JP5200517B2 - Reaction vessel - Google Patents

Description

  The present invention relates to a reaction vessel that encloses and stores a thermal reaction material that reacts by heating.

In recent years, the demand for gene diagnosis has increased, and various gene diagnosis methods and gene diagnosis kits have been released by various companies. In genetic diagnosis, tens to hundreds of microliters (μl) of specimens and reagents are introduced into a plurality of reaction chambers (wells, chambers), and predetermined enzyme reactions are performed to determine the genotype (gene type). Has been implemented.
Various types of such reaction containers (chips) are supplied by gene diagnosis methods and gene diagnosis apparatuses.
For example, as disclosed in Patent Document 1, samples and reagents are accommodated in a plurality of small rooms as necessary, and when the reaction is performed, the partitioning unit is opened to combine the small rooms so that the sample and the reagent are reacted. There is something configured.
Patent Document 1 exemplifies performing PCR (polymerase chain reaction) by applying heat to a reaction vessel and a sample processing apparatus by a heating means and performing temperature control.
In Patent Document 1, a reagent or the like for one specimen sample is enclosed in a series of tubes and used for a PCR reaction or the like, but many tubes are required to react a plurality of samples.
JP 2006-256939 A

The heat efficiency of the heat generating member and the heated member has been centered on the close contact between hard solids so far, and the spots related to the adhesion affect the heat conduction efficiency. Therefore, as a means to compensate for it, increase the heat conductivity of the contact member. So we have suppressed the effects of plaques.
As shown in Patent Document 1, when a reaction vessel or a sample processing apparatus (hereinafter referred to as reaction vessel or the like) itself is heated by a heating means, in addition to the difference in thermal conductivity depending on the material of the reaction vessel or the like, In addition, due to poor contact (adhesion) properties with a reaction vessel or the like, there were problems that heating could not be sufficiently performed, and heating took time.
In order to improve the contact between the heating means and the reaction vessel, a cushioning material or a metal plate having elasticity or adhesion or both may be sandwiched between the heating means and the reaction vessel that are temperature-controlled. Has been done. However, these measures increase the number of components.
In addition, when an attempt is made to stir the solution in the reaction vessel during heating, the lid must be opened once and the stirring operation must be performed. In this case, there are problems such as contamination, bumping of the solution, and evaporation. Concerned.

  The present invention has been made in view of such circumstances. The purpose of the present invention is to make the reaction chamber flexible along the shape of the temperature control member when the reaction chamber containing the reaction sample comes into contact with the temperature control member. By deforming, it is possible to efficiently transmit heat to the temperature control member, thereby providing a reaction vessel capable of promoting the reaction of the reaction sample.

Multiple order to achieve the above object, a reaction vessel of the present invention includes a plate-like body, comprising: a film member, and a lid member, the plate-like body, which is formed through the rear surface from the surface of the plate-like body has an opening, said film member has a flat portion which is placed on the surface of the plate-like body, and a plurality of the bag to be connected to the plane portion is inserted into the respective opening, the respective The bag body has a portion connected to the plane portion in a state where the plane portion is placed on the surface and inserted into each opening, and the upper lid member is used for the reaction sample to each bag body. After dispensing , the film member is configured to be placed through a sealing portion formed for each bag body so as to surround each insertion port on the flat surface portion and to seal the insertion port, , when the bag is in contact with the temperature adjusting member, the bag is deformed have along the shape of the temperature adjusting member Characterized in that it is constituted by a flexible material to expand the contact area between the serial temperature control member.

According to this invention, a film member is mounted on a plate-shaped body, and each bag body of a film member is each positioned inside each opening of a plate-shaped body. The reaction samples each bag is dispensed, the insertion opening of the bag after dispensing of the reaction sample into each bag body, upper lid member via a seal which is formed to surround the periphery thereof Rimitsu is closed. Film member when the bag is in contact with the temperature control member is constituted by a flexible material to expand the contact area between the temperature adjusting member and along had deformed to the shape of the bag body temperature adjusting member.
Therefore, since the bag body can be brought into close contact with the temperature control member by increasing the contact area with the temperature control member, the temperature of the temperature control member can be efficiently absorbed and the reaction of the reaction sample can be promoted.

(Embodiment 1)
<Reaction vessel>
Hereinafter, a reaction vessel according to an embodiment of the present invention will be described with reference to the drawings.
FIG. 1 is a diagram showing the configuration of the reaction vessel of the first embodiment.
As shown in FIG. 1, the reaction vessel includes a plate-like body 10, a film member 20, and an upper lid member 30.
A plurality of openings 11 are formed through the plate-like body 10 from the front surface 10A to the back surface 10B.
The film member 20 includes a flat part 21 placed on the surface 10 </ b> A of the plate-like body 10 and a bag body 22 connected to the flat part 21. The bag body 22 is located inside the opening 11 in a state where the flat surface portion 21 is placed on the surface 10A, and a portion connected to the flat surface portion 21 serves as an insertion port 22A. The bag body 22 protrudes downward from the back surface 10 </ b> B of the plate-like body 10.
The upper lid member 30 is placed on the flat surface portion 21 and configured to seal the insertion port 22A of the bag body 22.
The film member 20 is made of a flexible material that deforms along the shape of the temperature adjustment member 40 when the bag body 22 contacts the temperature adjustment member 40. Therefore, this reaction container can efficiently absorb the temperature of the temperature control member 40 and promote the reaction of the reaction sample.
This reaction vessel is suitable for reacting a small amount of reaction sample of several μl (microliter) to several ml (milliliter).

The film member 20 is placed on the plate-like body 10. Film member 20 is in close contact with the plate-shaped body 10, in addition that can be sealed by applying a physical pressure, by bonding, welding or the like, it can be fixed so as not to move the film member 20 .
The film member 20 has desired properties such as material, thickness, etc. suitable for the purpose of use, such as not inhibiting the intended reaction, no denaturation at the reaction temperature, and no reaction between the reaction solution and the container. Can be selected as appropriate.
Moreover, when forming a reaction container by welding, it is preferable to have the welding temperature which can be sealed with a commercially available heat sealer.
When forming a reaction vessel by bonding, it is necessary to confirm the required physical properties such as that the adhesive does not affect the reaction, the adhesive does not peel off during the reaction, and the time required for the adhesive to cure is short. There is.

  The film member 20 can be configured using, for example, polypropylene, vinyl chloride, silicon, Teflon (registered trademark), vinyl, synthetic rubber, silicon rubber, or polyethylene resin. As the film member 20, a thermosetting resin (for example, a phenol resin, an epoxy resin, or a urethane resin) can also be used as long as the resin itself does not react (modify) at the reaction temperature.

As the plate-like body 10, a desired material that suits the use conditions can be appropriately selected from materials that can be bonded to the film member 20, materials that do not denature at the reaction temperature, and mechanical strength at the time of handling.
As the upper lid member 30, for example, a heat insulating material can be used. The heat insulating material can be appropriately selected from commercially available materials as long as the heat necessary for the reaction does not escape.
In addition, when performing optical measurements such as fluorescence measurement, a heat-insulating material with light transmittance is adopted, or a heat-insulating material at the top of an individual reaction chamber can be removed, or the relevant part of the heat-insulating material is notched. Keep it.

<Reaction preparation>
Reactions that change temperature (heating / cooling) include biomolecule extraction reactions, binding reactions, amplification reactions, qualitative reactions, and quantitative reactions. Examples of using this flexible reaction vessel for nucleic acid amplification reactions include PCR (Polymerase chain reaction), LAMP (Loop-Mediated Isothermal Amplification), SMAP (SMart Amplification Process), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic) acids) etc.

The required amount of enzyme, primer, buffer, template DNA, dNTP, reporter molecule (fluorescent labeling agent), etc. necessary for the above reaction is dispensed into individual reaction chambers to prepare a reaction solution.
After dispensing, a film member made of the same material as the film member 20 can be placed on top of the film member 20 and heated to form the sealing portion (seal portion) 31.
About the sealing part 31, you may seal by sealing by using the welding and adhesion | attachment of film members, or pressing a part of plate-like body 10 from the outer side of a reaction container strongly.

  Mixing (stirring) of the reaction solution can be performed by deforming the bag body 22 by pressing or releasing the bag body (reaction chamber) 22 of the reaction vessel against a flat surface. It is also possible to stir the bag body 22 by deforming it with a finger or the like. Furthermore, mixing (stirring) can also be performed using ultrasonic vibration or air vibration by a member.

<Reaction>
After preparing the reaction, the reaction container is set on the temperature adjustment member 40. In FIG. 1, the fixture of the plate-like body 10 is not shown.
The temperature control required for the reaction is performed in the state of FIG.
When using the reaction product after the reaction is completed, if the reaction vessel has already been separated into separate reaction systems by welding, bonding, etc., break the vessel with a dispenser with a sharp tip, etc. When used or divided into individual reaction systems by external pressure or the like, the sealing pressure can be released sequentially, and the reaction products can be collected in order from the end.
Further, when measuring the reaction result with fluorescence, the heat insulating material 30 is removed or the photometry is performed from an angle at which the heat insulating material 30 does not get in the way.

<Modification 1>
FIG. 2 is a diagram showing a first modification of the reaction vessel according to the first embodiment.
As shown in FIG. 2, this reaction container is obtained by increasing the thickness of the plate-like body 10 shown in FIG.
The bag body 22 has a bottom portion 22B facing the insertion port 22A, and the bottom portion 22B is located on the same plane as the back surface 110B of the plate-shaped body 110 or between the front surface 110A and the back surface 110B of the plate-shaped body 110. Is located.
The film member 20 of the reaction container of Modification 1 is made of a material that expands following the reaction sample when the volume of the reaction sample accommodated in the bag body 22 expands.
When the temperature adjustment member 40 is in contact with the back surface 110B of the plate-like body 110, the temperature control member 40 closes the opening 111 of the plate-like body 110 at the back surface 110B. The bag body 22 expands during the reaction of the reaction sample, but the expansion is suppressed in a space surrounded by the temperature adjustment member 40, the plate-like body 110, and the heat insulating material 30. Thus, by making the individual reaction chambers have a constant shape as described above, the optical path length is constant when performing optical measurement, and the measurement accuracy can be improved.

<Modification 2>
FIG. 3 is a diagram showing a second modification of the reaction vessel of the first embodiment.
In the second modification, as in the first modification, the individual reaction chambers (bags) 22 of the reaction vessel are formed into a fixed shape.
As shown in FIG. 3, this reaction container is a plate-like body 210 provided with a recess 210C on the back surface 110B side of the plate-like body 110 shown in FIG. In this reaction container, when the temperature control member 40 is brought into contact with the back surface 210B of the plate-like body 210, the bottom 22B of the bag body 22 gets over the back surface 210B of the plate-like body 210 and is folded into the recess 210C of the plate-like body 210. It is.
Also in this example, the bag body 22 expands during the reaction of the reaction sample, but the expansion is suppressed in a space surrounded by the temperature control member 40, the plate-like body 210, and the heat insulating material 30.

<Modification 3>
FIG. 4 is a diagram showing a third modification of the reaction vessel according to the first embodiment.
Although this reaction container is the same as the reaction container shown in FIG. 1, it is not heated by the temperature adjustment member 40, but is heated in a non-contact manner using hot air, microwaves (electromagnetic waves) or the like. At the time of cooling, cold air, air cooling, etc. are similarly used in a non-contact manner. Thus, temperature control is implemented.

<Another usage pattern of the flexible reaction vessel>
As another use form of the reaction container of the present invention, it can be used for reactions and mixing other than the enzyme reaction shown in the following Example 1. Specifically, it is possible to carry out a chemical reaction accompanied by heat generation and endotherm in a reaction vessel.
It is also possible to carry out only mixing without reaction.

(Embodiment 2)
FIG. 5 is a diagram showing the configuration of the reaction vessel of the second embodiment.
As shown in FIG. 5, in the reaction container of the second embodiment, the reaction sample 301 is put into the tube 50 from the insertion port 50A of the tube 50 whose one end is closed by the sealing portion 51 and the other end is the insertion port 50A. It is a reaction container in which a reaction chamber 60 of a reaction sample is formed which is inserted and closed by sealing a tube portion on the side of the insertion port 50A with a sealing portion 51. The tube 50 is made of the same material as the film member 20 of the first embodiment. When the reaction chamber 60 comes into contact with the temperature adjustment member (the Peltier element 71 and the metal buffer material 72), the reaction chamber 60 is the temperature adjustment member 71. And it deform | transforms along the shape of 72, and closely_contact | adheres to a temperature control member. Therefore, this reaction container can efficiently absorb the temperature of the temperature control members 71 and 72 and promote the reaction of the reaction sample.

Examination of nucleic acid amplification reaction <Experiment>
Verification container material:
The following two types were selected as reaction containers of the second embodiment having flexible heat resistance for nucleic acid amplification demonstration tests.
A) Polypropylene sheet (thickness 70 μm) / Toray Film Processing Co., Ltd. ZK99
B) Polypropylene sheet (thickness 70 μm) / Toray Film Processing Co., Ltd. 3951
C) Commercially available polypropylene tube (200 μL size) / Applied Biosystems, MicroAmp Optical tubes
In the present example, two reaction chambers 60 were provided as the reaction container of the second embodiment.

experimental method:
First, a tube 50 was formed from a sheet of flexible material, and 50 μL of a PCR reaction solution 301 prepared according to the following nucleic acid amplification reaction solution composition was injected into the tube 50. Next, 50 μL of mineral oil 302 was added, and the cross section of the tube 50 was sealed by a thermal welding method. Continuing from the sealed portion 51, the same operation as described above was performed to form a flexible container in which the reaction solution was sealed so as to have a continuous structure, and a flow path sealing chamber was virtually assumed. Here, the flow path sealing chamber injects a reaction solution into a micro flow path such as a DNA chip, seals the flow path at an appropriate position, if necessary, and a space space between the seals. Is used as a reaction chamber (reaction vessel).

  Next, before installing them in a PCR (Polymerase chain reaction) apparatus, they were stirred and mixed by manually holding the reaction vessel, and then set in the apparatus. In order to shut off the heat of the dew point preventing upper heater 81 mounted on the reaction apparatus (Biometra, T-Gradient), a glass wool heat insulating material 82 is interposed between the upper portion of the reaction vessel and the upper heater 81, and the following The PCR reaction was performed according to the nucleic acid amplification reaction temperature cycle of (Fig. 5).

Nucleic acid amplification reaction solution composition:
Human genomic DNA (200 ng / μL): 1 μL
PRIMER-β3AR-FORWARD (10 pmoL / μL): 5 μL
PRIMER-β3AR-REVERSE (10 pmoL/μL): 5μL
dNTP Mixture (2.5 mM each): 5 μL
10XBuffer: 5μL
Taq Polymerase (5unit / μL): 1μL
DDW: 28μL
------------------------------------------
TOTAL 50μL / TEST VOLUME

Nucleic acid amplification reaction temperature cycle:
1) 95 ° C, 3min
2) 95 ° C, 30sec
3) 65 ° C, 30sec
4) 72 ° C, 30sec
5) 40 cycles from 2) to 4) 6) 72 ° C., 5 min

Gel electrophoresis analysis:
The reaction solution (3 μL) after the nucleic acid amplification reaction was electrophoresed on a 9% acrylamide gel, and the amplification behavior of the target nucleic acid chain was confirmed. A 20 base ladder marker was used as the electrophoresis marker (M).

result:
6 is a diagram showing gel electrophoresis of the nucleic acid amplification reaction product of Example 1. FIG.
From the result of gel electrophoresis analysis of the nucleic acid amplification reaction shown in FIG. 6, nucleic acid amplification was observed in each of the reaction vessels (A) and (B) as well as the control (C) of the nucleic acid amplification reaction.
From this result, by using the reaction container of the present invention, it is not necessary to bother to provide the same type as the reaction container on the temperature control member side as in the prior art. As the critical nucleic acid amplification reaction proceeds well, it is considered that there is no problem in thermal conductivity or reagent mixing, and there are also problems such as container rupture due to thermal expansion. Not. From the above, it has been clarified that the reaction vessel having this flexibility is excellent in various fields such as adhesion and thermal conductivity at the time of temperature control, mixing property, and thermal expansion durability.

FIG. 2 is a diagram showing a configuration of a reaction vessel according to the first embodiment. FIG. 6 is a diagram showing a first modification of the reaction container in the first embodiment. 6 is a diagram showing a second modification of the reaction vessel in the first embodiment. FIG. FIG. 10 is a diagram showing a third modification of the reaction container in the first embodiment. FIG. 4 is a diagram showing a configuration of a reaction vessel according to a second embodiment. 1 is a diagram showing gel electrophoresis of a nucleic acid amplification reaction product of Example 1. FIG.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 ... Plate-shaped body, 11 ... Opening, 20 ... Film member, 21 ... Planar part, 22 ... Bag body, 30 ... Upper lid member.

Claims (7)

Comprising a plate-like body, and the film member, and a top cover member,
The plate-like body has a plurality of apertures formed through from the front surface to the back surface of the plate-like body,
It said film member has a flat portion which is placed on the surface of the plate-like body, and a plurality of bag to be inserted to the connected each opening in the planar portion, each bag, the plane The portion connected to the flat surface portion in a state where the portion is placed on the surface and inserted into each opening is an insertion port,
The upper lid member is placed via a sealing portion formed for each bag body so as to surround the periphery of each insertion port on the flat surface portion after dispensing the reaction sample to each bag body. Configured to seal the insertion port;
Said film member, when the bag is in contact with the temperature control member is constituted by a flexible material which the bag is to enlarge the contact area between the temperature adjusting member deforms have along the shape of the temperature adjusting member ing,
A reaction vessel characterized by that.   The reaction container according to claim 1, wherein the bag body protrudes from the back surface of the plate-like body. The bag body has a bottom portion facing the insertion port,
The reaction container according to claim 1, wherein the bottom portion is located on the same plane as the back surface, or is located between the front surface and the back surface.   The said film member is comprised by the material which the volume expands following the said reaction sample, when the volume of the reaction sample accommodated in the inside of the said bag body expand | swells. The reaction vessel as described.   The reaction container according to claim 1, wherein the temperature adjustment member is in contact with a back surface of the plate-like body and closes the opening at the back surface.   2. The film member is made of polypropylene, vinyl chloride, silicon, Teflon (registered trademark), vinyl, synthetic rubber, silicon rubber, or polyethylene resin. Reaction vessel. The upper cover member, a reaction vessel according to claim 1 Symbol mounting, characterized in that it is constituted by a light transmitting member to allow optical measurement of the reaction sample.

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