JP2017042103A - Nucleic acid amplification reaction container - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide techniques of achieving facilitation of nucleic acid amplification reactions.SOLUTION: A nucleic acid amplification reaction container 100 comprises: a substrate 102 having a concave portion 102a; a cover film covering the concave portion 102a; a reaction chamber 108 which is defined with the concave portion 102a and the cover film, has a reaction chamber entrance 108a and a reaction chamber exit 108b, and in which a nucleic acid amplification reaction is performed; a first communication path 110 which is defined with the concave portion 102a and the cover film and communicates the reaction chamber entrance 108a and the outside of the substrate; a second communication path 112 which is defined with the concave portion 102a and the cover film and communicates the reaction chamber exit 108b and the outside of the substrate; and a heat welding part 120 which seals the first communication path 110 and the second communication path 112 by the hot welding between the parts defining the first communication path 110 of the substrate 102 and the cover film, and between the parts defining the second communication path 112 of the substrate 102 and the cover film respectively, in the state that a reaction solution flows into the reaction chamber 108.SELECTED DRAWING: Figure 3

Description

  The present application relates to a nucleic acid amplification reaction vessel used in a nucleic acid amplification apparatus.

  Conventionally, a nucleic acid amplification technique is known in which a nucleic acid such as DNA (Deoxyribonucleic Acid) is caused to undergo a polymerase chain reaction (PCR) to amplify the nucleic acid. As one of such nucleic acid amplification techniques, a reaction solution containing nucleic acid is accommodated in a nucleic acid amplification reaction vessel, and the nucleic acid amplification reaction vessel is moved with respect to a heat source at a different temperature to give a thermal cycle to the reaction solution. An apparatus is known (see, for example, Patent Document 1).

International Publication 2008/146754 Pamphlet

  As a result of intensive studies on the nucleic acid amplification technique described above, the present inventors have come up with a technique for simplifying the nucleic acid amplification reaction.

  The present application has been made in view of such circumstances, and an object thereof is to provide a technique for simplifying a nucleic acid amplification reaction.

  In order to solve the above problems, a nucleic acid amplification reaction vessel according to an aspect of the present application is defined by a substrate having a recess, a cover film attached to the substrate so as to cover the recess, a recess and a cover film, and a nucleic acid A reaction chamber inlet into which the reaction liquid containing the liquid flows in, and a reaction chamber outlet through which the gas in the reaction chamber is discharged as the reaction liquid flows into the reaction chamber. A reaction chamber that is thermally connected to a temperature control unit of the apparatus and in which a nucleic acid amplification reaction is performed in the reaction chamber; a first communication path that is defined by a recess and a cover film and that communicates the reaction chamber inlet and the outside of the substrate; The second communication path is defined by the recess and the cover film and communicates between the reaction chamber outlet and the outside of the substrate, and the first communication path of the substrate and the cover film is defined with the reaction solution flowing into the reaction chamber. Parts to do, substrates, and In part with each other to define a second communication path bars films are thermally welded, respectively, comprising a heat seal portion where the first communication passage and second communication passage is sealed, the.

  According to the present application, a technique for simplifying the nucleic acid amplification reaction can be provided.

1 is a perspective view showing a schematic structure of a nucleic acid amplification device according to an embodiment. It is a side view which shows schematic structure of a nucleic acid amplification reaction container, the cartridge for nucleic acid amplification apparatuses, and a temperature control part. It is a top view which shows the schematic structure inside a nucleic acid amplification reaction container. 4A is a cross-sectional view taken along line AA in FIG. FIG. 4B is a perspective view showing a schematic structure of the nucleic acid amplification reaction vessel. FIG. 5A is a plan view showing a schematic structure of the inside of the nucleic acid amplification reaction container according to the first modification. FIG. 5B is a cross-sectional view taken along the line BB in FIG. FIG. 5C is a cross-sectional view showing a schematic structure of a nucleic acid amplification reaction container according to Modification 2. FIG. 5D is a plan view showing a schematic structure of the inside of the nucleic acid amplification reaction container according to Modification 3. FIG. 5E is a cross-sectional view taken along the line CC in FIG. It is a perspective view which shows schematic structure of a nucleic acid amplification reaction container, the cartridge for nucleic acid amplification apparatuses, and a temperature control part. FIG. 7A is a side view schematically showing a preprocessing unit according to Modification 4. FIG. 7B is a cross-sectional view taken along the line DD in FIG. FIG. 7C is a side view schematically showing the preprocessing unit according to the fifth modification. FIG. 7D is a cross-sectional view taken along line EE in FIG. FIG. 8A is a perspective view showing a schematic structure of the cartridge stage, the holder rotation motor 304, and the holder vertical movement motor in the drive unit. FIG. 8B is a perspective view showing a schematic structure of a pump and a magnet drive motor in the drive unit. FIG. 8C is an enlarged perspective view showing the vicinity of the magnet connected to the magnet drive motor. It is a perspective view which shows schematic structure of a temperature control part. FIG. 10A is a schematic diagram for explaining an example of a method for measuring a nucleic acid amplification reaction by a measurement unit. FIG. 10B is a schematic diagram for explaining another example of the measuring method of the nucleic acid amplification reaction by the measuring unit. FIG. 11A and FIG. 11B are schematic diagrams for explaining the control of the control unit in the pretreatment of the reaction liquid. FIGS. 12A and 12B are schematic diagrams for explaining the control of the control unit in the pretreatment of the reaction liquid. FIGS. 13A and 13B are schematic diagrams for explaining the control of the control unit in the nucleic acid amplification reaction. FIG. 14A to FIG. 14C are schematic diagrams for explaining the control of the control unit in the nucleic acid amplification reaction. FIG. 15A to FIG. 15F are schematic diagrams for explaining a pretreatment process of a reaction solution performed in the nucleic acid amplification device according to the embodiment. FIGS. 16A to 16C are schematic diagrams for explaining nucleic acid dispersion when using the pretreatment units according to Modification 4 and Modification 5. FIG.

  The present invention will be described below based on preferred embodiments with reference to the drawings. The embodiments do not limit the invention but are exemplifications, and all features and combinations described in the embodiments are not necessarily essential to the invention.

  FIG. 1 is a perspective view showing a schematic structure of a nucleic acid amplification device 1 according to an embodiment. FIG. 2 is a side view showing a schematic structure of the nucleic acid amplification reaction vessel 100, the cartridge 200 for nucleic acid amplification device, and the temperature control unit 400. In FIG. 2, the cylindrical portion 214 of the cartridge 200 for nucleic acid amplification device is not shown. The nucleic acid amplification device 1 according to the present embodiment performs predetermined pretreatment on nucleic acid such as DNA or RNA, and amplifies the nucleic acid by controlling the temperature of the reaction solution containing the pretreated nucleic acid and a fluorescent substance. An apparatus capable of detecting nucleic acid amplification by an optical measurement method. That is, the nucleic acid amplification device 1 is a nucleic acid amplification device in which a pretreatment mechanism and a measurement mechanism are integrated. The nucleic acid amplifying apparatus 1 is a highly versatile apparatus that can perform a nucleic acid amplification reaction more quickly and easily than in the past.

  The nucleic acid amplification device 1 according to the present embodiment includes a plate-like base portion 2 and a cover 4 attached to the base portion 2. The base unit 2 constitutes the bottom surface of the casing of the nucleic acid amplification device 1, and the cover 4 constitutes the other surface excluding the bottom surface of the casing. In the interior of the nucleic acid amplification device 1, that is, in a space defined by the base portion 2 and the cover 4, a nucleic acid amplification reaction vessel 100, a nucleic acid amplification device cartridge 200, a drive unit 300, a temperature adjustment unit 400, and a measurement The unit 500 and the control unit 600 are accommodated.

  The cover 4 has an opening on the front side of the apparatus. The opening 6 is provided with a lid 6 that can be opened and closed. The user of the nucleic acid amplification device 1 can insert the nucleic acid amplification device cartridge 200 into or out of the device through the opening of the cover 4. The cover 4 is provided with a display unit 8 that displays the measurement result of the measurement unit 500. The display unit 8 may be provided with an operation unit for the user of the nucleic acid amplification device 1 to input various instructions to the control unit 600.

  A drive unit 300, a temperature adjustment unit 400, a measurement unit 500 and a control unit 600 are mounted on the base unit 2. The cartridge stage 302 of the driving unit 300 is provided with a cartridge 200 for nucleic acid amplification apparatus to which the nucleic acid amplification reaction vessel 100 is attached. Hereinafter, the configuration of each unit will be described in detail.

(Nucleic acid amplification reaction vessel)
FIG. 3 is a plan view showing a schematic structure inside the nucleic acid amplification reaction vessel 100. 4A is a cross-sectional view taken along line AA in FIG. FIG. 4B is a perspective view showing a schematic structure of the nucleic acid amplification reaction vessel 100. FIG. 3 shows the nucleic acid amplification reaction vessel 100 viewed from the cover film 104 side. As shown in FIGS. 2, 3, 4 (A) and 4 (B), the nucleic acid amplification reaction vessel 100 includes a substrate 102 and a cover film 104. Substrate 102 has a flat plate shape and has a recess 102a on one main surface side. The depth of the recess 102a is, for example, about 150 μm. Further, the substrate 102 has a connecting portion 106 on the other main surface side. Connection portion 106 protrudes obliquely on one end side of the other main surface. The connection unit 106 is inserted into the container support unit 212 of the holder unit 202 provided in the cartridge 200 for nucleic acid amplification device.

  The cover film 104 is attached to the substrate 102 so as to cover the recess 102a. The substrate 102 and the cover film 104 are formed of a resin material such as polypropylene resin, for example. Preferably, the cover film 104 is formed of a material having a lower melting point than the substrate 102. For example, the substrate 102 is formed of a polypropylene resin having a melting point of about 160 ° C., and the cover film 104 is formed of a polypropylene resin having a melting point of about 140 ° C. The cover film 104 may have a multilayer structure. For example, the cover film 104 has a multilayer film structure in which a metal film such as an aluminum film and a resin film such as polypropylene resin are laminated. A resin film is arrange | positioned at the side which contacts the reaction liquid mentioned later, and a metal film is arrange | positioned at the outer surface side of reaction container. According to such a structure, the temperature uniformity in the surface direction of the cover film 104 can be improved by the metal film while suppressing the influence of the metal film on the reaction solution by the resin film.

  The nucleic acid amplification reaction vessel 100 includes a reaction chamber 108 defined by a recess 102 a and a cover film 104. The reaction chamber 108 is a space surrounded by the bottom and side surfaces of the recess 102a and the cover film 104 that covers the opening of the recess 102a. The reaction chamber 108 stores a reaction solution containing nucleic acid. The reaction solution will be described in detail later. Further, the reaction chamber 108 is thermally connected to the temperature control unit 400 on the cover film 104 side, and a nucleic acid amplification reaction is performed in the reaction chamber 108. Note that although one reaction chamber 108 is provided on the substrate 102 in this embodiment mode, a plurality of reaction chambers 108 may be provided on the substrate 102.

  The nucleic acid amplification reaction vessel 100 has a structure in which the thickness M2 of the portion 104a that defines the reaction chamber 108 in the cover film 104 is thinner than the thickness M1 of the portion 102b that forms the bottom surface of the recess 102a in the substrate 102. Thereby, the temperature of the reaction solution can be adjusted with higher accuracy. For this reason, the nucleic acid amplification reaction can be stabilized. In the present embodiment, the entire portion 104a that defines the reaction chamber 108 of the cover film 104 is thinner than the thickness M1 of the portion 102b that forms the bottom surface of the recess 102a, but only a portion may be thin. .

  The reaction chamber 108 has a reaction chamber inlet 108a and a reaction chamber outlet 108b. The nucleic acid amplification reaction vessel 100 also includes a first communication path 110 that communicates the reaction chamber inlet 108a with the outside of the substrate, and a second communication path 112 that communicates the reaction chamber outlet 108b with the outside of the substrate. Both the first communication path 110 and the second communication path 112 are defined by the recess 102 a and the cover film 104. An end of the first communication path 110 on the outside of the substrate constitutes a substrate inlet 114. An end of the second communication path 112 on the outside of the substrate constitutes a substrate outlet 116. The substrate inlet 114 and the substrate outlet 116 are both provided at the distal end of the connecting portion 106.

  The reaction solution is injected from the outside of the substrate into the substrate through the substrate inlet 114. Therefore, the substrate inlet 114 corresponds to the reaction liquid inlet of the nucleic acid amplification reaction vessel 100. The reaction liquid injected from the substrate inlet 114 flows into the reaction chamber 108 from the reaction chamber inlet 108 a through the first communication path 110. Further, as the reaction liquid flows into the reaction chamber 108, the gas in the reaction chamber 108 is discharged from the reaction chamber outlet 108 b to the second communication path 112. The gas discharged to the second communication path 112 passes through the second communication path 112 and is discharged outside the substrate.

  Further, in the present embodiment, in order to more reliably fill the reaction chamber 108 with the reaction solution, and to more reliably remove bubbles in the reaction chamber 108, the first communication path 110 and the reaction chamber 108 An amount of the reaction liquid exceeding the total volume is injected. For this reason, a part of the reaction solution flowing into the reaction chamber 108 is discharged from the reaction chamber outlet 108 b to the second communication path 112 and reaches the substrate outlet 116.

  The reaction chamber inlet 108a and the reaction chamber outlet 108b are positioned so that the reaction chamber outlet 108b is positioned vertically above the reaction chamber inlet 108a in a state where the nucleic acid amplification reaction vessel 100 is mounted on the nucleic acid amplification apparatus 1. A relationship is established. In the nucleic acid amplification reaction container 100, the connecting portion 106 is arranged in the vertical direction while being attached to the nucleic acid amplification device 1. Therefore, the reaction chamber outlet 108b is disposed closer to the connecting portion 106 than the reaction chamber inlet 108a. By disposing the reaction chamber outlet 108b vertically above the reaction chamber inlet 108a, bubbles generated when the reaction chamber 108 is filled with the reaction liquid are discharged from the reaction chamber outlet 108b to the second communication path 112 side. Can be made easier.

  If bubbles exist in the reaction chamber 108, the bubbles expand in the course of the nucleic acid amplification reaction, and the temperature of the entire reaction solution may become uneven. If the temperature of the entire reaction solution becomes uneven, the stability of the nucleic acid amplification reaction is impaired. On the other hand, by disposing the reaction chamber outlet 108b vertically above the reaction chamber inlet 108a, the bubbles in the reaction chamber 108 can be more reliably eliminated, so that the nucleic acid amplification reaction can be stabilized. Can do.

  Preferably, the reaction chamber inlet 108 a is located at the lowest vertical direction in the reaction chamber 108, and the reaction chamber outlet 108 b is located at the uppermost vertical direction in the reaction chamber 108. Thereby, the bubbles in the reaction chamber 108 can be more easily removed.

  As shown in FIG. 3, the two side surfaces 108c and 108d of the reaction chamber 108 connecting the reaction chamber inlet 108a and the reaction chamber outlet 108b have concave curved surface portions 108c1 and 108d1, and tapered portions 108c2 and 108d2. The side surfaces 108c and 108d are configured by the side surfaces of the concave portion 102a and connect the cover film 104 and the bottom surface of the concave portion 102a.

  The concave curved surface portions 108c1 and 108d1 are concave curved surfaces that swell toward the outer edge side of the substrate 102, and extend in a region from the reaction chamber inlet 108a to the predetermined position P. The tapered portions 108c2 and 108d2 are tapered such that the opposing side surfaces 108c and 108d approach each other as they approach the reaction chamber outlet 108b, that is, the tapered portion 108c2 and the tapered portion 108d2 approach each other, and are provided from the predetermined position P to the reaction chamber outlet 108b. Extend to the area. The tapered portions 108c2 and 108d2 are planar.

  In the present embodiment, the concave curved surface portions 108c1 and 108c2 extend over the entire region from the reaction chamber inlet 108a to the predetermined position P. Further, the tapered portion 108c2 extends over the entire region from the predetermined position P to the reaction chamber outlet 108b. The tapered portion 108d2 extends from the predetermined position P to the front of the reaction chamber outlet 108b. Accordingly, the tapered portions 108c2 and 108d2 and the concave curved surface portions 108c1 and 108d1 are connected at the predetermined position P. On the side surface 108d, a horizontal portion 108d3 is interposed between the tapered portion 108d2 and the reaction chamber outlet 108b. Note that the side surface 108d may not have the horizontal portion 108d3. In this case, the tapered portion 108d2 extends over the entire region from the predetermined position P to the reaction chamber outlet 108b.

  By providing the concave curved surface portions 108c1 and 108d1, the volume of the reaction chamber 108 can be secured. Further, by arranging the tapered portions 108c2 and 108d2 vertically above the concave curved surface portions 108c1 and 108d1, it adheres to the side surfaces 108c and 108d as compared with the case where the concave curved surface portions 108c1 and 108d1 are arranged vertically above. The bubbles can be easily discharged from the reaction chamber outlet 108b to the second communication path 112 side. Furthermore, since the tapered portions 108c2 and 108d2 are connected to the ends of the concave curved surface portions 108c1 and 108d1 in the vertical direction, the refraction angle of the side surfaces 108c and 108d at the predetermined position P becomes an obtuse angle. Thereby, it is possible to prevent bubbles from being trapped at the end portions of the concave curved surface portions 108c1 and 108d1 on the upper side in the vertical direction.

  Further, the opening area of the reaction chamber inlet 108a is smaller than the opening area of the reaction chamber outlet 108b. That is, the flow path width of the first communication passage 110 at the connection portion between the first communication passage 110 and the reaction chamber inlet 108a is equal to the flow rate of the second communication passage 112 at the connection portion between the second communication passage 112 and the reaction chamber outlet 108b. It is smaller than the road width. As a result, the flow rate of the reaction liquid at the reaction chamber outlet 108b can be made larger than the flow rate of the reaction liquid at the reaction chamber inlet 108a, so that the bubbles in the reaction chamber 108 can be easily discharged outside the reaction chamber 108. . Further, the opening area of the substrate outlet 116 is larger than the opening area of the reaction chamber outlet 108b. This also makes it easier to discharge the bubbles in the reaction chamber 108.

  Further, the substrate 102 has a reaction liquid storage unit 118 at the substrate outlet 116. That is, the second communication path 112 has a larger opening at the substrate outlet 116 side than the end at the reaction chamber outlet 108 b side, and this opening portion constitutes the reaction liquid reservoir 118. The reaction liquid reservoir 118 stores the reaction liquid flowing out from the reaction chamber outlet 108 b to the second communication path 112. Thereby, it can suppress that the reaction liquid scatters out of the nucleic acid amplification reaction container 100 and the nucleic acid amplification apparatus 1 is contaminated. In the present embodiment, a reaction liquid storage unit 118 is also provided at the substrate inlet 114. Thereby, when a reaction liquid is inject | poured into the board | substrate inlet 114, it can prevent that a reaction liquid is scattered outside and the nucleic acid amplification apparatus 1 is contaminated.

  In addition, the nucleic acid amplification reaction vessel 100 includes a thermal welding part 120 for sealing the first communication path 110 and the second communication path 112. With the reaction liquid flowing into the reaction chamber 108, heat is applied to the heat welding unit 120 from the fourth temperature unit 410 of the temperature adjustment unit 400. Thereby, at least the cover film 104 is melted in the heat-welded portion 120, and the portions defining the first communication passage 110 of the substrate 102 and the cover film 104 and the second communication passage 112 of the substrate 102 and the cover film 104 are communicated. The parts to be defined are thermally welded to each other. As a result, the first communication path 110 and the second communication path 112 are sealed, and leakage of the reaction liquid in the reaction chamber 108 to the outside of the substrate is suppressed. In the nucleic acid amplification reaction container 100 according to the present embodiment, the first communication path 110 and the second communication path 112 are sealed by thermal welding of the cover film 104 and the substrate 102. Therefore, each communication path can be easily sealed. Therefore, the nucleic acid amplification reaction can be carried out easily.

  In the present embodiment, the first communication path 110 and the second communication path 112 are arranged so as to pass through one thermal welding part 120. That is, the heat welding part for sealing the 1st communicating path 110 and the heat welding part for sealing the 2nd communicating path 112 continue, and form the one heat welding part 120. FIG. Thereby, since the 1st communicating path 110 and the 2nd communicating path 112 can be sealed simultaneously, the operation | work required for a nucleic acid amplification reaction can be simplified. In addition, the heat welding part which seals each communicating path may be provided independently, respectively.

  Further, the heat welding part 120 includes a second recess 122 provided in the vicinity of the first communication path 110 and a third recess 124 provided in the vicinity of the second communication path 112. For example, in the second recess 122, the distance D1 between the second recess 122 and the first communication path 110 in the heat welded portion 120 is smaller than the flow path width W1 of the first communication path 110 in the heat welded portion 120. It is arranged to become. Further, in the third recess 124, the distance D2 between the third recess 124 and the second communication path 112 in the heat welding part 120 is smaller than the flow path width W2 of the second communication path 112 in the heat welding part 120. It is arranged to become.

  Further, in the heat welding part 120, the first communication path 110 and the second communication path 112 are such that the distance D3 between the first communication path 110 and the second communication path 112 is the second communication path in the heat welding part 120. It is arranged to be smaller than the channel width W2 of 112. Furthermore, the first communication path 110 and the second communication path 112 are arranged such that the distance D3 is smaller than the flow path width W1. The second recess 122, the first communication path 110, the second communication path 112, and the third recess 124 are arranged in this order in the heat welding part 120.

  By disposing the second concave portion 122 in the vicinity of the first communication passage 110, one partition wall of the first communication passage 110 in the heat welded portion 120 can be thinned. Similarly, by disposing the third recess 124 in the vicinity of the second communication path 112, one partition wall of the second communication path 112 in the heat welded portion 120 can be thinned. Moreover, the other partition of the 1st communicating path 110 and the 2nd communicating path 112 in the heat welding part 120 can be made thin by making the 1st communicating path 110 and the 2nd communicating path 112 adjoin.

  In the present embodiment, the first communication path 110 and the second communication path 112 extend close to each other in the heat welding portion 120. For this reason, the second communication path 112 functions as the second recess 122, and the first communication path 110 functions as the third recess 124. When the heat welding part of the first communication path 110 and the heat welding part of the second communication path 112 are provided apart from each other, the first recesses 122 are provided on both sides of the first communication path 110 to provide the first Both partition walls of the communication path 110 can be made thin. Similarly, by providing the third recesses 124 on both sides of the second communication path 112, both partition walls of the second communication path 112 can be made thin.

  Since both the partition walls of the first communication path 110 and the both partition walls of the second communication path 112 in the heat welding part 120 are thin, the partition walls of the communication paths are deformed by the contact of the fourth temperature part 410. Can be crushed. Thereby, the cover film 104 and the board | substrate 102 can be more reliably melt-bonded, As a result, the 1st communicating path 110 and the 2nd communicating path 112 can be sealed more reliably.

  Note that only the cover film 104 may be melted to seal each communication path. In this case, for example, convex portions that match the shapes of the first communication passage 110 and the second communication passage 112 are provided in the fourth temperature portion 410 of the temperature adjustment unit 400. And while making the 4th temperature part 410 contact | abut to the heat welding part 120, a convex part is engage | inserted in the 1st communicating path 110 and the 2nd communicating path 112. FIG. Thereby, the cover film 104 can be welded along the wall surfaces of the first communication path 110 and the second communication path 112. When only the cover film 104 is melted, the second recess 122 and the third recess 124 can be omitted. Moreover, the distance between the 1st communicating path 110 and the 2nd communicating path 112 can be set freely.

  At least a part of the portion defining the reaction chamber 108 in the substrate 102 and the cover film 104 is subjected to a hydrophilic treatment. As a result, the reaction solution can smoothly flow into the reaction chamber 108, and the bubbles in the reaction chamber 108 can be easily discharged. Note that at least a part of the substrate 102 and the cover film 104 that define the reaction chamber 108 may be subjected to water repellent treatment. Thereby, it can suppress that the nucleic acid in a reaction liquid adheres to the wall surface of the reaction chamber 108, and can raise the amplification efficiency of a nucleic acid.

  The nucleic acid amplification reaction vessel 100 can include the following modifications. FIG. 5A is a plan view showing a schematic structure of the inside of the nucleic acid amplification reaction vessel 100 according to the first modification. FIG. 5B is a cross-sectional view taken along the line BB in FIG. FIG. 5C is a cross-sectional view showing a schematic structure of the nucleic acid amplification reaction vessel 100 according to Modification 2. FIG. 5D is a plan view showing a schematic structure inside the nucleic acid amplification reaction vessel 100 according to Modification 3. FIG. 5E is a cross-sectional view taken along the line CC in FIG. In the following, each modified example will be described focusing on the configuration different from the nucleic acid amplification reaction vessel 100 according to Embodiment 1, and the common configuration will be briefly described or not described.

(Modification 1)
As shown in FIGS. 5 (A) and 5 (B), the nucleic acid amplification reaction vessel 100 according to Modification 1 has a protrusion that protrudes toward the cover film 104 at a portion that defines the reaction chamber 108 in the substrate 102. 126. By supporting the cover film 104 with the protrusion 126, it is possible to suppress the cover film 104 from approaching the bottom surface side of the recess 102a and reducing the volume of the reaction chamber 108. Note that a protrusion protruding toward the substrate 102 may be provided on the cover film 104. That is, at least one of the portions defining the reaction chamber 108 in the substrate 102 and the cover film 104 is provided with the projection 126 that protrudes to the other side, so that the volume of the reaction chamber 108 can be ensured more reliably. it can. The number and arrangement of the protrusions 126 are not limited to those illustrated.

(Modification 2)
As shown in FIG. 5C, the nucleic acid amplification reaction vessel 100 according to the modified example 2 includes a porous body 128 disposed in the reaction chamber 108. By disposing the porous body 128 in the reaction chamber 108, it is possible to prevent the cover film 104 from approaching the bottom surface side of the recess 102a and reducing the volume of the reaction chamber 108. The material used as the porous body 128 is not particularly limited as long as it does not inhibit the nucleic acid amplification reaction of the reaction solution and the measurement by the measurement unit 500, and for example, a conventionally known porous polymer or the like can be used. Note that the porous body 128 may be spread over the entire reaction chamber 108 or may be disposed in part.

(Modification 3)
As shown in FIGS. 5D and 5E, the nucleic acid amplification reaction vessel 100 according to the modified example 3 includes a melt film 130 that is a separate member from the cover film 104 in the heat welding portion 120. The molten film 130 is made of a material having a melting point lower than that of the cover film 104, for example. Alternatively, the molten film 130 is thinner than the cover film 104. The molten film 130 is melted together with the cover film 104 by heating at the fourth temperature unit 410. Thereby, in the heat welding part 120, the 1st communicating path 110 and the 2nd communicating path 112 can be sealed more reliably.

(Nucleic acid amplifier cartridge)
FIG. 6 is a perspective view showing a schematic structure of the nucleic acid amplification reaction vessel 100, the cartridge 200 for nucleic acid amplification device, and the temperature control unit 400. As shown in FIGS. 2 and 6, the cartridge 200 for nucleic acid amplification device includes a holder unit 202 and a pretreatment unit 204.

  The holder part 202 has a main body part 206 having a bottomed cylindrical shape. The central axis of the main body 206 coincides with the rotation axis X <b> 1 when the holder unit 202 is rotated by the driving unit 300. A plurality of partition walls 208 extending radially from the central axis (rotation axis X1) are provided inside the main body 206. A space defined by the bottom surface and the inner side surface of the main body 206 and the two partition walls 208 constitutes a storage chamber 210 that stores a processing liquid used for pretreatment of the reaction liquid. Therefore, the holder part 202 has a plurality of storage chambers 210 arranged in the circumferential direction of the rotation axis X1. A plurality of pretreatment liquids are individually stored in the storage chambers 210. The number of storage chambers 210 is not limited to that illustrated. The pretreatment of the reaction solution and the treatment solution will be described in detail later.

  Further, the holder part 202 has a container support part 212 and a cylindrical part 214. The container support part 212 and the cylindrical part 214 are arranged in the circumferential direction of the rotation axis X <b> 1 together with the plurality of storage chambers 210. The container support portion 212 includes a container fitting portion 216 disposed in the main body portion 206 and a container abutting portion 218 that protrudes downward from the main body portion 206 in the vertical direction. The container fitting part 216 has a recessed part opened downward in the vertical direction, and the connecting part 106 of the nucleic acid amplification reaction container 100 is inserted into this recessed part. As a result, the nucleic acid amplification reaction vessel 100 is supported by the vessel support 212.

  The container fitting portion 216 has openings 216a and 216b extending from the bottom surface of the recess into which the connection portion 106 is inserted to the surface on the upper side in the vertical direction of the container fitting portion 216. Therefore, the openings 216a and 216b are arranged in the circumferential direction of the rotation axis X1 together with the plurality of storage chambers 210. The opening 216a is connected to the substrate inlet 114 of the nucleic acid amplification reaction vessel 100 in a state where the nucleic acid amplification reaction vessel 100 is supported by the vessel support 212. The opening 216b is connected to the substrate outlet 116 of the nucleic acid amplification reaction vessel 100 in a state where the nucleic acid amplification reaction vessel 100 is supported by the vessel support 212.

  The container abutting portion 218 has an inclined surface 218a that extends downward in the vertical direction from the container fitting portion 216 and tilts in a direction away from the rotation axis X1 as it goes downward in the vertical direction. In the nucleic acid amplification reaction container 100, the main surface of the substrate 102 abuts on the inclined surface 218 a in a state where the connection part 106 is inserted into the container fitting part 216. Therefore, the container support part 212 supports the nucleic acid amplification reaction container 100 in a state inclined with respect to the vertical direction. With the nucleic acid amplification reaction vessel 100 supported by the vessel support 212, the cover film 104 becomes the lower surface and the substrate 102 becomes the upper surface. By supporting the nucleic acid amplification reaction vessel 100 in an inclined state, bubbles in the reaction chamber 108 can be easily discharged compared to the case where the nucleic acid amplification reaction vessel 100 is supported horizontally. A window portion 220 is provided on the inclined surface 218 a of the container abutting portion 218 at a position overlapping the reaction chamber 108 in a state where the nucleic acid amplification reaction vessel 100 is supported by the container support portion 212.

  The cylindrical portion 214 projects upward in the vertical direction from the wall surfaces of the plurality of storage chambers 210, that is, the partition walls 208. The cylindrical part 214 opens upward in the vertical direction, and the preprocessing unit 204 is inserted into the cylindrical part 214 through this opening. Thereby, the pre-processing unit 204 is supported by the holder unit 202. Since the holder unit 202 holds the pretreatment unit 204 in a state where the pretreatment unit 204, particularly the needle 226 is accommodated in the cylindrical portion 214, the possibility that the user of the nucleic acid amplification device 1 touches the needle 226 is reduced. Can do. For this reason, handling of the nucleic acid amplification device 1 can be simplified.

  The cylindrical part 214 has a notch part 222 through which the needle 226 passes when the holder part 202 and the pretreatment part 204 move relative to each other around the rotation axis X1. In the present embodiment, the plurality of storage chambers 210 are arranged with the tubular portion 214 interposed therebetween in the relative movement direction of the holder portion 202 and the pretreatment portion 204, that is, in the circumferential direction of the rotation axis X1. It includes a first storage chamber 210a and a second storage chamber 210b. The notch 222 includes a first notch 222a provided on the surface of the cylindrical portion 214 facing the first storage chamber 210a in the circumferential direction of the rotation axis X1, and the cylindrical portion 214 in the circumferential direction of the rotation axis X1. 2nd notch part 222b provided in the surface which faces the 2nd storage chamber 210b side.

  By providing the cylindrical portion 214 with the notch 222, the amount of vertical movement of the holder portion 202 and the pretreatment portion 204 when the holder portion 202 and the pretreatment portion 204 are relatively rotated is reduced. Can do. Thereby, the time required for the pretreatment of the reaction solution can be shortened.

  The pretreatment unit 204 includes a pretreatment chamber 224, a needle 226, and a syringe connection unit 228. The pretreatment chamber 224 has a substantially conical shape, and is arranged such that the apex faces downward in the vertical direction. The pretreatment chamber 224 is hollow, and the reaction liquid is accommodated in the internal space. The needle 226 is a cylindrical member in which the flow path extends from one end side to the other end side, and is arranged so that the flow path extends in the vertical direction. One end of the needle 226 located in the upper part in the vertical direction is connected to the lower end portion (the apex of the cone) of the pretreatment chamber 224. Thereby, one end of the flow path of the needle 226 and the internal space of the pretreatment chamber 224 are communicated with each other. The other end of the needle 226 located below the vertical direction faces the holder portion 202 side.

  The syringe connection part 228 is a cylindrical member in which the flow path extends from one end side to the other end side, and is arranged so that the flow path extends in the vertical direction. One end of the syringe connection portion 228 positioned vertically below is connected to the upper end portion (bottom surface of the cone) of the pretreatment chamber 224. As a result, one end of the flow path of the syringe connection part 228 communicates with the internal space of the pretreatment chamber 224. The syringe connection unit 228 is fitted to the tip of the syringe 320 of the drive unit 300.

  When the pretreatment unit 204 is attached to the syringe 320 and the tip of the needle 226 is inserted into the storage chamber 210, when the pump 308 of the drive unit 300 is driven, the processing liquid in the storage chamber 210 is removed from the tip of the needle 226. Can be aspirated. Further, when the pump 308 of the drive unit 300 is driven in a state where the tip of the needle 226 is inserted into the opening 216 a of the container fitting part 216, the reaction liquid in the pretreatment chamber 224 is opened from the tip of the needle 226 to the opening. 216a can be discharged.

  A cover film (not shown) is provided on the upper surface of the main body portion 206 in an area excluding the cylindrical portion 214. As a result, the plurality of storage chambers 210 and the openings 216a and 216b of the container fitting portion 216 are sealed. The needle 226 breaks through the cover film, and the tip is inserted into the storage chamber 210 or the opening 216a.

  The pre-processing unit 204 can include the following modifications. FIG. 7A is a side view schematically showing the preprocessing unit 204 according to the fourth modification. FIG. 7B is a cross-sectional view taken along the line DD in FIG. FIG. 7C is a side view schematically showing the preprocessing unit 204 according to the fifth modification. FIG. 7D is a cross-sectional view taken along line EE in FIG. In addition, illustration of the syringe connection part 228 is abbreviate | omitted in FIG. 7 (A) and FIG.7 (C). In the following, each modification will be described with a focus on a different configuration from the preprocessing unit 204 according to the first embodiment, and the common configuration will be briefly described or omitted.

(Modification 4)
As shown in FIGS. 7A and 7B, the pretreatment unit 204 according to the modification 4 includes a pretreatment chamber 224 that extends in a range of a half circumference in the circumferential direction of the needle 226. That is, the pretreatment chamber 224 has a shape in which half of a cone is cut out. The pretreatment chamber 224 has a first inclined surface 224 a and a second inclined surface 224 b that are in contact with one end side of the needle 226. The first inclined surface 224a has a relatively large inclination with respect to the extending direction Z of the needle 226 (the direction indicated by the arrow Z in FIG. 7A), and the second inclined surface 224b is the extending direction Z of the needle 226. The inclination with respect to is relatively small. That is, the second inclined surface 224b is steeper than the first inclined surface 224a. For example, the second inclined surface 224b extends substantially parallel to the vertical direction.

(Modification 5)
As shown in FIGS. 7C and 7D, the pretreatment unit 204 according to the modified example 5 includes a pretreatment chamber 224 having a shape in which a part of a cone is notched in the circumferential direction of the needle 226. . Similar to the fourth modification, the pretreatment chamber 224 includes a first inclined surface 224 a and a second inclined surface 224 b that are in contact with one end side of the needle 226. The first inclined surface 224a has a relatively large inclination with respect to the extending direction Z of the needle 226 (the direction indicated by the arrow Z in FIG. 7C), and the second inclined surface 224b is the extending direction Z of the needle 226. The inclination with respect to is relatively small. That is, the second inclined surface 224b is steeper than the first inclined surface 224a.

  By providing the second inclined surface 224b whose inclination is steeper than that of the first inclined surface 224a as in the modified examples 4 and 5, the nucleic acid can be easily dispersed in the treatment liquid. Therefore, nucleic acid pretreatment can be performed more reliably. Further, by providing the first inclined surface 224a having a gentler inclination than the second inclined surface 224b, it is possible to secure the capacity of the pretreatment chamber 224 while suppressing an increase in the vertical dimension of the pretreatment unit 204. . The procedure of nucleic acid pretreatment and the effects of Modifications 4 and 5 will be described in detail later.

(Drive part)
FIG. 8A is a perspective view showing a schematic structure of the cartridge stage 302, the holder rotation motor 304, and the holder vertical movement motor 306 in the driving unit 300. FIG. FIG. 8B is a perspective view showing a schematic structure of the pump 308 and the magnet drive motor 310 in the drive unit 300. FIG. 8C is an enlarged perspective view showing the vicinity of the magnet 322 connected to the magnet drive motor 310. As shown in FIGS. 1 and 8A to 8C, the drive unit 300 includes a cartridge stage 302, a holder rotation motor 304, a holder vertical movement motor 306, a pump 308, and a magnet drive motor 310.

  The cartridge stage 302 includes a substrate 312, a rotation base 314, and a substrate support portion 316. The substrate 312 is substantially flat and has an opening 312a at the center. The rotating pedestal 314 is annular, has an opening 314a in the center, and has a gear 314b on the outer peripheral surface. The rotation pedestal 314 is disposed so that the opening 314 a overlaps the opening 312 a of the substrate 312, and is installed to be rotatable with respect to the substrate 312. The holder 202 of the nucleic acid amplification device cartridge 200 is fitted into the opening 314a. In a state where the holder portion 202 is fitted in the opening 314a, the container abutting portion 218 protrudes downward in the vertical direction from the substrate 312 through the opening 312a.

  The substrate support unit 316 includes two arms 316a, an arm connection unit 316b, and a rack 316c. The two arms 316 a extend in the front-rear direction of the nucleic acid amplification device 1, are disposed on the left and right sides of the substrate 312, and are fixed to the left and right side surfaces of the substrate 312. The arm connection portion 316b extends in the left-right direction of the nucleic acid amplification device 1, and is connected to the end portions of the two arms 316a on the device rear side. A rack 316c is provided on the surface of the arm connecting portion 316b facing the lamp rear side. The rack 316c is disposed so as to extend vertically up and down.

  The holder rotation motor 304 is configured by a DC motor or the like, and is arranged so that the output shaft is parallel to the normal direction of the substrate 312. A gear 304 a is provided on the output shaft of the holder rotation motor 304. The holder rotation motor 304 is disposed so that the gear 304 a meshes with the gear 314 b of the rotation base 314. The drive unit 300 can rotate the holder unit 202 mounted on the rotation base 314 around the rotation axis X <b> 1 (see FIG. 6) by driving the holder rotation motor 304.

  The holder vertical movement motor 306 is configured by a DC motor or the like, and is arranged so that the output shaft extends in the left-right direction of the nucleic acid amplification device 1. A pinion 306 a is provided on the output shaft of the holder vertical movement motor 306. The holder vertical movement motor 306 is disposed so that the pinion 306 a meshes with the rack 316 c of the substrate support portion 316. The drive unit 300 can move the holder unit 202 mounted on the rotation base 314 up and down by driving the holder up-and-down moving motor 306.

  The pump 308 is a conventionally known pump that generates suction force and discharge force. One end side of the tube 318 is connected to the suction / discharge port of the pump 308. A syringe 320 is connected to the other end side of the tube 318. A pretreatment unit 204 is connected to the tip of the syringe 320. The driving unit 300 can suck the reaction liquid and the processing liquid from the tip of the needle 226 by driving the pump 308, and can discharge the reaction liquid and the processing liquid in the pretreatment chamber 224 from the tip of the needle 226. it can.

  The magnet drive motor 310 is constituted by a DC motor or the like, and a magnet 322 is provided on the output shaft. The magnet drive motor 310 is disposed in the vicinity of the syringe 320, and can move the magnet 322 to the first position and the second position by being driven. The first position is a position where the magnet 322 is close to the pretreatment chamber 224 of the pretreatment unit 204 (position shown in FIG. 8B). The second position is a position where the magnet 322 is farther from the pretreatment chamber 224 than the first position (position shown in FIG. 8C). As will be described later, the nucleic acid is present in the reaction solution in a state of being fixed to the magnetic beads. For this reason, the nucleic acid in the pretreatment chamber 224 can be captured by arranging the magnet 322 at the first position. Moreover, the captured nucleic acid can be released by arranging the magnet 322 at the second position.

  The drive unit 300 rotates the holder unit 202 by the holder rotation motor 304 to relatively rotate the holder unit 202 (the storage chamber 210) and the pretreatment unit 204 around the rotation axis X1. In addition, the drive unit 300 moves the holder unit 202 up and down by the holder up-and-down movement motor 306, thereby moving the holder unit 202 (the storage chamber 210) and the pretreatment unit 204 up and down relatively close to each other. Moreover, the holder part 202 (container support part 212) and the temperature control part 400 are relatively spaced apart and moved closer together by the vertical movement of the holder part 202. In addition, the drive part 300 may rotate and move the holder part 202 and the pre-processing part 204 relatively by displacing the pre-processing part 204 instead of or in addition to the holder part 202. Similarly, the drive unit 300 may move the holder unit 202 and the temperature adjustment unit 400 up and down relatively by displacing the temperature adjustment unit 400 instead of or in addition to the holder unit 202.

(Temperature adjuster)
FIG. 9 is a perspective view showing a schematic structure of the temperature adjustment unit 400. As shown in FIGS. 2, 6, and 9, the temperature adjustment unit 400 includes a pedestal 402, a first temperature unit 404, a second temperature unit 406, a third temperature unit 408, and a fourth temperature unit 410. Have The pedestal 402 is a flat member, and each temperature unit is placed on the pedestal 402. The pedestal 402 is disposed so as to overlap the holder portion 202 in the vertical direction. The first temperature unit 404, the second temperature unit 406, the third temperature unit 408, and the fourth temperature unit 410 are arranged in the circumferential direction of the rotation axis X1. In the present embodiment, the temperature parts are arranged in the order of the fourth temperature part 410, the first temperature part 404, the third temperature part 408, and the second temperature part 406 in the circumferential direction of the rotation axis X1.

  The first temperature unit 404 includes, for example, a conventionally known heater (not shown), and the heater is heated by power supplied from an external power source, and is maintained at a predetermined first temperature. The first temperature is a temperature for causing a heat denaturation reaction to the nucleic acid in the reaction solution, and is, for example, about 90 ° C to 95 ° C. The first temperature may be slightly higher than the heat denaturation temperature, for example, about 110 ° C. Thereby, the time until the reaction solution reaches the heat denaturation temperature can be shortened, and the nucleic acid amplification efficiency can be increased. The first temperature unit 404 has a lower surface of the nucleic acid amplification reaction vessel 100 supported by the vessel support unit 212, that is, an inclined surface 404 a extending in parallel to the cover film 104. In the first temperature unit 404, at least the inclined surface 404a is maintained at the first temperature.

  The 2nd temperature part 406 has a heater like the 1st temperature part 404, and is maintained at the 2nd temperature lower than the 1st temperature. The second temperature is a temperature for causing an annealing reaction on the nucleic acid in the reaction solution, and is, for example, about 65 ° C to 70 ° C. The second temperature unit 406 has an inclined surface 406 a that extends in parallel to the lower surface of the nucleic acid amplification reaction vessel 100 supported by the vessel support unit 212. In the second temperature unit 406, at least the inclined surface 406a is maintained at the second temperature. The temperature adjustment unit 400 can change the temperature of the reaction solution to be a temperature cycle of the nucleic acid amplification reaction by the first temperature unit 404 and the second temperature unit 406.

  The 3rd temperature part 408 has a heater like the 1st temperature part 404, and is maintained at the 3rd temperature lower than the 2nd temperature. The third temperature is slightly lower than the annealing temperature, and is about 40 ° C., for example. In the temperature cycle applied to the reaction solution, by providing the third temperature between the first temperature and the second temperature, the time required to reach the second temperature from the first temperature can be shortened, and nucleic acid amplification. Efficiency can be increased. The third temperature unit 408 has an inclined surface 408 a extending in parallel to the lower surface of the nucleic acid amplification reaction vessel 100 supported by the vessel support unit 212. In the third temperature unit 408, at least the inclined surface 408a is maintained at the third temperature.

  The fourth temperature unit 410 includes a heater similarly to the first temperature unit 404 and is maintained at a fourth temperature that is at least higher than the melting point of the cover film 104. The fourth temperature is, for example, about 165 ° C. The fourth temperature unit 410 has a heat melting part 410a at the top in the vertical direction. In the fourth temperature part 410, at least the heat melting part 410a is maintained at the fourth temperature. The thermal melting part 410a is disposed so as to contact the thermal welding part 120 in the nucleic acid amplification reaction vessel 100 supported by the container support part 212.

(Measurement part)
FIG. 10A is a schematic diagram for explaining an example of a method for measuring a nucleic acid amplification reaction by the measurement unit 500. FIG. 10B is a schematic diagram for explaining another example of a method for measuring a nucleic acid amplification reaction by the measurement unit 500. As the measurement unit 500, a general measurement unit used in a nucleic acid amplification apparatus can be employed. For example, the measurement unit 500 includes a light emitting unit 502 and a light receiving unit 504 as shown in FIG.

  First, the reaction solution used in the nucleic acid amplification device 1 according to the present embodiment includes a fluorescent probe that binds complementarily to a predetermined nucleic acid sequence. The fluorescent probe bound to the nucleic acid emits fluorescence upon receiving excitation light. Since such a fluorescent probe is conventionally known, detailed description thereof is omitted. In addition, the nucleic acid amplification reaction vessel 100 has a light transmitting part at least partially in contact with the reaction solution. In the present embodiment, the substrate 102 and the cover film 104 are both formed of a light-transmitting material, and therefore the entire nucleic acid amplification reaction vessel 100 corresponds to the light-transmitting portion. The light transmitting part can transmit the excitation light emitted from the light emitting part 502 and the fluorescence emitted from the fluorescent probe.

  The light emitting unit 502 includes a light source such as an LED, and irradiates the reaction liquid with excitation light that excites the fluorescent probe through the light transmitting unit. The fluorescence emitted when the fluorescent probe is excited by the excitation light is emitted to the outside of the nucleic acid amplification reaction vessel 100 through the light transmitting part. The light receiving unit 504 includes a light receiving element and receives fluorescence emitted from the nucleic acid amplification reaction vessel 100. The light receiving unit 504 separates incident light using a Savart plate or the like and receives target fluorescence. Thereby, the measurement part 500 can measure the nucleic acid amplification reaction of the reaction liquid in the nucleic acid amplification reaction vessel 100. Measurement unit 500 transmits a signal indicating the measurement result to control unit 600. The control unit 600 that has received the signal displays the measurement result on the display unit 8.

  The measurement unit 500 can measure the nucleic acid amplification reaction in a state where the nucleic acid amplification reaction vessel 100 is in contact with any one of the temperature control units 400. Thereby, the nucleic acid amplification reaction can be easily measured.

  For example, as shown in FIG. 10A, in the state where the nucleic acid amplification reaction vessel 100 is in contact with the inclined surface 406a of the second temperature portion 406, the upper surface of the nucleic acid amplification reaction vessel 100 from the light emitting portion 502, that is, the substrate 102. The main surface is irradiated with excitation light L1. Excitation light L1 emitted from the light emitting unit 502 is applied to the nucleic acid amplification reaction vessel 100 through the window 220 of the vessel support unit 212. The excitation light L1 enters the reaction chamber 108, and fluorescence L2 is emitted from the fluorescent probe bonded to the nucleic acid. The light receiving unit 504 receives the fluorescence L2 emitted to the outside from the side surface connecting the lower surface and the upper surface of the nucleic acid amplification reaction vessel 100, that is, the side surface of the substrate 102.

  Alternatively, as shown in FIG. 10B, excitation light L1 is emitted from the light emitting unit 502 to the upper surface of the nucleic acid amplification reaction vessel 100 in a state where the nucleic acid amplification reaction vessel 100 is in contact with the inclined surface 406a of the second temperature unit 406. Is irradiated. Thereby, the excitation light L1 is irradiated into the reaction chamber 108, and the fluorescence L2 is emitted from the fluorescent probe bonded to the nucleic acid. The light receiving unit 504 receives the fluorescence L2 emitted from the upper surface of the nucleic acid amplification reaction vessel 100 to the outside.

  One end of a light guide member (not shown) such as an optical fiber is installed at the light exit of the light emitting unit 502. The other end of the light guide member is disposed in the vicinity of the nucleic acid amplification reaction vessel 100. The excitation light L1 emitted from the light emitting unit 502 is irradiated to the nucleic acid amplification reaction vessel 100 through the light guide member. One end of a light guide member (not shown) such as an optical fiber is installed at the light incident port of the light receiving unit 504. The other end of the light guide member is disposed in the vicinity of the nucleic acid amplification reaction vessel 100. The fluorescence L2 emitted from the reaction chamber 108 enters the light receiving unit 504 through the light guide member. By interposing a light guide member between the light emitting unit 502 and the light receiving unit 504 and the nucleic acid amplification reaction vessel 100, the degree of freedom of installation of the light emitting unit 502 and the light receiving unit 504 can be increased.

  The light reception unit 504 may receive the fluorescence L2 from either the upper surface or the side surface of the nucleic acid amplification reaction vessel 100, but the light reception from the side surface can measure the nucleic acid amplification reaction with higher accuracy. In the reaction chamber 108, the distance from the cover film 104 to the bottom surface of the recess 102a is extremely short compared to the distance between two opposing side surfaces of the recess 102a. For this reason, when the volume of the reaction chamber 108 decreases due to deformation of the cover film 104 or the like, the change in the intensity of the fluorescence L2 emitted from the upper surface of the nucleic acid amplification reaction vessel 100 is the change in the intensity of the fluorescence L2 emitted from the side surface. Bigger than For this reason, it is possible to measure with higher accuracy by receiving the fluorescence L2 from the side surface of the nucleic acid amplification reaction vessel 100.

  When the excitation light L1 is irradiated from the upper surface of the nucleic acid amplification reaction vessel 100 and the fluorescence L2 is received from the side surface, the nucleic acid amplification reaction vessel 100 may have a light transmitting portion on the upper surface and the side surface. Further, when the excitation light L1 is irradiated from the upper surface of the nucleic acid amplification reaction vessel 100 and the fluorescence L2 is received from the upper surface, the nucleic acid amplification reaction vessel 100 only needs to have a translucent portion on the upper surface. Note that the measurement unit 500 may measure the nucleic acid amplification reaction using a known measurement method other than the fluorescence detection method.

(Control part)
The control unit 600 is realized by elements and circuits including a CPU and a memory of a computer as a hardware configuration, and is realized by a computer program and the like as a software configuration. The control unit 600 stores various programs and data. The control unit 600 executes each stored program to control each unit of the nucleic acid amplification device 1.

  For example, the control unit 600 controls the drive of the holder rotation motor 304, the holder vertical movement motor 306, and the pump 308 as follows according to a predetermined preprocessing program. FIG. 11A, FIG. 11B, FIG. 12A, and FIG. 12B are schematic diagrams for explaining the control of the control unit 600 in the pretreatment of the reaction liquid. In addition, in FIG. 11 (A) and FIG. 12 (A), illustration of the holder rotation motor 304 is abbreviate | omitted.

  In the pretreatment of the reaction solution, the reaction solution is stored in the pretreatment chamber 224 in advance. As shown in FIGS. 11A and 11B, when the processing liquid stored in the predetermined storage chamber 210 is sucked into the preprocessing chamber 224, the needle 226 of the preprocessing unit 204 is set in the predetermined storage chamber. In a state facing 210, the control unit 600 drives the holder vertical movement motor 306 of the driving unit 300 to move the substrate 312 upward in the vertical direction. As a result, the needle 226 of the pretreatment unit 204 is inserted into the storage chamber 210. Then, the pump 308 is driven, and the needle 226 sucks the processing liquid stored in the opposing storage chamber 210 from the tip. Thereby, the processing liquid is transferred to the preprocessing chamber 224. Then, in the pretreatment chamber 224, a pretreatment for reacting the reaction solution with the sucked treatment solution is performed.

  When the pretreatment of the reaction liquid using the predetermined processing liquid is completed, the holder vertical movement motor 306 of the driving unit 300 is driven by the control unit 600 as shown in FIGS. 12 (A) and 12 (B). The substrate 312 moves downward in the vertical direction. As a result, the needle 226 stored in the storage chamber 210 is pulled out of the storage chamber 210. The substrate 312 moves downward until the tip of the needle 226 reaches a position vertically above the lower end of the notch 222. Then, the holder rotation motor 304 is driven, the holder unit 202 and the pretreatment unit 204 are relatively rotated, and the needle 226 moves to a position facing the other storage chamber 210. Then, the holder vertical movement motor 306 is driven again to move the substrate 312 upward in the vertical direction, and the needle 226 is inserted into the storage chamber 210.

  Thereafter, this operation is repeated, and the needle 226 is sequentially moved to a position facing each of the plurality of storage chambers 210 to suck the processing liquid in each storage chamber 210. Then, pretreatment of the reaction solution using the treatment solution accommodated in each accommodation chamber 210 is performed. Therefore, according to the nucleic acid amplification device 1 or the cartridge 200 for nucleic acid amplification device according to the present embodiment, the pretreatment of the reaction solution can be performed more simply, and thus the nucleic acid amplification reaction can be performed more simply. Can do.

  Further, the control unit 600 controls the driving of the holder rotation motor 304 and the holder vertical movement motor 306 as follows in accordance with a predetermined nucleic acid amplification reaction program. FIGS. 13A, 13B, and 14A to 14C are schematic diagrams for explaining the control of the control unit 600 in the nucleic acid amplification reaction. In FIG. 13A, the holder rotation motor 304 is not shown. 14A to 14C, the cylindrical portion 214 is not shown.

  As shown in FIG. 13A, FIG. 13B, and FIG. 14A, in the nucleic acid amplification reaction, the holder vertical movement motor 306 is driven by the control unit 600 and the holder unit 202 mounted on the substrate 312. Moves vertically downward. The holder unit 202 moves downward until the lower surface of the nucleic acid amplification reaction vessel 100 supported by the vessel support unit 212 comes into contact with a predetermined temperature unit. Thereby, the reaction liquid in the reaction chamber 108 is heated to a predetermined temperature.

  When the time specified by the nucleic acid amplification reaction program elapses, as shown in FIG. 14B, the control unit 600 drives the holder up / down movement motor 306 to move the holder unit 202 upward in the vertical direction. Next, as shown in FIG. 14C, the holder rotation motor 304 is driven by the controller 600, and the nucleic acid amplification reaction vessel 100 is positioned above the temperature portion where the nucleic acid amplification reaction vessel 100 should be brought into contact next. Until it does, the holder part 202 and the temperature control part 400 are rotated relatively. Then, the holder up-and-down movement motor 306 is driven again, the holder part 202 moves downward in the vertical direction, and the nucleic acid amplification reaction vessel 100 comes into contact with any other temperature part.

  Thereafter, when this operation is repeated and the nucleic acid amplification reaction vessel 100 comes into contact with the first temperature unit 404 to the third temperature unit 408, the reaction solution in the nucleic acid amplification reaction vessel 100 corresponds to the temperature cycle of the nucleic acid amplification reaction. Temperature change is added. Further, when the nucleic acid amplification reaction vessel 100 is brought into contact with the fourth temperature unit 410, the substrate 102 and the cover film 104 are thermally welded to each other in the heat welding portion 120 of the nucleic acid amplification reaction vessel 100.

  In the present embodiment, the nucleic acid amplification reaction vessel 100 is supported by the vessel support 212 in a state inclined with respect to the vertical direction. As a result, the bubbles in the reaction chamber 108 can be easily discharged. Therefore, stabilization of the nucleic acid amplification reaction can be achieved. In addition, the container support part 212 and the temperature control part 400 are relatively spaced apart from each other and approached so that the lower surface of the nucleic acid amplification reaction container 100 is in contact with the first temperature part 404 and in contact with the second temperature part 406. The state, the state of contacting the third temperature unit 408, and the state of contacting the fourth temperature unit 410 are switched. For this reason, the lower surface of the nucleic acid amplification reaction vessel 100 can be reliably brought into contact with each temperature part. As a result, the nucleic acid amplification reaction can be carried out stably.

  Also, compared to a structure in which the temperature unit that moves the nucleic acid amplification reaction vessel 100 in the horizontal direction and contacts the lower surface of the nucleic acid amplification reaction vessel 100 is switched, the cartridge 200 for the nucleic acid amplification device and the temperature adjustment unit 400 are required. Position accuracy can be lowered. For this reason, the manufacturing process of the nucleic acid amplification device 1 can be simplified.

  Further, the driving unit 300 rotates the holder unit 202 in the nucleic acid amplification reaction by using the holder rotation motor 304 that rotates the holder unit 202 in the pretreatment process. That is, a rotation axis for relatively rotating the holder unit 202 and the temperature adjusting unit 400 and a rotation axis for relatively rotating the holder unit 202 and the pretreatment unit 204 are common. , Both are rotation axes X1. For this reason, the structure of the nucleic acid amplification device 1 can be simplified.

  In addition, the controller 600 controls the temperatures of the first temperature unit 404 to the fourth temperature unit 410. For example, a temperature sensor is provided in each temperature part. The control part 600 can maintain the temperature of each temperature part by controlling the electric power feeding to the heater of each temperature part based on the information obtained from this temperature sensor. In addition, the control unit 600 controls turning on / off of the light emitting unit 502.

(Operation of nucleic acid amplifier)
Next, the operation of the nucleic acid amplification device 1 will be described in detail. First, a sample containing a nucleic acid that is a reaction target of a nucleic acid amplification reaction is collected. Samples include nasal swabs, pharyngeal swabs, whole blood, plasma, serum, stool, urine, tissue, cervical swabs, sputum, culture fluid, cerebrospinal fluid, formalin-fixed paraffin-embedded tissue (FFPE), etc. Can do. The collected specimen is accommodated in the pretreatment chamber 224 of the pretreatment unit 204 as a reaction solution. The reaction solution is introduced into the pretreatment chamber 224 through the flow path of the syringe connection unit 228, for example. Depending on the type of sample collected, pretreatment such as viscosity adjustment may be performed.

  The pretreatment unit 204 into which the reaction solution has been charged is fixed to the cylindrical unit 214. An unused nucleic acid amplification reaction vessel 100 is attached to the vessel support 212. A predetermined processing liquid is stored in each storage chamber 210 of the holder unit 202.

  An example of the processing liquid stored in the storage chamber 210 is a processing liquid containing magnetic silica particles. Magnetic silica particles are particles obtained by applying a silica coating on the surface of a magnetic material, and nucleic acids are adsorbed on silica on the surface of the magnetic material. The nucleic acid can be captured by the magnet 322 by supporting the nucleic acid on the magnetic silica particles. Accordingly, it is possible to prevent the nucleic acid from flowing out toward the storage chamber 210 when the processing solution is discharged from the pretreatment chamber 224. In addition, as a nucleic acid capture method, a boom method using a silica membrane, a SPRI (Solid Phase Reversible Immobilization) method, filtration, or the like may be employed.

  Examples of the processing liquid stored in the storage chamber 210 include a processing liquid for dissolving a film of cells, viruses, bacteria, or the like contained in a specimen. Examples of such treatment liquid include chaotropic agents, surfactants, proteases, reducing agents, sodium hydroxide, organic solvents, and the like. Other treatment solutions include those for inactivating nucleases that degrade nucleic acids and those for denaturing proteins that may inhibit nucleic acid amplification reactions. Examples of such treatment liquid include a combination of a chaotropic agent and a surfactant, a protease, a nuclease inhibitor, and the like. Further, in the storage chamber 210, reagents necessary for nucleic acid amplification reaction and measurement, such as DNA polymerase, primer, fluorescent probe, and reverse transcriptase as required, are stored.

  In the holder portion 202, a plurality of storage chambers 210 are arranged in the circumferential direction of the rotation axis X1. The treatment liquids are stored in the storage chambers 210 so as to be arranged in the order of use in the pretreatment process. For example, the drive unit 300 first moves the preprocessing unit 204 located in the cylindrical portion 214 to a position facing the first storage chamber 210a. Then, it moves to the adjacent storage chamber 210 sequentially, and finally moves to the 2nd storage chamber 210b. Accordingly, the processing liquids are stored in the storage chambers 210 so as to be arranged from the first storage chamber 210a toward the second storage chamber 210b in the order of use in the pretreatment process. Preferably, the treatment liquid containing magnetic silica particles is stored in the first storage chamber 210a. Note that the processing liquid does not have to be stored in all the storage chambers 210.

  The pretreatment unit 204 containing the reaction solution and the unused nucleic acid amplification reaction vessel 100 are mounted, and the nucleic acid amplification device cartridge 200 containing the treatment solution in each accommodation chamber 210 is inserted into the nucleic acid amplification device 1. Is done. The user of the nucleic acid amplification device 1 can insert the nucleic acid amplification device cartridge 200 into the device through the opening of the cover 4 by opening the lid 6. The cartridge 200 for nucleic acid amplification device is fitted into the opening 314a of the rotation base 314. After the lid 6 is closed, pretreatment of the reaction solution is started.

  When the pretreatment is started, the holder vertical movement motor 306 is first driven to move the substrate 312 upward in the vertical direction, and the syringe connection portion 228 of the pretreatment portion 204 approaches the tip of the syringe 320. Then, the syringe connection part 228 is fitted into the tip of the syringe 320. Thereafter, pretreatment of the reaction solution using each treatment solution is performed.

  Here, a procedure of pretreatment using an arbitrary treatment liquid TL1 will be described. FIG. 15A to FIG. 15F are schematic diagrams for explaining a pretreatment process of a reaction solution performed in the nucleic acid amplification device 1 according to the embodiment.

  First, as shown in FIG. 15A, the reaction solution, that is, the nucleic acid N adsorbed on the magnetic silica particles is separated from the wall surface of the pretreatment chamber 224 by the magnet 322 arranged at the first position by the magnet drive motor 310. Captured. Further, the needle 226 is inserted into the storage chamber 210.

  Next, as illustrated in FIG. 15B, the processing liquid TL <b> 1 in the storage chamber 210 is sucked and transferred into the preprocessing chamber 224. Further, the magnet 322 is disposed at the second position by the magnet drive motor 310. Thereby, the captured nucleic acid N is released and begins to be dispersed in the treatment liquid TL1. Subsequently, as shown in FIGS. 15C and 15D, the treatment liquid TL1 containing the nucleic acid N is discharged into the storage chamber 210 and then sucked into the pretreatment chamber 224 again. By this procedure, the nucleic acid N can be reliably dispersed in the treatment liquid TL1, and further stirred in the treatment liquid TL1. Thus, the reaction between the nucleic acid N and the treatment liquid TL1 can be promoted.

  When the pretreatment of the reaction liquid using the treatment liquid TL1 is completed by repeating the discharge and suction a predetermined number of times, the magnet 322 is arranged at the first position by the magnet drive motor 310 as shown in FIG. The Thereby, the nucleic acid N is captured on the wall surface of the pretreatment chamber 224. Then, as shown in FIG. 15F, only the treatment liquid TL1 is discharged into the storage chamber 210. By the above procedure, the pretreatment of the reaction solution using the treatment solution TL1 is completed.

  When the pretreatment using the treatment liquid TL1 is completed, the needle 226 is pulled out of the storage chamber 210 by driving the holder vertical movement motor 306. Then, by driving the holder rotating motor 304, the preprocessing unit 204 is moved to a position facing the storage chamber 210 that stores the processing liquid for the next preprocessing. Thereafter, this operation is repeated, and pretreatment with each treatment liquid is performed.

  When the pretreatment unit 204 according to Modification 4 or Modification 5 is used as the pretreatment unit 204, the nucleic acid N captured on the wall surface of the pretreatment chamber 224 can be more reliably dispersed in the treatment liquid TL1. . FIG. 16A to FIG. 16C are schematic diagrams for explaining nucleic acid dispersion when the pretreatment unit 204 according to Modification 4 and Modification 5 is used. Note that the pre-processing unit 204 illustrated in FIGS. 16A to 16C is a cross-sectional view taken along line FF in FIG. 7B or a line GG in FIG. 7D. It corresponds to a cross-sectional view along the line. FIG. 16A shows a state immediately after the treatment liquid TL1 is sucked into the pretreatment chamber 224. FIGS. 16B and 16C show a state where the processing liquid TL1 is discharged from the pretreatment chamber 224. FIG.

  The pretreatment unit 204 of the cartridge 200 for nucleic acid amplification device according to the first embodiment includes a pretreatment chamber 224 having a substantially conical or funnel shape. Thereby, the volume of the pretreatment chamber 224 can be secured while avoiding an increase in the vertical dimension of the pretreatment unit 204. On the other hand, when the nucleic acid N is captured by the magnet 322, the nucleic acids N (magnetic silica particles) are likely to gather together to form a granule. This agglomerate tends to adhere to an inclined surface with a shallow angle in the pretreatment chamber 224.

  Since the nucleic acid N agglomerates are attached to the wall of the pretreatment chamber 224 where the inclination is gentle, even if the suction and discharge of the treatment liquid TL1 is repeated while the magnet 322 is moved to the second position, it is easily treated. In some cases, the liquid TL1 may not be dispersed. For this reason, in order to reliably perform the reaction between the nucleic acid N and the treatment liquid, it is necessary to increase the number of ejection and suction steps shown in FIGS. 15C and 15D.

  On the other hand, in the pre-processing unit 204 according to the modified examples 4 and 5, as shown in FIG. 16A, the magnet 322 is disposed in the vicinity of the second inclined surface 224b, and the nucleic acid N is placed on the second inclined surface 224b. Can be captured. Then, with the nucleic acid N captured by the second inclined surface 224b, the processing liquid TL1 is sucked and transferred into the pretreatment chamber 224. Next, as shown in FIG. 16B, after the magnet 322 moves to the second position and the nucleic acid N is released, the treatment liquid TL1 is discharged into the storage chamber 210.

  Since the nucleic acid N agglomerates adhere to the second inclined surface 224b that is steeper than the first inclined surface 224a, a strong water flow may be applied to the agglomerates when the processing liquid TL1 is discharged. it can. As a result, as shown in FIG. 16C, the nucleic acid N can be broken and the nucleic acid N can be dispersed in the treatment liquid TL1. As a result, the time required for preprocessing can be shortened.

  The magnet 322 is preferably disposed in the vicinity of the connection portion between the pretreatment chamber 224 and the needle 226 on the second inclined surface 224b. That is, it is preferable that the magnet 322 is disposed at a position facing the bent portion formed by connecting the first inclined surface 224a and the needle 226 in the second inclined surface 224b. In this case, the nucleic acid N is captured at a position where the cross-sectional area (the cross-sectional area in the horizontal direction) of the internal space of the pretreatment unit 204 changes rapidly. Thereby, a stronger water flow can be applied to the agglomerates of the nucleic acid N. Further, since the vortex is likely to occur at a position where the cross-sectional area of the internal space changes rapidly, the nucleic acid N can be more reliably dispersed and stirred.

  Note that the arrangement of the magnets 322 may be changed according to the amount of the processing liquid to be sucked, for example. That is, the required amount of the processing liquid used for the pretreatment may vary greatly depending on the type. When a series of pretreatments are performed in the same pretreatment unit 204, in the pretreatment using a small amount of treatment liquid, the treatment liquid reaches only the lower end portion of the pretreatment chamber 224, or further, the pretreatment chamber 224. May not reach. In this case, it becomes difficult to sufficiently capture the nucleic acid N dispersed in the treatment liquid.

  On the other hand, according to the pre-processing part 204 provided with the 2nd inclined surface 224b, the range which can arrange | position the magnet 322 can be expanded. That is, by providing the pretreatment chamber 224 with the second inclined surface 224b extending substantially parallel to the needle 226, the magnet 322 can be freely brought into contact with the lower end of the needle 226 from the upper end of the second inclined surface 224b. Can do. For this reason, the magnet 322 can be brought into contact with the optimum position according to the amount of the processing liquid.

  After pretreatment with all treatment solutions is completed, DNA polymerase, primers, and fluorescent probes are mixed in the reaction solution. Thereafter, the pretreatment unit 204 is moved to a position facing the opening 216 a of the container fitting unit 216. Then, the reaction liquid that has been pretreated in the pretreatment chamber 224 is discharged from the pretreatment unit 204 to the opening 216a. The discharged reaction solution is injected into the nucleic acid amplification reaction vessel 100 from the substrate inlet 114 connected to the opening 216a. Thereby, the reaction solution can be easily injected into the nucleic acid amplification reaction vessel 100. The reaction solution is injected into the nucleic acid amplification reaction vessel 100 until it reaches the substrate outlet 116. Thereby, the reaction chamber 108 can be more reliably filled with the reaction solution, and bubbles in the reaction chamber 108 can be removed.

  When the reaction solution is injected into the nucleic acid amplification reaction vessel 100, the drive of the pump 308 is controlled so that the average injection speed from the start of injection to a predetermined time is slower than the average injection speed from the predetermined time to the end of injection. Is done. Thereby, generation | occurrence | production of a bubble can be suppressed. The pump 308 is controlled so as to suck a part of the injected reaction solution after a predetermined amount of the reaction solution is injected into the nucleic acid amplification reaction vessel 100. Thereby, the bubbles in the reaction chamber 108 can be more reliably removed. The amount of the reaction solution injected into the nucleic acid amplification reaction vessel 100 before the suction is, for example, an amount that is equal to or greater than the total volume of the first communication path 110 and the reaction chamber 108.

  When the injection of the reaction solution into the nucleic acid amplification reaction vessel 100 is completed, the holder rotation motor 304 is driven by the control unit 600, and the holder unit 202 is rotated so that the nucleic acid amplification reaction vessel 100 and the fourth temperature unit 410 face each other. Moved. In addition, when each part is aligned so that the nucleic acid amplification reaction container 100 and the fourth temperature part 410 face each other at a position where the pretreatment part 204 and the opening part 216a of the container fitting part 216 face each other, This operation can be omitted.

  Subsequently, the holder vertical movement motor 306 is driven in a state where the nucleic acid amplification reaction vessel 100 and the fourth temperature unit 410 face each other, and the nucleic acid amplification reaction vessel 100 is moved downward in the vertical direction. And the cover film 104 located in the heat welding part 120 of the nucleic acid amplification reaction container 100 contacts the heat melting part 410a of the fourth temperature part 410. Thereby, in the heat welding part 120, the part which defines the 1st communicating path 110 of the board | substrate 102 and the cover film 104, and the parts which define the 2nd communicating path 112 are heat-welded, and the 1st communicating path 110 is comprised. And the 2nd communicating path 112 is sealed. Since the nucleic acid amplification device 1 includes the fourth temperature unit 410, the first communication path 110 and the second communication path 112 can be easily sealed, and thus the procedure required for the nucleic acid amplification reaction can be simplified. .

  After the first communication path 110 and the second communication path 112 are sealed, the nucleic acid amplification reaction is started. In the nucleic acid amplification reaction, the driving unit 300 causes the lower surface of the nucleic acid amplification reaction vessel 100 to contact each temperature unit in the order of the first temperature unit 404, the third temperature unit 408, and the second temperature unit 406. That is, first, the nucleic acid amplification reaction vessel 100 is brought into contact with the first temperature unit 404. Thereby, the temperature of the reaction solution becomes the heat denaturation temperature, and the nucleic acid in the reaction solution is heat denatured. The time for the heat denaturation reaction is, for example, about 2 seconds. Next, the nucleic acid amplification reaction vessel 100 is brought into contact with the third temperature unit 408. As a result, the temperature of the reaction solution decreases and approaches the temperature at which the annealing reaction occurs.

  After a predetermined time has elapsed since the nucleic acid amplification reaction vessel 100 was brought into contact with the third temperature unit 408, the nucleic acid amplification reaction vessel 100 is brought into contact with the second temperature unit 406. Thereby, the temperature of the reaction solution is maintained at the annealing temperature, and the nucleic acid in the reaction solution is annealed. The annealing reaction time is, for example, about 5 seconds. The above process is set as one cycle, for example, 30 cycles are repeated. The nucleic acid amplification reaction is completed by the above steps. The entire nucleic acid amplification reaction takes about 5 minutes.

  After the nucleic acid amplification reaction is completed, the light source of the light emitting unit 502 is turned on by the control unit 600. As a result, excitation light is emitted from the light emitting unit 502 and irradiated onto the nucleic acid amplification reaction vessel 100. When the nucleic acid amplification reaction vessel 100 is irradiated with excitation light, the fluorescent probe in the reaction solution is excited to emit fluorescence. This fluorescence is emitted to the outside of the nucleic acid amplification reaction vessel 100 and received by the light receiving unit 504. The measurement result acquired by the measurement unit 500 is sent to the control unit 600 and displayed on the display unit 8. Through the above steps, the user of the nucleic acid amplification device 1 can acquire information such as the amount of amplified nucleic acid.

  After the measurement of the nucleic acid amplification reaction is accommodated, the pretreatment unit 204 is removed from the syringe 320 by a desorption mechanism (not shown) and inserted into the cylindrical unit 214. The user of the nucleic acid amplification device 1 can open the lid 6 and take out the cartridge 200 for nucleic acid amplification device from the device. The removed cartridge 200 for nucleic acid amplification device is discarded. When the nucleic acid amplification device cartridge 200 is taken out of the device, the needle 226 is inserted into the cylindrical portion 214, so that the user can safely remove the nucleic acid amplification device cartridge 200 and discard it.

  As described above, the nucleic acid amplification reaction vessel 100 according to the present embodiment has the reaction chamber defined by the substrate 102 having the recess 102a, the cover film 104 covering the recess 102a, and the recess 102a and the cover film 104. 108. In the reaction chamber 108, the cover film 104 side is thermally connected to each temperature part of the temperature adjustment unit 400, whereby a nucleic acid amplification reaction is performed in the reaction chamber 108. The thickness M2 of the portion 104a that defines the reaction chamber 108 in the cover film 104 is smaller than the thickness M1 of the portion 102b that forms the bottom surface of the recess 102a in the substrate 102. Thereby, the temperature of the reaction solution can be adjusted with higher accuracy. For this reason, the nucleic acid amplification reaction can be stabilized. In addition, the temperature adjustment of the reaction solution by the temperature adjustment unit 400 can be speeded up and made uniform. For this reason, nucleic acid amplification efficiency can be improved.

  Further, the nucleic acid amplification reaction vessel 100 has a heat welding part 120. Then, with the reaction liquid flowing into the reaction chamber 108, the substrate 102 and the cover film 104 are thermally welded in the heat welding portion 120, and the first communication passage 110 and the second communication passage 112 are sealed. Thereby, the procedure at the time of implementing a nucleic acid amplification reaction can be simplified. Therefore, the nucleic acid amplification reaction can be carried out easily. As a result, the time required for the nucleic acid amplification reaction can be shortened.

  In addition, the nucleic acid amplification device 1 according to the present embodiment has a container support unit 212 that supports the nucleic acid amplification reaction container 100 in a state inclined with respect to the vertical direction, and the temperature of the reaction solution becomes a temperature cycle of the nucleic acid amplification reaction. The temperature control unit 400 to be changed in this manner, the container support unit 212, and the temperature control unit 400 are relatively spaced apart from each other in the vertical direction, and the drive unit 300 that makes the lower surface of the nucleic acid amplification reaction vessel 100 contact the temperature control unit 400. With. Thereby, it is possible to easily remove bubbles in the nucleic acid amplification reaction vessel 100 and to bring the nucleic acid amplification reaction vessel 100 and the temperature control unit 400 into contact with each other more accurately. Therefore, the nucleic acid amplification reaction can be stabilized. Further, the nucleic acid amplification reaction can be performed with high accuracy, and the nucleic acid amplification efficiency can be improved.

  Further, in the nucleic acid amplification device 1, the driving unit 300 relatively moves the holder unit 202 having the plurality of storage chambers 210 for storing the processing liquid and the preprocessing unit 204 having the preprocessing chamber 224 around the rotation axis X1. To rotate. Accordingly, the pretreatment unit 204 is sequentially moved to a position facing each of the plurality of storage chambers 210, and the pretreatment of the reaction liquid using the treatment liquid stored in each of the storage chambers 210 is performed. For this reason, the pretreatment of the reaction solution can be performed more easily. As a result, the nucleic acid amplification reaction can be carried out more easily.

  The present invention is not limited to the above-described embodiments and modifications, and various modifications such as various design changes can be added based on the knowledge of those skilled in the art, and such modifications are added. The embodiments and modifications are also included in the scope of the present invention. New embodiments resulting from addition of further modifications to the above-described embodiments and modifications have the effects of the combined embodiments, modifications, and further modifications.

  DESCRIPTION OF SYMBOLS 1 Nucleic acid amplification apparatus, 100 Nucleic acid amplification reaction container, 102 Substrate, 102a Concave part, 104 Cover film, 108 Reaction chamber, 120 Thermal welding part, 200 Nucleic acid amplification apparatus cartridge, 202 Holder part, 204 Pre-processing part, 210 Storage chamber, 212 container support section, 214 cylindrical section, 224 pretreatment chamber, 226 needle, 300 drive section, 400 temperature control section, 404 first temperature section, 406 second temperature section, 408 third temperature section, 410 fourth temperature section , 500 measuring unit, 502 light emitting unit, 504 light receiving unit.

Claims (5)

A substrate having a recess;
A cover film affixed to the substrate so as to cover the recess,
The reaction chamber is defined by the recess and the cover film, and has a reaction chamber inlet through which a reaction solution containing nucleic acid flows, and a reaction chamber outlet through which the gas in the reaction chamber is discharged as the reaction solution flows into the reaction chamber. And storing a reaction solution containing nucleic acid, and a reaction chamber in which a nucleic acid amplification reaction is performed in the reaction chamber by being thermally connected to the temperature control unit of the nucleic acid amplification device;
A first communication path that is defined by the recess and the cover film and that communicates the reaction chamber inlet and the outside of the substrate;
A second communication path that is defined by the recess and the cover film and that communicates the reaction chamber outlet and the outside of the substrate;
The portions defining the first communication passages of the substrate and the cover film and the portions defining the second communication passages of the substrate and the cover film with the reaction liquid flowing into the reaction chamber. A heat welding portion in which the first communication path and the second communication path are sealed,
A nucleic acid amplification reaction vessel comprising:   2. The nucleic acid amplification reaction container according to claim 1, wherein an opening area of a substrate outlet which is an end portion of the second communication path on the outside of the substrate is larger than an opening area of the reaction chamber outlet.   The nucleic acid amplification reaction container according to claim 2, wherein the substrate has a reaction solution reservoir at the substrate outlet.   4. The heat welding portion according to claim 1, further comprising: a second recess provided in the vicinity of the first communication path; and a third recess provided in the vicinity of the second communication path. 2. A nucleic acid amplification reaction container according to item 1.   The nucleic acid amplification reaction container according to claim 4, wherein a distance between the first communication path and the second communication path in the thermal welding part is smaller than a flow path width of the second communication path in the thermal welding part. .

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