be sent out from the holding flow path 111 to another flow path. That is, as shown in FIG. 6, pads as a part of the pressing block 4 are arranged on the flexible sheet 2, and are formed at the flow path start end and the flow path end end of the substantially rectangular holding flow path 111. Are connected to the access channels 114 and 115, respectively. These access channels 114 and 115 are used to inject a sample containing a reagent and a nucleic acid into the nucleic acid detection cassette 100 or to discharge the sample out of the nucleic acid detection cassette 100. Further, a connecting channel 116 is further connected to the channel start end, and a connecting channel 117 is further connected to the channel end. These connection flow paths 116 and 117 extend to the peripheral edge of the intermediate block 101 and are configured to be connectable to flow paths provided in other blocks that are adjacently connected. A channel in which the connection channel 116, the holding channel 111, and the connection channel 117 are connected in this order is configured. That is, through one of the connection channels 116 and 117, a liquid or gas (hereinafter simply referred to as a fluid) from another block is received and sent to the holding channel 111, and through the other of the connection channels 116 and 117, The fluid in the holding channel 111 can be sent out to another block.

  A central pad 401, a left wing pad 402, and a right wing pad 403 are disposed on the flexible sheet 2 above the holding channel 111. The left wing pad 402 and the right wing pad 403 are positioned at and near the flow path start end and the flow path end end of the holding flow path 111, respectively. The center pad 401 is located in a portion where the flow path width is widened and in contact with the left wing pad 402 and the right wing pad 403. These pads 401, 402, and 403 substantially cover the upper part of the holding channel 111. In the present embodiment, the left wing pad 402 and the right wing pad 403 have substantially the same area, and the center pad 401 is set to the extent that the areas of the left wing pad 402 and the right wing pad 403 are combined.

  A left / inside pad 404 and a right in / out pad 405 are arranged above the in / out channels 114 and 115, respectively. A left connection pad 406 and a right connection pad 407 are disposed above the connection channels 116 and 117, respectively. Each of these pads is pressed almost vertically from the surface of the flexible sheet 2 toward the fixed substrate 1, so that the flexible sheet 2 comes into close contact with the corresponding groove of the fixed substrate 1 and the volume is almost zero. It has a shape to become.

  By operating these pads 401 to 405 by the pressing block 4, the fluid in the flow path 111 can be sent out.

  Next, the detailed configuration of the heat transfer unit 200 shown in FIG. 1 will be described with reference to FIGS.

  FIG. 7 is a perspective view illustrating an example of a configuration at the time of heat transfer to the nucleic acid detection cassette 100 of the heat transfer unit 200 including the two heat transfer blocks 200a and 200b. FIG. 8 is a cross-sectional view showing a state in which the heat transfer blocks 200 a and 200 b are separated from the nucleic acid detection cassette 100. FIG. 9 is a cross-sectional view of the heat transfer blocks 200a and 200b in contact with the nucleic acid detection cassette 100.

  The nucleic acid detection cassette 100 is a completely closed system in order to prevent genes other than the specimen from entering the sample. It should be noted that the pressing block 4 is omitted in FIGS. 8 to 12 because it is not used during temperature control. That is, when the nucleic acid detection cassette 100 is heated or cooled by the heat transfer unit 200, the pressure block 4 is removed or moved from the flexible sheet 2 together with the pads 401 to 405, and the heat transfer blocks 200a and 200b are moved. Direct contact is made with the flexible sheet 2 and the fixed substrate 1.

  The heat transfer unit 200 performs temperature control necessary for the amplification reaction on the reagent in the nucleic acid detection cassette 100. Therefore, as shown in FIG. 8, the heat transfer unit 200 can perform heating and cooling. The heat transfer block 200a includes a Peltier element 201a, a contact pad 204a made of a metal having good thermal conductivity such as aluminum or copper, a heat sink 202a, and a cooling fan 203a. The Peltier element 201a and the contact pad 204a, and the Peltier element 201a and the heat sink 202a are joined together with a grease in which silicone oil is used as a base oil and powder having good thermal conductivity such as alumina.

  Reference numerals 205a and 205b denote temperature sensors that measure the temperature of predetermined portions of the heat transfer blocks 200a and 200b and control the temperature of the heat transfer blocks 200a and 200b. The sample temperature in the flow path 111 is controlled by controlling the temperature of the heat transfer blocks 200a and 200b. The structure of the heat transfer block 200b is the same as that of 200a, and includes a Peltier element 201b, a contact pad 204b, a heat sink 202b, and a cooling fan 203b.

  In controlling the temperature of the nucleic acid cassette 100, the auxiliary member 300 is disposed between the nucleic acid detection cassette 100 and the heat transfer block 200a.

  The above is the outline of the apparatus. Next, a case where the apparatus performs temperature control on the cassette will be described.

  FIG. 9 is a cross-sectional view in a heat transfer state according to the first embodiment of the present invention. An auxiliary member 300 disposed between the nucleic acid detection cassette 100 and the heat transfer block 200a is sandwiched therebetween. The auxiliary member 300 has a plane portion 301 and a frame portion 302. The planar portion 301 is in contact with the contact pad 204a and conducts heat. The frame portion 302 forms a closed space 310 between the nucleic acid detection cassette 100 and the contact pad 204a. The closed space 310 seals the heat of the planar portion 301 and transfers heat to the reagent held in the flow path 111 of the nucleic acid detection cassette 100, and is determined to have a size that does not exceed the internal volume of the flow path. . For example, the closed space is defined as 10 mm × 10 mm × 0.3 mm with respect to the volume in the flow path of 10 mm × 10 mm × 0.5 mm. According to experiments, it has been found that when the distance between the nucleic acid detection cassette 100 and the contact pad 204a is 0.5 mm or more, the reagent in the channel 111 cannot be raised to the target temperature.

  FIG. 10 is a cross-sectional view when the temperature inside the flow path 111 becomes high during execution of temperature control. When the liquid inside the flow channel thermally expands, the volume inside the flow channel 111 increases, the flexible sheet 2 expands outside the nucleic acid detection cassette 100, and the volume of the closed space 310 decreases. In the structure shown in FIG. 9, the closed space 310 allows the volume inside the flow path 111 to increase, and the situation where the pressure inside the flow path 111 increases and liquid leakage occurs can be prevented.

  FIG. 11A and FIG. 11B are graphs showing the gas temperature in the flow path 111. The object to be measured is air held in the flow path 111, and two measurement points are the central portion and the end portion of the flow path 111. The set temperature was changed in the order of 98 ° C., 60 ° C., and 72 ° C., and the temperature in the flow path 111 at each set value was measured using a K thermocouple.

  FIG. 11A shows the result when the temperature control is performed without the auxiliary member 300 as a comparative example, and FIG. 11B shows the case where the temperature control is performed with the auxiliary member 300 inserted. Shows the results. In the comparative example in which the auxiliary member 300 is not inserted, when the set temperature reaches 98 ° C., a temperature difference of 10 ° C. or more is generated between the center and the end in the flow path 111. On the other hand, when the auxiliary member 300 is inserted, the difference is within 2 ° C. regardless of the set temperature. It is considered that the closed space 310 was formed by inserting the auxiliary member 300, and the heat escaping into the air was reduced.

  Further, in the comparative example in which the auxiliary member 300 is not provided, when the set temperature is 95 ° C. or higher, liquid leakage occurs in the middle, whereas in the structure in which the auxiliary member 300 is provided as shown in FIG. There was no leakage.

  As described above, by using the auxiliary member 300 according to the first embodiment of the present invention, liquid leakage that may occur in temperature control such as a polymerase chain reaction including a set temperature close to 100 ° C. and the center-end portion The temperature difference between them can be reduced. In addition, since the temperature in the flow path 111 becomes uniform, the reaction efficiency related to the nucleic acid amplification reaction and other nucleic acid detection increases.

  The present invention is not limited to a nucleic acid amplification reaction, and can be applied to any reaction such as DNA extraction or single strand as long as it requires a high-temperature state near 100 ° C.

  FIG. 12 is a cross-sectional view schematically showing the reaction apparatus in the heat transfer state according to the second embodiment of the present invention. The nucleic acid detection cassette 100 and the heat transfer block 200a are in contact with each other. The contact pad 204a has a recess that forms a closed space 310 having a size not exceeding the internal volume of the flow channel with the nucleic acid detection cassette 100. The peripheral portion of the contact pad 204a is in contact with the nucleic acid detection cassette 100. Even in such a structure, even if the flexible sheet 2 swells outside the nucleic acid detection cassette 100 as in the auxiliary member 300 shown in FIG. 9, the closed space 310 allows the volume inside the flow path 111 to increase. Further, it is possible to prevent a situation in which the pressure inside the flow path 111 increases and liquid leakage occurs.

  The shape of the recess is formed in a rectangular recess as shown in FIG. 13 (a), a circular recess as shown in FIG. 13 (b), or a polygonal recess as shown in FIG. 13 (c). As long as it is possible to form a closed space 310 having a size that does not exceed the internal volume of the flow channel with the nucleic acid detection cassette 100, any type may be used.

  As described above, the present invention is a closed-type nucleic acid detection cassette used for consistently and automatically processing from input of a sample containing nucleic acid to nucleic acid amplification and other necessary processing and detection of a target nucleic acid. It is effective in the technical field of reactors and systems using

1 is a perspective view showing an overview of a reaction apparatus according to a first embodiment of the present invention. FIG. 2 is a perspective view schematically showing an automatic control mechanism in which the reaction apparatus shown in FIG. 1 is incorporated. It is a perspective view which isolate | separates and shows each structure of the automatic control mechanism shown by FIG. It is a perspective view which isolate | separates and shows each structure of the nucleic acid detection cassette shown by FIG. FIG. 5 is a perspective view showing an overview of the nucleic acid detection cassette shown in FIG. 4. FIG. 5 is an exploded perspective view schematically showing an intermediate block of the nucleic acid detection cassette shown in FIG. 4. It is a perspective view which shows an example of the structure at the time of the heat transfer to the nucleic acid detection cassette of the closed system of the heat transfer unit shown by FIG. It is sectional drawing which shows the state which spaced apart the heat transfer unit shown by FIG. 7 from the nucleic acid detection cassette. It is sectional drawing of the state which made the heat transfer unit shown by FIG. 8 contact the nucleic acid detection cassette. It is sectional drawing which shows the state which the inside of the flow path expanded at the time of the heat transfer from the heat transfer unit shown in FIG. 8 to the nucleic acid detection cassette. (A) And (b) is a graph which shows the experimental result of the temperature in a flow path with respect to setting temperature in the reaction apparatus which concerns on a comparative example, and the reaction apparatus shown in FIG. It is sectional drawing which shows an example of the state at the time of the heat transfer from the heat transfer unit which concerns on 2nd Embodiment of this invention to a nucleic acid detection cassette. (A), (b) and (c) is a perspective view which shows the various examples of the recessed part of the contact pad shown in FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Fixed substrate, 2 ... Flexible sheet, 3 ... Cover plate, 4 ... Press block 100 ... Nucleic acid detection cassette, 111 ... Flow path, 200, 210 ... Heat transfer unit, 200a, b ... Heat transfer block, 201a, b: Peltier element, 202a, b ... heat sink, 203a, b ... cooling fan, 204a, b ... contact pad, 205a, b ... thermocouple, 300 ... auxiliary member, 301 ... flat part, 302 ... frame part, 310 ... closed Space, 701, 702 ... drive unit, 703 ... electric connector, 711 ... Z drive, 713a ... movable Y stage, 713b ... movable mounting plate, 714 ... X drive, 720 ... support, 721 ... cassette holder, 721a, 721b ... Rail, 722 ... Fixed X stage, 723 ... Movable X stage, 724 ... Z stage mounting plate, 725 ... Fixed Z stage Stage, 726 ... movable Z stage, 727 ... mounting plate, 732 ... Y drive unit, 741 ... mounting base

Claims (4)

A closed-type nucleic acid detection cassette comprising a closed channel formed between a fixed substrate and a flexible sheet and including a holding channel for holding a sample and a reagent;
A first heat transfer block that transfers heat to the sample and reagent in the holding flow path via the flexible sheet ;
A second heat transfer block for sandwiching the nucleic acid detection cassette with the first heat transfer block, wherein the nucleic acid detection cassette is in contact with the fixed substrate and transfers heat to the sample and reagent in the holding channel. Two heat transfer blocks ;
A contact member having a recess and forming a closed space on the flexible sheet for contacting the flexible sheet and transferring heat from the first heat transfer block to the sample and the reagent in the recess. A contact member having a volume allowing the deformation of the flexible sheet accompanying the thermal expansion of the sample and the reagent .
A reaction apparatus comprising: The contact member is disposed between the flexible sheet and the first heat transfer block, and is provided in an auxiliary member including a frame portion that forms the closed space, or provided in the first heat transfer block. The reaction apparatus according to claim 1 , further comprising a contact pad including a recess that forms the closed space . The reaction device according to claim 1 , wherein the contact member has a peripheral portion of a concave portion of the contact member in contact with the flexible member .     The reaction apparatus according to claim 1, wherein the closed space has a volume that does not exceed a volume inside the holding flow path.

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