JP2018033414A - Nucleic acid amplification reaction vessel, nucleic acid amplification reaction device, and nucleic acid amplification reaction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid amplification reaction vessel which can reduce the probability of contaminants being mixed therein.SOLUTION: The nucleic acid amplification reaction vessel carries out a nucleic acid amplification reaction by transferring a reaction liquid containing a reagent back and forth between a first region and a second region, and comprises a third region which is different from the first region and the second region and is located in a region other than the flow path in which the reaction liquid is transferred back and forth between the first region and the second region, with the reagent being placed in the third region.SELECTED DRAWING: Figure 1

Description

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

  In recent years, gene-based medical care such as gene diagnosis and gene therapy has attracted attention due to the development of gene utilization technology, and many methods using genes for variety discrimination and variety improvement have been developed in the field of agriculture and livestock. . As a technique for utilizing a gene, a technique such as a PCR (Polymerase Chain Reaction) method is widely used. Today, PCR has become an indispensable technique for elucidating information on biological materials.

  The PCR method is a method of amplifying a target nucleic acid by subjecting a solution (reaction solution) containing a nucleic acid (target nucleic acid) to be amplified and a reagent to thermal cycling. The thermal cycle is a process in which two or more temperatures are periodically applied to the reaction solution. In the PCR method, a method of applying a two-stage or three-stage thermal cycle is common. Nucleic acid amplification can be quantified, for example, by using a probe.

  The speeding up of PCR is a technique necessary for shortening the testing time of genetic testing, and is highly expected in the genetic testing industry.

  For example, Patent Document 1 describes a method in which a polymerase is provided at a concentration of at least 0.5 μM and a primer is at least 2 μM, and is completed in a cycle time of less than 20 seconds per cycle.

Special table 2015-520614

  As a result of intensive studies, the inventors have found that the probe inhibits the nucleic acid amplification reaction and the speeding up of PCR becomes insufficient. Therefore, it is conceivable to mix the probe with the reaction solution after performing the nucleic acid amplification reaction.

  However, when the nucleic acid amplification reaction vessel is opened after the nucleic acid amplification reaction is performed and the probe is mixed with the reaction solution, there is a concern that foreign matter may be mixed into the nucleic acid amplification reaction vessel.

  One of the objects according to some aspects of the present invention is to provide a nucleic acid amplification reaction vessel that can reduce the possibility of contamination. Another object of some aspects of the present invention is to provide a nucleic acid amplification reaction apparatus to which the nucleic acid amplification reaction vessel can be attached. Another object of some aspects of the present invention is to provide a nucleic acid amplification reaction method using the nucleic acid amplification reaction vessel.

The nucleic acid amplification reaction vessel according to the present invention is
A nucleic acid amplification reaction vessel for performing a nucleic acid amplification reaction by reciprocating a reaction solution containing a reagent between a first region and a second region,
A third region that is different from the first region and the second region, and is located between the first region and the second region;
A reagent is arranged in the third region.

  In such a nucleic acid amplification reaction vessel, it is not necessary to open the lid of the nucleic acid amplification reaction vessel and introduce the reagent into the nucleic acid amplification reaction vessel after performing the nucleic acid amplification reaction. Therefore, in such a nucleic acid amplification reaction container, the possibility that foreign substances are mixed can be reduced.

In the nucleic acid amplification reaction container according to the present invention,
The reagent arranged in the third region may include a probe.

  In such a nucleic acid amplification reaction container, it can suppress that a probe inhibits nucleic acid amplification reaction.

In the nucleic acid amplification reaction container according to the present invention,
In the second region, a reagent is disposed,
The reagent arranged in the second region may include a primer, a polymerase, and dNTP.

  In such a nucleic acid amplification reaction vessel, a nucleic acid amplification reaction can be performed using a reagent arranged in the second region.

In the nucleic acid amplification reaction container according to the present invention,
The reagent disposed in the second region may be disposed in a lyophilized state.

  In such a nucleic acid amplification reaction vessel, the reagent arranged in the second region can be attached to the inner wall of the container body defining the second region, and the reagent arranged in the second region and the third region It is possible to arrange the reagents arranged in the state in a state of being separated from each other more reliably.

In the nucleic acid amplification reaction container according to the present invention,
The reagent arranged in the third region may be arranged in a lyophilized state.

  In such a nucleic acid amplification reaction vessel, the reagent arranged in the third region can be attached to the inner wall of the container body defining the third region, and the reagent arranged in the second region and the third region It is possible to arrange the reagents arranged in the state in a state of being separated from each other more reliably.

In the nucleic acid amplification reaction container according to the present invention,
The reagent arranged in the third region may include a polymerase.

  In such a nucleic acid amplification reaction vessel, for example, the probe can be more reliably decomposed than when the probe is decomposed only by the polymerase contained in the reagent arranged in the second region.

In the nucleic acid amplification reaction container according to the present invention,
Including a flow path through which the reaction solution can flow,
The flow path is
A first flow path having the first region and the second region;
A second channel having the third region and bent and connected to the first channel;
You may have.

  In such a nucleic acid amplification reaction vessel, the reaction solution may move to the third region while the reaction solution is reciprocated between the first region and the second region to perform the nucleic acid amplification reaction. Lower.

In the nucleic acid amplification reaction container according to the present invention,
Including a flow path through which the reaction solution can flow,
The flow path is sandwiched between a first inner wall and a second inner wall facing the first inner wall,
The distance between the first inner wall and the second inner wall is such a length that the reaction solution contacts the first inner wall and the second inner wall when the reaction solution is introduced into the flow path. May be.

  In such a nucleic acid amplification reaction vessel, the heat of the heating part can be sufficiently transferred to the reaction solution.

The nucleic acid amplification reaction apparatus according to the present invention comprises:
A mounting part to which the nucleic acid amplification reaction container according to the present invention can be mounted;
A first heating unit for heating the first region to a first temperature;
A second heating unit for heating the second region to a second temperature higher than the first temperature;
A third heating unit for heating the third region to a third temperature lower than the second temperature;
A drive mechanism for moving the nucleic acid amplification reaction vessel so that the reaction solution moves to the first region, the second region, and the third region;
including.

  In such a nucleic acid amplification reaction apparatus, since the nucleic acid amplification reaction container according to the present invention can be mounted, the possibility of foreign matters being mixed can be reduced.

In the nucleic acid amplification reaction apparatus according to the present invention,
A third heating unit that heats the third region to a third temperature lower than the second temperature may be included.

  In such a nucleic acid amplification reaction apparatus, the third region can be heated to the third temperature by the third heating unit.

In the nucleic acid amplification reaction apparatus according to the present invention,
A detection mechanism for detecting fluorescence from the reaction solution mixed with the reagent arranged in the third region may be included.

  In such a nucleic acid amplification reaction apparatus, it is possible to detect fluorescence from a reaction solution in which reagents arranged in the third region are mixed.

The nucleic acid amplification reaction method according to the present invention comprises:
A nucleic acid amplification reaction method using the nucleic acid amplification reaction vessel according to the present invention,
A nucleic acid amplification reaction step of performing a nucleic acid amplification reaction by reciprocating the reaction solution between the first region at a first temperature and the second region at a second temperature higher than the first temperature;
After the nucleic acid amplification reaction step, the reaction solution is moved to a third region having a third temperature lower than the second temperature, and the reagent arranged in the third region is mixed with the reaction solution. Process,
A first moving step of moving the reaction solution to the second region after the mixing step;
A second moving step of moving the reaction solution to the first region or the third region after the first moving step;
A detection step of detecting fluorescence from the reaction solution after the second movement step;
including.

  In such a nucleic acid amplification reaction method, since the nucleic acid amplification reaction container according to the present invention is used, the possibility of foreign matters being mixed can be reduced.

Sectional drawing which shows typically the nucleic acid amplification reaction container which concerns on this embodiment. Sectional drawing which shows typically the nucleic acid amplification reaction container which concerns on this embodiment. Sectional drawing which shows typically the nucleic acid amplification reaction apparatus which concerns on this embodiment. Sectional drawing which shows typically the nucleic acid amplification reaction apparatus which concerns on this embodiment. The functional block diagram of the nucleic acid amplification reaction apparatus which concerns on this embodiment. The flowchart for demonstrating the nucleic acid amplification reaction method which concerns on this embodiment. Sectional drawing for demonstrating the nucleic acid amplification reaction method which concerns on this embodiment. Sectional drawing for demonstrating the nucleic acid amplification reaction method which concerns on this embodiment. Sectional drawing for demonstrating the nucleic acid amplification reaction method which concerns on this embodiment. Sectional drawing for demonstrating the nucleic acid amplification reaction method which concerns on this embodiment. Sectional drawing which shows typically the nucleic acid amplification reaction container which concerns on the 1st modification of this embodiment. Sectional drawing which shows typically the nucleic acid amplification reaction container which concerns on the 2nd modification of this embodiment. Sectional drawing which shows typically the nucleic acid amplification reaction container which concerns on the 3rd modification of this embodiment. Sectional drawing which shows typically the nucleic acid amplification reaction container which concerns on the 4th modification of this embodiment. Sectional drawing which shows typically the nucleic acid amplification reaction container which concerns on the 4th modification of this embodiment.

  DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. The embodiments described below do not unduly limit the contents of the present invention described in the claims. In addition, not all of the configurations described below are essential constituent requirements of the present invention.

1. Nucleic Acid Amplification Reaction Container First, a nucleic acid amplification reaction container according to this embodiment will be described with reference to the drawings. FIG. 1 is a cross-sectional view schematically showing a nucleic acid amplification reaction vessel 100 according to this embodiment. FIG. 2 is a cross-sectional view taken along line II-II in FIG. 1 schematically showing the nucleic acid amplification reaction vessel 100 according to the present embodiment.

  The nucleic acid amplification reaction container 100 includes a container body 10 as shown in FIGS. 1 and 2. The material of the container body 10 is, for example, glass, polymer, metal or the like. The nucleic acid amplification reaction container 100 includes a flow path 20 formed by the container body 10. The flow path 20 is provided with the first reagent 2 and the second reagent 4.

1.1. Channel The channel 20 is a channel through which the reaction solution 6 can flow. The flow path 20 is sealed. FIG. 1 shows a state before the reaction solution 6 is arranged in the flow path 20, and FIG. 2 shows a state where the reaction solution 6 is arranged in the flow path 20.

  The reaction solution 6 is adjusted (generated) by contacting a template nucleic acid solution containing a target nucleic acid (template nucleic acid) and the first reagent 2 disposed in the container body 10. Accordingly, the reaction solution 6 contains the template nucleic acid and the first reagent 2 (including the template nucleic acid component and the first reagent 2 component), and serves as a place for proceeding with the nucleic acid amplification reaction. The template nucleic acid may be DNA (Deoxyribo Nucleic Acid) or RNA (Ribo Nucleic Acid).

  A liquid (not shown) is accommodated in the flow path 20. The liquid is a liquid that is not miscible with the reaction liquid 6, that is, does not mix. The liquid has a specific gravity different from that of the reaction liquid 6. Specifically, the specific gravity of the liquid is smaller than the specific gravity of the reaction liquid 6. Examples of the liquid include dimethyl silicone oil and paraffin oil. The liquid covers the inner wall of the container body 10 and covers the surface of the reaction solution 6 when the reaction solution 6 is disposed in the flow path 20. The liquid is not filled in the flow path 20. Thereby, in the nucleic acid amplification reaction vessel 100, the reaction solution 6 can be moved faster than in the case where the liquid fills the flow path 20.

  As shown in FIG. 1, the flow path 20 has a first region 30, a second region 32, and a third region 34. The first region 30 of the flow path 20 is a region heated to the first temperature. For example, the reaction liquid 6 positioned in the first region 30 is heated to the first temperature. The first temperature is a temperature suitable for primer annealing reaction and extension reaction, and is, for example, 55 ° C. or higher and 75 ° C. or lower. The first region 30 is, for example, a portion of the flow path 20 that is located in the opening 40a provided in the first heating unit 40 (see FIG. 3).

  The second region 32 of the flow path 20 is a region different from the first region 30. The second region 32 is a region that is heated to a second temperature that is higher than the first temperature. For example, the reaction solution 6 located in the second region 32 is heated to the second temperature. The second temperature is a temperature suitable for the dissociation (denaturation reaction) of double-stranded DNA, and is, for example, 85 ° C. or higher and 105 ° C. or lower. The second region 32 is, for example, a portion of the flow path 20 that is located in the opening 42a provided in the second heating unit 42 (see FIG. 3).

  The third region 34 of the flow path 20 is a region different from the first region 30 and the second region 32, and is a region other than the route through which the reaction solution 6 reciprocates between the first region 30 and the second region 32. To position. In the example illustrated in FIG. 1, the third region 34 is a region that is different from the first region 30 and the second region 32 and is located other than between the first region 30 and the second region 32. The third region 34 is a region heated to a third temperature lower than the second temperature. The reaction solution 6 located in the third region 34 is heated to, for example, a third temperature. The third temperature may be higher than the first temperature, may be the same as the first temperature, or may be lower than the first temperature. The third temperature is a temperature suitable for detecting fluorescence from the reaction solution 6 mixed with the probe. For example, the third region 34 is a portion of the flow path 20 that is located in an opening 44 a provided in the third heating unit 44 (see FIG. 3).

  For example, when the Tm (Melting Temperature) value of the probe is lower than the Tm value of the primer, the third temperature is preferably lower than the first temperature. When the third temperature is lower than the first temperature, the temperature of the reaction solution 6 is decreased when the reaction solution 6 is moved from the second region 32 to the third region 34 as compared with the case where the third temperature is equal to or higher than the first temperature. The speed can be increased. This can reduce the probability of primer annealing and increase the probability of probe hybridization. Therefore, the fluorescence intensity from the reaction solution 6 can be increased. The third temperature is, for example, 40 ° C. or higher and 105 ° C. or lower.

  As shown in FIG. 1, the flow path 20 includes a first flow path 22 and a second flow path 24. The first flow path 22 has a first region 30 and a second region 32. In the illustrated example, the first flow path 22 has a first region 30 and a part of the second region 32. The first flow path 22 is a linear flow path. The shape of the first flow path 22 is, for example, a rectangular parallelepiped. The first region 30 is a region at one end of the first flow path 22, and the second region 32 is a region at the other end of the first flow path 22.

  The second flow path 24 has a third region 34. The second flow path 24 is a linear flow path. The shape of the second flow path 24 is, for example, a rectangular parallelepiped. The third region 34 is a region at one end of the second flow path 24. The second flow path 24 is connected to the first flow path 22. In the illustrated example, the first channel 22 and the second channel 24 are connected to each other at the other end of the first channel 22 and the other end of the second channel 24. Yes. The first flow path 22 is connected to the second flow path 24 in the second region 32.

  The second flow path 24 is bent and connected to the first flow path 22. That is, the extending direction of the first flow path 22 and the extending direction of the second flow path 24 are not parallel to each other, and the flow path 20 is formed by connecting the first flow path 22 and the second flow path 24. And having a bent portion. In the illustrated example, the extending direction of the first flow path 22 and the extending direction of the second flow path 24 are orthogonal to each other, and the flow path 20 has an L shape (substantially L shape). doing. The angle θ formed by the extending direction of the first flow path 22 and the extending direction of the second flow path 24 may be not less than 70 ° and not more than 110 °.

  As shown in FIG. 2, the flow path 20 is sandwiched between a first inner wall 10a and a second inner wall 10b that faces the first inner wall 10a. The first inner wall 10 a and the second inner wall 10 b are inner walls of the container body 10. The flow path 20 is formed by the inner walls 10a and 10b. The distance D between the first inner wall 10a and the second inner wall 10b is such a length that the reaction liquid 6 contacts the first inner wall 10a and the second inner wall 10b when the reaction liquid 6 is introduced into the flow path 20. is there. Specifically, the distance D is 0.5 mm or more and 3 mm or less. By making the distance D 0.5 mm or more, it can be suppressed that the manufacture of the nucleic acid amplification reaction vessel 100 becomes difficult. By setting the distance D to 3 mm or less, the heat of the heating part can be sufficiently transferred to the reaction solution 6.

  The nucleic acid amplification reaction container 100 is a container for performing the nucleic acid amplification reaction by reciprocating the reaction solution 6 between the first region 30 and the second region 32. Furthermore, the nucleic acid amplification reaction vessel 100 moves the reaction solution 6 to the third region 34 after reciprocating the reaction solution 6 between the first region 30 and the second region 32, and fluorescence from the reaction solution 6. It is a container for detecting.

1.2. First Reagent First reagent 2 is arranged in second region 32 as shown in FIG. The first reagent 2 is arranged in a state of being separated from the second reagent 4. The first reagent 2 may be introduced into the second region 32 from the introduction port 33. For example, the first reagent 2 is disposed in a lyophilized state (a lyophilized state). Thereby, the first reagent 2 can be attached to the inner wall of the container body 10 that defines the second region 32. The first reagent 2 is a reagent for amplifying nucleic acid. The first reagent 2 includes, for example, a primer, a polymerase, dNTP, and a buffer.

  The primer is designed to anneal to the template nucleic acid. Annealing means that a primer binds to DNA. The first reagent 2 is a forward primer (Forward Primer) that anneals to one single-stranded template nucleic acid (single-stranded DNA) as a primer after the double-stranded template nucleic acid (double-stranded DNA) is denatured. And a reverse primer that anneals to the other single-stranded DNA. The concentrations of the forward primer and the reverse primer contained in the reaction solution 6 mixed with the second reagent 4 are, for example, 0.4 μM or more and 12.8 μM or less, preferably 1.6 μM or more and 9.6 μM or less, respectively. .

  Although it does not specifically limit as a polymerase, DNA polymerase is mentioned. The DNA polymerase polymerizes a nucleotide complementary to the base of the template nucleic acid at the end of the primer annealed to the template nucleic acid having a single-stranded structure (single-stranded DNA). As the DNA polymerase, a heat-resistant enzyme or a PCR enzyme is preferable. For example, there are a large number of commercially available products such as Taq polymerase, KOD polymerase, Tfi polymerase, Tth polymerase, or improved versions thereof. A DNA polymerase that can be used is preferred. The amount of polymerase contained in the reaction solution 6 mixed with the second reagent 4 is, for example, 0.5 U or more.

  dNTP represents a mixture of four types of deoxyribonucleotide triphosphates. That is, dNTP represents a mixture of dATP, dCTP, dGTP, and dTTP. DNA polymerase joins dATP, dCTP, dGTP, and dTTP to the ends of the annealed primers to form new DNA (extension reaction). The concentration of dNTP contained in the reaction solution 6 mixed with the second reagent 4 is, for example, 0.06 mM or more and 0.75 mM or less, preferably 0.125 mM or more and 0.5 mM or less.

  The buffer is, for example, a buffer containing a salt. Examples of the salt contained in the buffer include salts of Tris, Hepes, Pipes, phosphoric acid and the like. With these salts, the pH of the buffer can be adjusted. The buffer contains a divalent cation. Examples of the divalent cation include Mn, Co, and Mg. The concentration of the divalent cation contained in the reaction solution 6 mixed with the second reagent 4 is, for example, 2 mM or more and 7.5 mM or less.

  When RNA is used as the template nucleic acid, the first reagent 2 further includes a reverse transcriptase. Examples of the reverse transcriptase include Avian Myeloblast Virus, Ras Associated Virus Type 2 (Ras Associated Virus Type 2), Mouse Moloney Murine Leukemia Virus (Mouse Molly Murine Leukemia Immunodeficiency) Reverse transcriptase derived from virus type 1 (Human Immunodefective Virus type 1) is used.

  When the first reagent 2 is lyophilized, the first reagent 2 contains a sugar. Examples of the sugar include non-reducing sugars such as sucrose, trehalose, raffinose, and melezitose among disaccharides and trisaccharides. Among disaccharides and trisaccharides, trehalose, in particular, has a high function as a cryoprotectant and prevents the lyophilized reagent from coming into contact with water molecules due to its strong hydration power, thus improving the storage stability of the lyophilized reagent. Can be improved. The lyophilized reagent can be prepared by lyophilizing a mixed reagent solution containing each component of the first reagent 2. The temperature at the time of freeze-drying is, for example, about −80 ° C. In addition, when the 2nd reagent 4 is freeze-dried, the 2nd reagent 4 may also contain saccharide | sugar.

1.3. Second Reagent Second reagent 4 is arranged in third region 34. The second reagent 4 may be introduced into the third region 34 from an inlet (not shown) communicating with the third region 34. The introduction port may be closed by a lid (not shown) after the second reagent 4 is introduced. For example, the second reagent 4 is disposed in a lyophilized state. Thereby, the second reagent 4 can be attached to the inner wall of the container body 10 that defines the third region 34. The second reagent 4 is a reagent for quantifying the nucleic acid amplification amount. The second reagent 4 includes, for example, a probe and a polymerase.

  The probe is a fluorescently labeled probe used for quantifying the amount of nucleic acid amplification. At least a portion defining the third region 34 of the container body 10 is made of a material that can transmit fluorescence from the reaction solution 6 mixed with the probe and excitation light for obtaining fluorescence. When the second reagent 4 is mixed with the reaction solution 6, the concentration of the probe contained in the reaction solution 6 is 0.5 μM or more and 7.2 μM or less, preferably 0.9 μM or more and 3.6 μM or less.

  The probe may be a hydrolysis probe comprising a reporter dye and a quencher dye. Specifically, the probe may be a TaqMan (registered trademark) probe. While the hydrolysis probe is hybridizing to single-stranded DNA to form a double-stranded structure, the reporter dye is emitted by the quencher dye in close proximity to the reporter dye (by the quenching effect). Is suppressed. However, when the probe is degraded by the exonuclease activity by polymerase, the quenching effect is eliminated and the reporter dye emits light. By this luminescence, the amount of nucleic acid amplification can be quantified. Hybridization means that a probe binds to DNA. When a hydrolysis probe is used as the probe, a polymerase having 5′-3 ′ exonuclease activity is used as the polymerase.

  The probe may be a non-hydrolyzed probe other than the hydrolyzed probe. Specifically, the probe may be a Q (Quenching) probe using a fluorescence quenching phenomenon. Specifically, the probe may be SYBR Green I. The Q probe emits light in a state where it is not hybridized to single-stranded DNA, and is quenched when hybridized to single-stranded DNA. The amount of nucleic acid amplification can be quantified based on the difference in emission intensity. When a Q probe is used as a probe, a polymerase having no 5′-3 ′ exonuclease activity is used as a polymerase. An example of a polymerase that does not have 5′-3 ′ exonuclease activity is KOD polymerase, but this enzyme has a higher rate of extension reaction than Taq polymerase, which is generally used, and speeds up thermal cycle. Can be achieved.

  For example, Taq polymerase is used as the polymerase contained in the second reagent 4 when a hydrolysis probe is used as the probe contained in the second reagent 4. When a Q probe is used as the probe contained in the second reagent 4, for example, KOD polymerase is used. In addition, the 2nd reagent 4 does not contain a polymerase and may be comprised only by the probe.

  The nucleic acid amplification reaction vessel 100 has, for example, the following characteristics.

  The nucleic acid amplification reaction vessel 100 is a region different from the first region 30 and the second region 32, and is located in a region other than the path where the reaction solution 6 reciprocates between the first region 30 and the second region 32. There are three regions 34, and the second reagent 4 is arranged in the third region 34. Therefore, in the nucleic acid amplification reaction vessel 100, when the nucleic acid amplification reaction is performed by reciprocating the reaction solution 6 between the first region 30 and the second region 32, the reaction solution 6 contacts the second reagent 4. Can be suppressed. Therefore, in the nucleic acid amplification reaction vessel 100, it is not necessary to open the lid of the nucleic acid amplification reaction vessel 100 and introduce the second reagent 4 into the nucleic acid amplification reaction vessel 100 after performing the nucleic acid amplification reaction. Therefore, in the nucleic acid amplification reaction vessel 100, it is possible to reduce the possibility that foreign matter is mixed. Furthermore, the operation can be simplified as compared with the case where the second reagent 4 is introduced into the container by opening the lid after the nucleic acid amplification reaction.

  In the nucleic acid amplification reaction vessel 100, the second reagent 4 includes a probe. Therefore, in the nucleic acid amplification reaction vessel 100, it is possible to suppress the probe from inhibiting the nucleic acid amplification reaction.

  In the nucleic acid amplification reaction container 100, the first reagent 2 is disposed in the second region 32, and the first reagent 2 includes a primer, a polymerase, and dNTP. Therefore, the nucleic acid amplification reaction vessel 100 can perform the nucleic acid amplification reaction using the first reagent 2.

  In the nucleic acid amplification reaction vessel 100, the first reagent 2 is placed in a lyophilized state. Therefore, in the nucleic acid amplification reaction container 100, the first reagent 2 can be attached to the inner wall of the container body 10 that defines the second region 32, and the first reagent 2 is disposed in a liquid state. As compared with the above, the first reagent 2 and the second reagent 4 can be arranged in a state of being more reliably separated from each other. Furthermore, in the nucleic acid amplification reaction vessel 100, the storage stability of the first reagent 2 can be enhanced.

  In the nucleic acid amplification reaction vessel 100, the second reagent 4 is disposed in a lyophilized state. Therefore, in the nucleic acid amplification reaction container 100, the second reagent 4 can be attached to the inner wall of the container body 10 that defines the third region 34, and the second reagent 4 is arranged in a liquid state. As compared with the above, the first reagent 2 and the second reagent 4 can be arranged in a state of being more reliably separated from each other. Furthermore, in the nucleic acid amplification reaction vessel 100, the second reagent 4 is transferred to the first channel while the reaction solution 6 is reciprocated between the first region 30 and the second region 32 to perform the nucleic acid amplification reaction. It can suppress falling to 22. Furthermore, in the nucleic acid amplification reaction vessel 100, the storage stability of the second reagent 4 can be enhanced.

  In the nucleic acid amplification reaction vessel 100, the second reagent 4 includes a polymerase. Therefore, in the nucleic acid amplification reaction vessel 100, for example, the probe can be more reliably decomposed than when the probe is decomposed only by the polymerase contained in the first reagent 2.

  In the nucleic acid amplification reaction container 100, the flow path 20 has a first flow path 22 having a first area 30 and a second area 32, and a third area 34, and is bent and connected to the first flow path 22. And a second flow path 24. Therefore, in the nucleic acid amplification reaction vessel 100, while the reaction solution 6 is reciprocated between the first region 30 and the second region 32 and the nucleic acid amplification reaction is performed, the reaction solution 6 is transferred to the third region 34. The possibility of moving to can be reduced.

  In the nucleic acid amplification reaction vessel 100, the flow path 20 is sandwiched between the first inner wall 10a and the second inner wall 10b facing the first inner wall 10a, and the distance D between the first inner wall 10a and the second inner wall 10b. Is the length that the reaction solution 6 contacts the first inner wall 10a and the second inner wall 10b when the reaction solution 6 is introduced into the flow path 20. Therefore, in the nucleic acid amplification reaction vessel 100, the heat of the heating unit can be sufficiently transferred to the reaction solution 6.

  In addition, the component which the 1st reagent 2 and the 2nd reagent 4 contain is not limited to said example. For example, the first reagent 2 may be a reagent for performing the first PCR in the nested PCR method, and the second reagent 4 is a reagent for performing the second PCR in the nested PCR method. Also good. The second reagent 4 may not include a probe.

2. Next, the nucleic acid amplification reaction apparatus according to the present embodiment will be described with reference to the drawings. FIG. 3 is a cross-sectional view schematically showing a nucleic acid amplification reaction apparatus 200 according to this embodiment. 4 is a cross-sectional view taken along the line IV-IV in FIG. 3 schematically showing the nucleic acid amplification reaction apparatus 200 according to the present embodiment. FIG. 5 is a functional block diagram of the nucleic acid amplification reaction apparatus 200 according to this embodiment.

  The nucleic acid amplification reaction apparatus according to the present invention can be equipped with the nucleic acid amplification reaction container according to the present invention. Below, the nucleic acid amplification reaction apparatus 200 which can mount | wear with the nucleic acid amplification reaction container 100 as a nucleic acid amplification reaction container concerning this invention is demonstrated.

  As shown in FIGS. 3 to 5, the nucleic acid amplification reaction apparatus 200 includes a first heating unit 40, a second heating unit 42, a third heating unit 44, a drive mechanism 50, a detection mechanism 60, and a processing unit. 70, an operation unit 72, a display unit 74, and a storage unit 76. The nucleic acid amplification reaction apparatus 200 may include a nucleic acid amplification reaction vessel 100.

  3 and 4, the drive mechanism 50, the detection mechanism 60, the processing unit 70, the operation unit 72, the display unit 74, and the storage unit 76 are not illustrated. Further, in FIG. 5, illustration of the nucleic acid amplification reaction vessel 100 and the heating units 40, 42, 44 is omitted.

  The first heating unit 40, the second heating unit 42, and the third heating unit 44 are provided in contact with the nucleic acid amplification reaction vessel 100. The heating units 40, 42, and 44 are aluminum heat blocks that transmit heat generated from a heater (not shown) to the nucleic acid amplification reaction vessel 100, for example. The temperature of the heating units 40, 42, 44 may be controlled by a temperature sensor and processing unit 70 (not shown).

  The first heating unit 40 heats the first region 30 of the flow path 20 to the first temperature. For example, the first heating unit 40 heats the reaction liquid 6 located in the first region 30 to the first temperature. The first heating unit 40 is provided with an opening 40a. In the illustrated example, the opening 40a is a hole with a bottom. The first region 30 is a region inserted into the opening 40a. The opening 40a is a mounting portion to which the nucleic acid amplification reaction vessel 100 can be mounted.

  The second heating unit 42 heats the second region 32 of the flow path 20 to the second temperature. For example, the second heating unit 42 heats the reaction solution 6 located in the second region 32 to the second temperature. The second heating unit 42 is provided with an opening 42a. In the illustrated example, the opening 42a is a through-hole penetrating the second heating unit 42 and has a bent shape. The second region 32 is a region inserted into the opening 42a. The opening 42a is a mounting part to which the nucleic acid amplification reaction vessel 100 can be mounted.

  The third heating unit 44 heats the third region 34 of the flow path 20 to the third temperature. For example, the third heating unit 44 heats the reaction liquid 6 located in the third region 34 to the third temperature. The third heating unit 44 is provided with an opening 44a. In the illustrated example, the opening 44a is a hole with a bottom. The third region 34 is a region inserted into the opening 44a. The opening 44a is a mounting part to which the nucleic acid amplification reaction vessel 100 can be mounted. The third heating unit 44 is provided with an opening 44b. The opening 44b communicates with the opening 44a. The opening 44 b is an opening for guiding excitation light from a detection mechanism 60 described later to the third region 34 and guiding fluorescence from the reaction liquid 6 mixed with the probe to the detection mechanism 60.

  The drive mechanism 50 is a mechanism that moves the nucleic acid amplification reaction vessel 100 and the heating units 40, 42, and 44 so that the reaction solution 6 moves to the first region 30, the second region 32, and the third region 34. For example, the drive mechanism 50 moves the nucleic acid amplification reaction vessel 100 and the heating units 40, 42, 44 based on an input signal from the processing unit 70. Although not shown, the drive mechanism 50 may include a support unit that supports the nucleic acid amplification reaction vessel 100 and the heating units 40, 42, and 44, and a motor that moves the support unit.

  The detection mechanism 60 is a mechanism for detecting fluorescence from the reaction solution 6 mixed with the probe. For example, based on an input signal from the processing unit 70, the detection mechanism 60 irradiates the reaction liquid 6 mixed with the probe with excitation light, and detects fluorescence from the reaction liquid 6. The detection mechanism 60 is not particularly limited as long as fluorescence can be detected. For example, a known fluorescence measuring device is used.

  The processing unit 70 performs processing for controlling the drive mechanism 50 and the detection mechanism 60 in accordance with, for example, a program stored in the storage unit 76. The processing unit 70 is realized by a processor such as a CPU (Central Processing Unit), for example.

  The operation unit 72 performs a process of acquiring an operation signal corresponding to an operation by the user and sending the operation signal to the processing unit 70. The operation unit 72 is realized by, for example, a button, a key, a touch panel display, a microphone, or the like.

  The display unit 74 displays the image generated by the processing unit 70. The display unit 74 is realized by, for example, an LCD (Liquid Crystal Display), a CRT (Cathode Ray Tube), or the like.

  The storage unit 76 stores programs, data, and the like for the processing unit 70 to perform various calculation processes and control processes. The storage unit 76 is used as a work area of the processing unit 70, and is also used for temporarily storing calculation results and the like executed by the processing unit 70 according to various programs. The storage unit 76 is realized by, for example, a RAM (Random Access Memory).

  In the nucleic acid amplification reaction apparatus 200, since the nucleic acid amplification reaction vessel 100 can be mounted, the possibility of foreign matters being mixed can be reduced.

3. Nucleic Acid Amplification Reaction Method Next, a nucleic acid amplification reaction method according to this embodiment will be described with reference to the drawings. FIG. 6 is a flowchart for explaining the nucleic acid amplification reaction method according to this embodiment. 7 to 9 are cross-sectional views for explaining the nucleic acid amplification reaction method according to this embodiment. For convenience, the drive mechanism 50, the detection mechanism 60, the processing unit 70, the operation unit 72, the display unit 74, and the storage unit 76 are not shown in FIGS. Moreover, in FIGS. 7-9, the direction (gravity action direction) where gravity acts is shown by the arrow g. Below, the nucleic acid amplification reaction method using the nucleic acid amplification reaction apparatus 200 equipped with the nucleic acid amplification reaction vessel 100 will be described as an example.

  First, the lid (not shown) of the nucleic acid amplification reaction vessel 100 is opened, the template nucleic acid solution is introduced into the second region 32 of the flow path 20, and the template nucleic acid solution and the first reagent 2 are brought into contact with each other. As shown in FIG. 7, a reaction solution 6 is prepared (step S1).

  Next, the nucleic acid amplification reaction vessel 100 is attached to the openings 40a, 42a, 44a of the heating units 40, 42, 44 (step S2). In this step, the arrangement of the nucleic acid amplification reaction vessel 100 is the first arrangement. In the first arrangement, as shown in FIG. 7, the second region 32 is located below the first region 30 and the third region 34 in the gravity action direction. Therefore, the reaction solution 6 is located in the second region 32.

  Next, when the processing unit 70 receives a signal to start processing from the operation unit 72, the processing unit 70 controls the heating units 40, 42, and 44 to set the first region 30 to the first temperature and set the second region 32 to the second region 32. A process of forming a temperature gradient in the flow path 20 is performed using the second temperature and the third region 34 as the third temperature (step S3). By this step, a temperature gradient in which the temperature gradually changes between the first temperature and the second temperature is formed between the first region 30 and the second region 32. Furthermore, a temperature gradient in which the temperature gradually changes between the second temperature and the third temperature is formed between the second region 32 and the third region 34. In this step, since the reaction solution 6 is located in the second region 32, it is heated to the second temperature. Thereby, a denaturation reaction can be performed on the reaction solution 6.

  Next, when the first time (first period) has elapsed after the second heating unit 42 reaches the second temperature, the processing unit 70 controls the drive mechanism 50 to arrange the nucleic acid amplification reaction vessel 100. Is switched from the first arrangement to the second arrangement (step S4). The processing unit 70 may incorporate a timer. The first time is a heating time for the denaturation reaction, and is, for example, 1 second or more and 10 seconds or less.

  In the second arrangement, as shown in FIG. 8, the first region 30 is located below the second region 32 and the third region 34 in the gravitational action direction. Therefore, the reaction solution 6 moves from the second region 32 to the first region 30. The reaction solution 6 is heated to the first temperature in the first region 30. Thereby, the primer annealing reaction and the extension reaction can be performed on the reaction solution 6. The processing unit 70 stops the operation of the drive mechanism 50 for the second time (second period) after the arrangement of the nucleic acid amplification reaction vessel 100 is changed to the second arrangement. Thereby, the arrangement of the nucleic acid amplification reaction vessel 100 is held in the second arrangement for the second time. The second time is a heating time for the annealing reaction and the extension reaction, and is 1 second or more and 10 seconds or less.

  Next, the processing unit 70 performs a process of determining whether or not the number of times of switching from the first arrangement to the second arrangement (the number of cycles) has reached a predetermined number stored in the storage unit 76 in advance (Step S70). S5). Each time the processing unit 70 switches from the first arrangement to the second arrangement, the processing unit 70 stores the number of cycles in the storage unit 76, and the cycle number and a predetermined number of times stored in the storage unit 76 in advance. Compare. The predetermined number of times stored in advance in the storage unit 76 is not particularly limited, and is, for example, 20 times or more and 60 times or less.

  If the processing unit 70 determines in step S5 that the number of cycles has reached a predetermined number (in the case of “Yes” in FIG. 6), the processing unit 70 proceeds to step S7.

  On the other hand, if the processing unit 70 determines in step S5 that the number of cycles has not reached the predetermined number (in the case of “No” in FIG. 6), the processing unit 70 proceeds to step S6. In step S <b> 6, the processing unit 70 controls the drive mechanism 50 to perform processing for switching the arrangement of the nucleic acid amplification reaction vessel 100 from the second arrangement to the first arrangement. For example, the processing unit 70 stops the operation of the drive mechanism 50 for a first time after setting the arrangement of the nucleic acid amplification reaction vessel 100 to the first arrangement.

  And the process part 70 transfers to step S4 again, and performs the process which switches arrangement | positioning of the nucleic acid amplification reaction container 100 from 1st arrangement to 2nd arrangement.

  As described above, the processing unit 70 performs a process of switching the arrangement of the nucleic acid amplification reaction vessel 100 between the first arrangement and the second arrangement until the number of cycles reaches a predetermined number. As a result, the reaction solution 6 can be reciprocated between the first region 30 and the second region 32 to give a thermal cycle to the reaction solution 6 and a nucleic acid amplification reaction can be performed (nucleic acid amplification reaction). Process).

  Next, the processing unit 70 controls the drive mechanism 50 to perform processing for switching the arrangement of the nucleic acid amplification reaction vessel 100 from the second arrangement to the first arrangement (step S7). Thereby, the reaction solution 6 can be moved to the second region 32 as shown in FIG.

  Next, the processing unit 70 controls the drive mechanism 50 to perform processing for switching the arrangement of the nucleic acid amplification reaction vessel 100 from the first arrangement to the third arrangement (step S8). In the third arrangement, as shown in FIG. 9, the third region 34 is located below the first region 30 and the second region 32 in the direction of gravity action. Therefore, the reaction solution 6 moves from the second region 32 to the third region 34. Thereby, the reaction liquid 6 can be moved to the 3rd area | region 34, and the 2nd reagent 4 can be mixed with the reaction liquid 6 (mixing process). The reaction solution 6 is heated to the third temperature. The processing unit 70 may stop the operation of the driving mechanism 50 for a third time (third period) after the arrangement of the nucleic acid amplification reaction vessel 100 is changed to the third arrangement. The third time is, for example, a time for reliably mixing the reaction solution 6 and the second reagent 4.

  Next, the processing unit 70 controls the drive mechanism 50 to perform processing for switching the arrangement of the nucleic acid amplification reaction vessel 100 from the third arrangement to the first arrangement (step S9). Thereby, as shown in FIG. 7, the reaction liquid 6 can be moved to the 2nd area | region 32 (1st movement process). The reaction solution 6 is heated at the second temperature in the second region 32. Thereby, a denaturation reaction can be performed on the reaction solution 6. The processing unit 70 stops the operation of the driving mechanism 50 for the first time after the arrangement of the nucleic acid amplification reaction vessel 100 is changed to the first arrangement.

  Next, the processing unit 70 controls the drive mechanism 50 to perform processing for switching the arrangement of the nucleic acid amplification reaction vessel 100 from the first arrangement to the third arrangement (step S10). Thereby, as shown in FIG. 9, the reaction liquid 6 can be moved to the 3rd area | region 34 (2nd transfer process). The reaction solution 6 is heated at the third temperature in the third region 34. Thereby, for example, hybridization of the probe and decomposition of the probe by the polymerase can be performed on the reaction solution 6. The processing unit 70 stops the operation of the drive mechanism 50 for the fourth time (fourth period) after the arrangement of the nucleic acid amplification reaction vessel 100 is changed to the third arrangement. The fourth time is, for example, a time period for performing probe hybridization and probe degradation by polymerase and detecting fluorescence from the reaction solution 6.

  Next, the processing unit 70 controls the detection mechanism 60 to irradiate the reaction solution 6 with excitation light for detecting fluorescence, and obtains fluorescence intensity from the detection mechanism 60 as a measurement result of the detection mechanism 60. Is performed (step S11). Thereby, the fluorescence from the reaction solution 6 can be detected (detection step). The processing unit 70 may perform the process for irradiating the excitation light at the same time as starting the process of step S10. The processing unit 70 may read the amplification curve stored in advance in the storage unit 76 and perform a process of calculating the nucleic acid amplification amount based on the amplification curve and the acquired fluorescence intensity. The processing unit 70 may perform processing for causing the display unit 74 to display the calculated nucleic acid amplification amount. Then, the processing unit 70 ends the process.

  Here, FIG. 10 is a cross-sectional view taken along line XX of FIG. 9 schematically showing the nucleic acid amplification reaction apparatus 200. As shown in FIG. 10, in the third arrangement, the third region 34 is at a position facing the detection mechanism 60. Excitation light from the detection mechanism 60 reaches the reaction solution 6 through the opening 44b, and fluorescence from the reaction solution 6 reaches the detection mechanism 60 through the opening 44b.

  In the nucleic acid amplification reaction method, since the nucleic acid amplification reaction vessel 100 is used, it is possible to reduce the possibility of contamination.

  In the above example, in the second transition step (step S10), the processing unit 70 performs a process of controlling the drive mechanism 50 to switch the arrangement of the nucleic acid amplification reaction vessel 100 from the first arrangement to the third arrangement. However, the processing unit 70 may perform a process of switching the arrangement of the nucleic acid amplification reaction vessel 100 from the first arrangement to the second arrangement by controlling the drive mechanism 50. Thereby, the reaction solution 6 can be moved to the first region 30, and the reaction solution 6 is heated at the first temperature in the first region 30. Then, for example, probe hybridization and probe decomposition by polymerase can be performed on the reaction solution 6. In this case, in the second arrangement, the first region 30 is at a position facing the detection mechanism 60, and the processing unit 70 controls the detection mechanism 60 to cause the reaction solution 6 to detect excitation light for detecting fluorescence. The process of irradiating is performed. Also. In this case, the third heating unit 44 may not be provided.

4). 4. Modification of nucleic acid amplification reaction vessel 4.1. First Modification Next, a nucleic acid amplification reaction container according to a first modification of the present embodiment will be described with reference to the drawings. FIG. 11 is a cross-sectional view schematically showing a nucleic acid amplification reaction vessel 110 according to a first modification of the present embodiment.

  Hereinafter, in the nucleic acid amplification reaction container 110 according to the first modified example of the present embodiment, members having the same functions as those of the constituent members of the nucleic acid amplification reaction container 100 according to the present embodiment described above are denoted by the same reference numerals, Detailed description thereof is omitted. This is the same in the nucleic acid amplification reaction containers according to the second to fourth modifications according to the present embodiment described below.

  In the nucleic acid amplification reaction vessel 100 described above, as shown in FIG. 1, at the other end of the first flow channel 22 and the other end of the second flow channel 24, the first flow channel 22 and The second flow paths 24 were connected to each other. In the nucleic acid amplification reaction container 100, the first flow path 22 is connected to the second flow path 24 in the second region 32. The flow path 20 had an L shape (substantially L shape).

  On the other hand, in the nucleic acid amplification reaction vessel 110, as shown in FIG. 11, the first channel 22 and the first channel 22 are formed at the center of the first channel 22 and the other end of the second channel 24. The two flow paths 24 are connected to each other. The first flow path 22 is connected to the second flow path 24 in a region different from the first region 30 and the second region 32. The channel 20 has a T-shape (substantially T-shape).

  In the nucleic acid amplification reaction method using the nucleic acid amplification reaction vessel 110, the reaction solution 6 is reciprocated between the first region 30 and the second region 32 to perform the nucleic acid amplification reaction. Next, the reaction solution 6 is moved to the third region 34, and the second reagent 4 is mixed with the reaction solution 6. Next, the reaction solution 6 is moved to the second region 32. Next, the reaction solution 6 is moved to the third region 34. Next, the fluorescence from the reaction solution 6 is detected. The above steps can be performed using the nucleic acid amplification reaction apparatus according to the present invention to which the nucleic acid amplification reaction vessel 110 can be attached. This is the same in the nucleic acid amplification reaction containers according to the second to fourth modifications according to this embodiment shown below.

4.2. Second Modified Example Next, a nucleic acid amplification reaction container according to a second modified example of the present embodiment will be described with reference to the drawings. FIG. 12 is a cross-sectional view schematically showing a nucleic acid amplification reaction vessel 120 according to a second modification of the present embodiment.

  The nucleic acid amplification reaction vessel 100 described above has an L-shaped (substantially L-shaped) cross-sectional shape as shown in FIG.

  On the other hand, the nucleic acid amplification reaction vessel 120 has a polygonal cross-sectional shape, specifically, a triangular cross-sectional shape, as shown in FIG. In the illustrated example, the nucleic acid amplification reaction vessel 120 has a cross-sectional shape of an isosceles triangle. Among the isosceles triangles, the sides 122 and 124 are variations having the same length, and the side 126 is a side connecting the sides 122 and 124. The sides 122, 124, and 126 are formed by the inner surface of the container body 10. In the illustrated example, the first region 30 is defined by the sides 124 and 126. The second region 32 is defined by the sides 122 and 126. The third region 34 is defined by the sides 122 and 124. Although not shown, the side of the nucleic acid amplification reaction vessel 120 having a polygonal cross-sectional shape may be curved.

4.3. Third Modification Next, a nucleic acid amplification reaction container according to a third modification of the present embodiment will be described with reference to the drawings. FIG. 13 is a cross-sectional view schematically showing a nucleic acid amplification reaction vessel 130 according to a third modification of the present embodiment.

  The nucleic acid amplification reaction vessel 100 described above has an L-shaped (substantially L-shaped) cross-sectional shape as shown in FIG.

  On the other hand, the nucleic acid amplification reaction vessel 130 has a polygonal cross-sectional shape as shown in FIG. 13, specifically, a quadrangular cross-sectional shape. In the illustrated example, the nucleic acid amplification reaction vessel 130 has a square cross-sectional shape. In the illustrated example, the first region 30, the second region 32, and the third region 34 are defined by square corners. The first region 30 and the third region 34 are located on a diagonal line. Although not shown, the side of the nucleic acid amplification reaction vessel 130 having a polygonal cross-sectional shape may be curved.

4.4. Fourth Modified Example Next, a nucleic acid amplification reaction container according to a fourth modified example of the present embodiment will be described with reference to the drawings. FIG. 14 is a cross-sectional view schematically showing a nucleic acid amplification reaction vessel 140 according to a fourth modification of the present embodiment.

  In the nucleic acid amplification reaction vessel 100 described above, as shown in FIG. 1, the first region 30, the second region 32, and the third region 34 are not arranged in a straight line.

  On the other hand, in the nucleic acid amplification reaction container 140, as shown in FIG. 14, the first region 30, the second region 32, and the third region 34 are arranged on a straight line. The nucleic acid amplification reaction container 140 has, for example, a rectangular parallelepiped shape. In the illustrated example, the first region 30 is provided on one end side of the nucleic acid amplification reaction vessel 140, the third region 34 is provided on the other end side of the nucleic acid amplification reaction vessel 140, The second region 32 is located between the third region 34.

  The nucleic acid amplification reaction container 140 has a protrusion 142 that protrudes from the inner surface of the container body 10 between the second region 32 and the third region 34. The protrusion 142 protrudes from the inner surface of the container body 10 in a direction orthogonal to the direction from the second region 32 to the third region 34. The second region 32 is provided adjacent to the protrusion 142.

  In the nucleic acid amplification reaction vessel 140, the reaction solution 6 is transferred to the third region by the protrusion 142 during the nucleic acid amplification reaction while the reaction solution 6 is reciprocated between the first region 30 and the second region 32. 34 can be prevented. When moving the reaction solution 6 to the third region 34, the nucleic acid amplification reaction vessel 140 is moved so that the reaction solution 6 gets over the protrusion 142.

  Note that the nucleic acid amplification reaction container 140 may have a protruding portion 144 that protrudes toward the first region 30 from the tip of the protruding portion 142, as shown in FIG. The second region 32 may be defined by the protrusion 142 and the protrusion 144. This ensures that the reaction solution 6 moves to the third region 34 during the nucleic acid amplification reaction by reciprocating the reaction solution 6 between the first region 30 and the second region 32. Can be prevented.

  In the present invention, a part of the configuration may be omitted within a range having the characteristics and effects described in the present application, or each embodiment or modification may be combined.

  The present invention includes configurations that are substantially the same as the configurations described in the embodiments (for example, configurations that have the same functions, methods, and results, or configurations that have the same objects and effects). In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that exhibits the same operational effects as the configuration described in the embodiment or a configuration that can achieve the same object. Further, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

DESCRIPTION OF SYMBOLS 2 ... 1st reagent, 4 ... 2nd reagent, 6 ... Reaction liquid, 10 ... Container main body, 10a ... 1st inner wall, 10b ... 2nd inner wall, 20 ... Flow path, 22 ... 1st flow path, 24 ... 2nd 30 ... 1st area | region, 32 ... 2nd area | region, 34 ... 3rd area | region, 40 ... 1st heating part, 40a ... Opening part, 42 ... 2nd heating part, 42a ... Opening part, 44 ... 3rd heating 44a, 44b ... opening, 50 ... drive mechanism, 60 ... detection mechanism, 70 ... processing part, 72 ... operation part, 74 ... display part, 76 ... storage part, 100, 110, 120 ... nucleic acid amplification reaction vessel, 122, 124, 126 ... side, 130, 140 ... nucleic acid amplification reaction vessel, 142, 144 ... projection, 200 ... nucleic acid amplification reaction device

Claims (12)

A nucleic acid amplification reaction vessel for performing a nucleic acid amplification reaction by reciprocating a reaction solution containing a reagent between a first region and a second region,
A third region that is different from the first region and the second region, and is located in a region other than a path in which the reaction solution reciprocates between the first region and the second region;
A nucleic acid amplification reaction vessel in which a reagent is disposed in the third region. In claim 1,
The reagent arranged in the third region is a nucleic acid amplification reaction vessel containing a probe. In claim 1 or 2,
In the second region, a reagent is disposed,
The nucleic acid amplification reaction vessel, wherein the reagent arranged in the second region includes a primer, a polymerase, and dNTP. In claim 3,
The nucleic acid amplification reaction vessel, wherein the reagent arranged in the second region is arranged in a lyophilized state. In any one of Claims 1 thru | or 4,
The nucleic acid amplification reaction container, wherein the reagent arranged in the third region is arranged in a lyophilized state. In any one of Claims 1 thru | or 5,
The reagent arranged in the third region is a nucleic acid amplification reaction vessel containing a polymerase. In any one of Claims 1 thru | or 6,
Including a flow path through which the reaction solution can flow,
The flow path is
A first flow path having the first region and the second region;
A second channel having the third region and bent and connected to the first channel;
A nucleic acid amplification reaction vessel. In any one of Claims 1 thru | or 7,
Including a flow path through which the reaction solution can flow,
The flow path is sandwiched between a first inner wall and a second inner wall facing the first inner wall,
The distance between the first inner wall and the second inner wall is such a length that the reaction liquid contacts the first inner wall and the second inner wall when the reaction liquid is introduced into the flow path. Nucleic acid amplification reaction vessel. A mounting part to which the nucleic acid amplification reaction container according to any one of claims 1 to 8 can be mounted;
A first heating unit for heating the first region to a first temperature;
A second heating unit for heating the second region to a second temperature higher than the first temperature;
A drive mechanism for moving the nucleic acid amplification reaction vessel so that the reaction solution moves to the first region, the second region, and the third region;
A nucleic acid amplification reaction apparatus comprising: In claim 9,
A nucleic acid amplification reaction apparatus comprising a third heating unit for heating the third region to a third temperature lower than the second temperature. In claim 9 or 10,
A nucleic acid amplification reaction apparatus comprising a detection mechanism for detecting fluorescence from the reaction solution mixed with the reagent arranged in the third region. A nucleic acid amplification reaction method using the nucleic acid amplification reaction container according to any one of claims 1 to 8,
A nucleic acid amplification reaction step of performing a nucleic acid amplification reaction by reciprocating the reaction solution between the first region at a first temperature and the second region at a second temperature higher than the first temperature;
After the nucleic acid amplification reaction step, the reaction solution is moved to a third region having a third temperature lower than the second temperature, and the reagent arranged in the third region is mixed with the reaction solution. Process,
A first moving step of moving the reaction solution to the second region after the mixing step;
A second moving step of moving the reaction solution to the first region or the third region after the first moving step;
A detection step of detecting fluorescence from the reaction solution after the second movement step;
A nucleic acid amplification reaction method comprising:

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