JPWO2003004596A1 - Reaction vessel and reactor - Google Patents

Abstract

When the reaction solution is accommodated in the reaction chamber, centrifugation is not essential and the reaction can be automated, the temperature of the reaction solution accommodated in the reaction chamber can be quickly controlled, and the reaction solution accommodated in the reaction chamber can be controlled. A reaction vessel, a reaction apparatus, and a method capable of realizing monitoring of a reaction occurring in a reaction chamber in real time even if the amount of The temperature control of the reaction solution 4a accommodated in S1a is performed via the bottom plate portion 22a constituting the reaction vessel main body 2a and the pressing portion 32a constituting the lid member 3a, and the irradiation of the reaction solution 4a with excitation light and Detection of the fluorescence emitted from the reaction solution 4a is performed via the first side plate 23a constituting the reaction vessel main body 2a.

Description

Technical field
The present invention relates to a reaction vessel, a reaction apparatus, and a method capable of rapidly controlling the temperature of a reaction solution and monitoring the reaction in real time.
Background art
The polymerase chain reaction (hereinafter, referred to as “PCR”) is a technology that uses a thermostable polymerase and primers to amplify a target nucleic acid by raising or lowering the temperature. The fields include genetic engineering, biological test methods and detection methods. Widely used in
The principle of PCR is to maintain the temperature at which the double-stranded DNA containing the target DNA sequence dissociates into single strands at the first stage, and the temperature at which the forward and reverse primers anneal to the dissociated single-stranded DNA. The second step is repeated a number of times, and the third step of maintaining a temperature at which a DNA strand complementary to the single-stranded DNA is synthesized by the DNA polymerase is repeated a number of times according to the thermal profile (temperature rise / fall) set in the three steps. In this case, the target DNA is geometrically amplified.
For example, a reaction solution containing a double-stranded DNA containing a target DNA sequence, an excessive amount of a pair of primers, and a thermostable polymerase is subjected to one cycle of 95 ° C. for 30 seconds, 65 ° C. for 30 seconds, and 72 ° C. for 1 minute. The reaction can be performed for 30 to 40 cycles to allow the PCR to proceed. At 95 ° C., the double-stranded DNA dissociates into single-stranded DNA. Next, when the primer is cooled to an appropriate temperature (65 ° C. in the above example) according to the base sequence of the primer, the primer and the single-stranded DNA are annealed. Next, when the reaction temperature of the polymerase is increased (72 ° C. in the above example), the DNA synthesis reaction by the polymerase proceeds.
As described above, since temperature control of a reaction solution is important in PCR, PCR is usually carried out using a thermostat that can be programmed for temperature control and a reaction vessel that can be used for the device.
Most commonly, a microtube is brought into close contact with a hole in a metal block equipped with a heating / cooling device, and the reaction solution in the microtube is heated (dissociation of double-stranded DNA) through the metal block, A device that repeats a cycle of cooling (annealing of primer) and heating (extension reaction by polymerase) is used. There are two types of metal block cooling systems, one using a compressor and the other using a Peltier cooling system. Recently, rather than using a metal block, the reaction solution in the microtube is moved by moving the microtube along with the rack and sequentially immersing it in an independent liquid or solid phase incubator at three temperatures. There is also an apparatus which repeats a cycle of heating (dissociation of double-stranded DNA), cooling (annealing of primer), and heating (extension reaction by polymerase).
When the number of specimens is large, such as when PCR is performed for screening purposes, PCR of 96 specimens is performed at once using a PCR microtiter plate (96 wells) in order to process many specimens at once. Devices that can do so have also been developed.
In recent years, in order to efficiently process a large number of samples in genetic diagnosis and genome projects, preparation of samples containing target nucleic acids (eg, extraction of nucleic acids from cells), amplification of target nucleic acids by PCR, and progress of PCR A series of operations such as monitoring (for example, presence or absence of target nucleic acid amplification, amount of PCR amplification product, etc.) (for example, detection, measurement, qualitative analysis, quantitative analysis, etc.) are automated, and a large number of samples can be efficiently processed in parallel. The need for processing is increasing. In order to automate these series of operations and efficiently process a large number of samples in parallel, firstly, the time required for PCR should be reduced as much as possible, and secondly, the amount of samples required for PCR can be reduced. Third, it is necessary to monitor the progress of the PCR in real time (ie, immediately during the progress of the PCR).
However, the conventional PCR reaction apparatus and PCR reaction container perform PCR by typical temperature control such as 30 to 40 cycles of 95 ° C. for 30 seconds, 65 ° C. for 30 seconds, and 72 ° C. for 1 minute. Therefore, it is difficult to achieve the object of shortening the time required for PCR as much as possible using a conventional PCR reaction device and PCR reaction container. For example, when a conventional PCR reaction apparatus and PCR reaction vessel are used to perform 30 to 40 cycles at 95 ° C. for 30 seconds, 65 ° C. for 30 seconds, and 72 ° C. for 1 minute, the PCR is performed. It takes about one hour to complete.
In addition, in the conventional PCR reaction apparatus and PCR reaction container, if the amount of the sample (reaction liquid) is too small, the solvent (usually water) in the reaction liquid evaporates during the PCR, and the reaction stops. In some cases. This is because, in a reaction chamber where PCR proceeds (for example, a microtube, a well of a microtiter plate), the contact area between the air and the reaction solution is large, so that the environment in which the solvent in the reaction solution tends to evaporate; Since the temperature of the reaction chamber wall is not uniform and there is a portion of the reaction chamber wall lower than the temperature of the reaction solution (for example, the upper part of a microtube or the upper part of a well of a microtiter plate), the evaporated solvent This is due to liquefaction at a part. Therefore, it is difficult to achieve the object of minimizing the amount of the reaction solution using a conventional PCR reaction apparatus and PCR reaction vessel.
Under such circumstances, a device has been developed in which a small amount of reaction solution is sealed in a microcapillary with a large surface area and good thermal conductivity, and heating and cooling are performed with hot air from a halogen lamp or the like and cold air at room temperature. Was. As this type of apparatus, for example, a light cycler (LightCycler) (manufactured by Roche Molecular Biochemicals) is commercially available. In this apparatus, by using a microcapillary having a large surface area and good thermal conductivity, it is possible to control the temperature at about 20 ° C./sec. One cycle requires only about 30 to 60 seconds, and 30 cycles require 15 to 15 seconds. It can be completed in about 30 minutes. Further, by using a microcapillary, PCR using a small amount of a reaction solution of about 5 to 20 μl can be realized. Furthermore, the fluorescence emitted from the reaction solution in accordance with the amount of the PCR amplification product can be measured quickly and with high sensitivity due to the characteristics of the glass capillary, such as that most of the irradiation light is focused on the tip of the capillary. Can be measured in real time.
As described above, the PCR reaction device using the microcapillary as the PCR reaction container can shorten the time required for PCR by rapid temperature control of the reaction solution, and can reduce the amount of the reaction solution required for PCR to a very small amount. Since it is possible to measure the progress of PCR in real time, it is extremely useful when performing PCR alone.
However, in this PCR reaction apparatus, when enclosing the reaction solution in the microcapillary, the reaction solution is added to a plastic container placed above the glass capillary, sealed with a plastic stopper, and then centrifuged. Is required to be moved from the plastic container into the glass capillary, and then each capillary is removed from the centrifuge and installed in the reactor. Also, if air is mixed in when enclosing the reaction solution in the microcapillary, the air expands due to heating in the course of PCR, and the reaction solution moves inside the microcapillary, causing a decrease in PCR amplification efficiency and the like. Great care must be taken when enclosing the reaction solution in the capillary.
Therefore, a PCR reaction device using a microcapillary as a PCR reaction container can be used to prepare a sample containing a target nucleic acid (eg, extraction of a nucleic acid from a cell), amplify a target nucleic acid by PCR, and the progress of PCR (eg, , Monitoring of target nucleic acid amplification, the amount of PCR amplification product, etc.) is difficult to use for automation.
In addition, no matter which PCR reaction apparatus or PCR reaction vessel is used, it is possible to prevent the reaction (usually water) in the reaction solution from evaporating during the PCR and stopping the reaction. For this purpose, it is necessary to cover a reaction chamber (e.g., a microtube, a well of a microtiter plate) in which a PCR proceeds by sealing a lid material on a reaction vessel main body containing a reaction solution. Therefore, in the conventional PCR reaction device and PCR reaction container, it is necessary to temporarily remove the lid material from the reaction container body in order to obtain the amplified fragment obtained by PCR, and to amplify the target nucleic acid by PCR. It is difficult to automate the operation from to the acquisition of an amplified fragment.
Disclosure of the invention
Therefore, a first object of the present invention is to enable the reaction to be automated without the necessity of centrifugation when accommodating the reaction solution in the reaction chamber, and to quickly control the temperature of the reaction solution accommodated in the reaction chamber. The reaction can proceed even if the amount of the reaction solution contained in the reaction chamber is very small, and the reaction occurring in the reaction chamber is monitored in real time (that is, immediately during the progress of the reaction). To provide a reaction vessel, a reaction apparatus and a method that can be used.
In addition, the second object of the present invention can achieve the first object, and after performing the reaction in a state where the lid member is attached to the reaction container body, without detaching the lid member from the reaction container body. It is an object of the present invention to provide a reaction container, a reaction apparatus, and a method capable of obtaining a reaction product contained in a reaction solution in a reaction container.
(1) In order to achieve the above object, the reaction container of the present invention can have a reaction chamber body having an opening and having a reaction chamber capable of containing a reaction solution, and can seal the opening of the reaction chamber. A reaction container provided with a lid member, wherein the lid member and the reaction chamber are in contact with a reaction solution contained in the reaction chamber when the lid member is attached to the reaction container body. Having a surface, so that light can be transmitted from the reaction solution contained in the reaction chamber to the outside of the reaction vessel through the contact surface of the lid material, and the lid material is made of a light-transmitting material. The reaction vessel main body is formed of a light-transmissive material so that light can be transmitted from the reaction solution contained in the reaction chamber to the outside of the reaction vessel through the contact surface of the reaction chamber. It is characterized by comprising.
The reaction chamber provided in the reaction vessel main body has an opening and can accommodate a reaction solution. The reaction solution is added from the opening of the reaction chamber and accommodated in the reaction chamber. The reaction chamber is a place where a desired reaction is generated, and a reaction solution contained in the reaction chamber contains components necessary for generating the desired reaction, reagents necessary for measuring the progress of the reaction ( For example, a light-emitting substance such as a fluorescent dye) is included.
The structure of the reaction chamber is not particularly limited as long as it has an opening and can accommodate a reaction solution. Generally, the reaction chamber is formed in the reaction vessel main body as a concave portion having an opening at the upper end. The reaction chamber is preferably formed in the main body of the reaction vessel as a recess formed by a thin plate. In this case, by performing heat transfer between the outside of the reaction chamber and the reaction liquid in the reaction chamber through the thin plate, the temperature of the reaction liquid can be quickly and efficiently controlled. In addition, by performing irradiation of light to the reaction chamber and detection of light emitted from the reaction solution through the thin plate, setting of irradiation conditions and light receiving conditions becomes easy.
The reaction chamber does not need to have a structure like a capillary (capillary tube), and centrifugation is not indispensable when accommodating the reaction solution in the reaction chamber. In the reaction vessel of the present invention, when the lid material is attached to the reaction vessel body, the lid material (for example, a convex portion provided on the lid material) enters the inside of the reaction chamber from the opening of the reaction chamber, and enters the reaction chamber. Since the reaction chamber comes into contact with the accommodated reaction liquid, it is preferable that the reaction chamber has a structure in which a lid (for example, a projection provided on the lid) can easily enter. In addition, in order to automate the dispensing of the reaction solution, the reaction solution added from the opening reaches the bottom of the reaction chamber as it is (without applying a force other than gravity downward to the reaction solution). It is preferable to have such a structure as to obtain. Therefore, in the reaction vessel of the present invention, it is rather inappropriate that the reaction chamber has a structure like a capillary.
When the lid is attached to the reaction container body, the opening of the reaction chamber is sealed with the lid. As a result, contamination with the reaction solution contained in the reaction chamber can be prevented, and a desired reaction can be accurately generated in the reaction chamber. When the reaction container body has a plurality of reaction chambers, the opening of each reaction chamber is sealed with a lid material, and the reaction solution contained in one reaction chamber is mixed with the reaction solution in another reaction chamber. Can be prevented, whereby the desired reaction can be accurately generated in each reaction chamber.
From the viewpoint of preventing contamination to the reaction solution accommodated in the reaction chamber, it is preferable that the lid member has a first contact portion that can adhere to the periphery of the opening of the reaction chamber. In this case, the first contact portion of the lid member and the peripheral portion of the opening of the reaction chamber are in close contact with each other, whereby the reaction chamber is sealed and contamination with the reaction solution can be prevented.
Further, from the viewpoint of preventing contamination of the reaction solution contained in the reaction chamber, the lid member can be in close contact with the inner peripheral surface of the reaction chamber (the inner peripheral surface of the concave portion formed in the reaction vessel main body). It is preferable to have two contact parts. In this case, the second contact portion of the lid member and the inner peripheral surface of the reaction chamber are in close contact with each other, and the reaction chamber is hermetically sealed, so that contamination to the reaction solution can be prevented.
The structure of the lid member is not particularly limited as long as the lid member has a contact surface that can come into contact with the reaction solution in a state where the lid member is attached to the reaction vessel main body. As a structure of the lid member, a flat plate having a convex portion can be exemplified. In this case, when the lid member is attached to the reaction container main body, the convex portion enters the inside of the reaction chamber from the opening of the reaction chamber, and comes into contact with the reaction solution contained in the reaction chamber. It is preferable that the projection formed on the lid member is formed of a thin plate. In this case, by performing heat transfer between the outside of the lid member and the reaction solution in the reaction chamber through the thin plate, the temperature of the reaction solution can be quickly and efficiently controlled. In addition, by performing irradiation of light to the reaction chamber and detection of light emitted from the reaction solution through the thin plate, setting of irradiation conditions and light receiving conditions becomes easy.
The lid member and the reaction chamber have a contact surface that comes into contact with the reaction solution contained in the reaction chamber when the lid member is attached to the reaction container body. In the present invention, the surface of the reaction chamber that comes into contact with the reaction solution is referred to as a “contact surface of the reaction chamber”, and the surface of the lid material that comes into contact with the reaction solution is referred to as a “contact surface of the lid material”. In addition, the contact surface of the reaction chamber and the contact surface of the lid material do not necessarily mean a fixed surface, but change depending on conditions (for example, the volume of the reaction solution accommodated in the reaction chamber) (for example, the contact area increases or decreases). ) In some cases. For example, when the convex portion of the lid material presses the reaction liquid, the liquid level of the reaction liquid may rise, and the contact surface of the reaction chamber and the contact surface of the lid material may increase.
In the reaction vessel of the present invention, when a desired reaction is caused in the reaction chamber, the temperature of the reaction solution is controlled as necessary. The temperature control of the reaction solution is usually performed after the lid material is attached to the reaction vessel main body. However, the temperature control of the reaction solution may be performed before and / or during the process in which the lid member is attached to the reaction vessel body. When the temperature of the reaction solution is controlled after the lid material is attached to the reaction vessel main body, the temperature of the reaction solution is controlled by heat transfer through the contact surface of the reaction chamber and / or the contact surface of the lid material. Can be. In the reaction vessel of the present invention, not only the heat transfer through the contact surface of the reaction chamber but also the heat transfer through the contact surface of the lid member is possible, so that the temperature of the reaction solution can be quickly controlled. .
In the reaction vessel of the present invention, the reaction generated in the reaction chamber is not particularly limited. However, the reaction vessel of the present invention requires a reaction to control the temperature of the reaction solution when starting, proceeding or stopping the reaction (for example, It can be suitably used for an enzyme reaction), and particularly preferably for a reaction (eg, PCR) in which the temperature of a reaction solution needs to be controlled periodically or over time when the reaction proceeds. Here, the “temperature control of the reaction solution” includes both changing (raising and lowering) the temperature of the reaction solution and maintaining the temperature of the reaction solution.
The reaction container of the present invention may further include a heat conductive metal block or a heat conductive metal plate provided so as to be in contact with the reaction container main body and / or the lid member. In this case, the temperature control of the reaction vessel main body is performed through the contact surface between the reaction vessel main body and the heat conductive metal block or the heat conductive metal plate, and the lid member and the heat conductive metal block or the heat conductive metal plate are connected to each other. The temperature of the lid is controlled via the contact surface. Then, the temperature of the reaction solution is controlled via the contact surface of the reaction chamber and / or the contact surface of the lid member. The heat conductive metal block or the heat conductive metal plate may be provided so as to be in contact with one of the reaction vessel main body and the lid member, or may be provided so as to be in contact with both. Since the heat conductive metal block or the heat conductive metal plate can be easily formed according to the respective shapes of the reaction vessel main body and the lid material, it is possible to increase the contact area with the reaction vessel main body and the lid material. Thereby, heat transfer through the heat conductive metal block or the heat conductive metal plate can be efficiently performed. The heat conductive metal block or the heat conductive metal plate can be used as a medium for heat transfer (heat exchanger), a member for holding the reaction vessel main body, and a lid material for attaching the lid material to the reaction vessel main body. Can also be used as a member for applying pressure.
In the reaction container of the present invention, when the lid member is attached to the reaction container body, the lid member (for example, a convex portion provided on the lid member) enters the inside of the reaction chamber from the opening of the reaction chamber, and Gas such as air existing in the chamber is pushed out of the reaction chamber, and the opening of the reaction chamber is sealed in that state. Therefore, the amount of gas such as air existing in the reaction chamber is smaller than before the lid material is attached. Further, since the lid material (for example, a convex portion provided on the lid material) that has entered the inside of the reaction chamber comes into contact with the reaction liquid accommodated in the reaction chamber, the gas such as air existing in the reaction chamber and the reaction liquid Is smaller than before the lid material is attached. As described above, when the lid member is attached to the reaction container main body, the gas such as air existing in the reaction chamber decreases, and the contact area between the gas such as air existing in the reaction chamber and the reaction solution decreases. Therefore, when a target reaction proceeds in the reaction chamber, evaporation of the reaction solution into a gas such as air existing in the reaction chamber can be suppressed. This allows the reaction to proceed even if the amount of the reaction solution contained in the reaction chamber is very small.
In the reaction vessel of the present invention, the lid is made of a light-transmitting material so that light can pass through the contact surface of the lid from the reaction solution contained in the reaction chamber to the outside of the reaction vessel. The reaction vessel main body is made of a light-transmitting material so that light can pass through the contact surface of the reaction chamber from the reaction solution contained in the reaction chamber to the outside of the reaction vessel.
Light from the reaction solution contained in the reaction chamber to the outside of the reaction vessel may be transmitted through a part of the contact surface of the lid member, or may be transmitted through the entire contact surface of the lid member. May be able to transmit light. Further, as long as light can be transmitted from the reaction solution contained in the reaction chamber to the outside of the reaction vessel through the contact surface of the lid member, only a part of the lid member may be made of a light transmitting material. Alternatively, the entire lid may be made of a light-transmitting material.
Light from the reaction solution accommodated in the reaction chamber to the outside of the reaction vessel may be transmitted through a part of the contact surface of the reaction chamber, or may be transmitted through the entire contact surface of the reaction chamber. May be able to transmit light. In addition, as long as light can be transmitted from the reaction solution contained in the reaction chamber to the outside of the reaction vessel through the contact surface of the reaction chamber, only a part of the reaction vessel main body may be made of a light transmitting material. However, the entire reaction vessel may be made of a light-transmitting material.
In the reaction container of the present invention, only one of the lid member and the reaction container body may be formed of a light-transmitting material, or both may be formed of a light-transmitting material. When only one of the lid member and the reaction vessel body is made of a light-transmitting material, the other is made of a light-impermeable material.
The type of the light-transmitting material is not particularly limited, and any material may be used as long as it is transparent or translucent and has the strength required for the lid member and the reaction container body. Examples of such a material include plastic and glass.
In the reaction vessel of the present invention, light (for example, fluorescence or chemiluminescence) emitted from the reaction solution contained in the reaction chamber is transmitted to the outside of the reaction vessel through the contact surface of the lid and / or the contact surface of the reaction chamber. I do. That is, in the reaction vessel of the present invention, light (for example, fluorescence or chemiluminescence) emitted from the reaction solution can be detected outside the reaction vessel while the reaction solution is contained in the reaction chamber. Therefore, when the light emitted from the reaction solution serves as an indicator of the progress of the reaction occurring in the reaction chamber, by detecting the light emitted from the reaction solution, the progress of the reaction can be monitored in real time (that is, the reaction progress). (Instantly during monitoring).
Here, “monitoring” includes quantitative or qualitative measurement or analysis performed continuously or intermittently during the progress of the reaction, as well as quantitative or qualitative measurement after the reaction reaches a steady state or after the reaction is completed. It also includes basic measurement and analysis. The “reaction progress” includes the presence or absence of the progress of the reaction and the degree thereof.
Detection of light emitted from the reaction solution may be performed via only one of the contact surface of the lid member and the contact surface of the reaction chamber, or may be performed through both. Also. Detection of light emitted from the reaction solution may be performed through a part of the lid and / or the contact surface of the reaction chamber, or may be performed through the whole.
(2) In the first embodiment of the reaction container of the present invention, the light is transmitted from the outside of the reaction container to the reaction solution accommodated in the reaction chamber through the contact surface of the lid member. The lid material is made of a light-transmitting material, or, from outside the reaction vessel to the reaction solution contained in the reaction chamber, so that light can be transmitted through the contact surface of the reaction chamber, The reaction container main body is made of a light transmissive material.
In the reaction vessel according to this aspect, the lid is formed of a light-transmitting material so that light can pass through the contact surface of the lid from the outside of the reaction vessel to the reaction solution contained in the reaction chamber. In such a case, the reaction liquid contained in the reaction chamber can be irradiated with excitation light emitted from a light source such as a laser provided outside the reaction container of the present invention via the contact surface of the lid member. If a fluorescent substance such as a fluorescent dye is added to the reaction liquid, the fluorescent substance is excited by irradiating the reaction liquid with excitation light, and the reaction liquid emits fluorescence. Since the fluorescence emitted from the reaction solution passes through the contact surface of the lid and / or the contact surface of the reaction chamber to the outside of the reaction vessel of the present invention, the fluorescence detector provided outside the reaction vessel of the present invention Can be detected by
Further, in the reaction vessel according to this aspect, the reaction vessel main body is made of a light-transmitting material so that light can be transmitted from the outside of the reaction vessel to the reaction solution contained in the reaction chamber via the contact surface of the reaction chamber. When configured, it is possible to irradiate the reaction liquid contained in the reaction chamber with the excitation light emitted from a light source such as a laser provided outside the reaction vessel of the present invention via the contact surface of the reaction chamber. it can. If a fluorescent substance such as a fluorescent dye is added to the reaction liquid, the fluorescent substance is excited by irradiating the reaction liquid with excitation light, and the reaction liquid emits fluorescence. Since the fluorescence emitted from the reaction solution passes through the contact surface of the lid and / or the contact surface of the reaction chamber to the outside of the reaction vessel of the present invention, the fluorescence detector provided outside the reaction vessel of the present invention Can be detected by
In the reaction vessel according to this aspect, light from the outside of the reaction vessel to the reaction solution contained in the reaction chamber can be transmitted through the lid member and / or a part of the contact surface of the reaction chamber. Alternatively, the light may be transmitted through the whole. Further, as long as light can be transmitted from the outside of the reaction vessel to the reaction solution contained in the reaction chamber via the contact surface of the lid member, only a part of the lid member may be made of a light transmitting material. Alternatively, the entire lid may be made of a light-transmitting material. Further, as long as light can be transmitted from the outside of the reaction vessel to the reaction solution contained in the reaction chamber via the contact surface of the reaction chamber, only a part of the reaction vessel main body may be made of a light transmitting material. However, the entire reaction container body may be made of a light-transmitting material.
In the reaction container according to this aspect, irradiation of the reaction solution with excitation light and detection of fluorescence emitted from the reaction solution can be performed from outside the reaction container while the reaction solution is contained in the reaction chamber. . Therefore, if a phosphor that can be an indicator of the progress of the reaction is added to the reaction solution, the progress of the reaction can be monitored in real time (ie, the progress of the reaction) by detecting the fluorescence emitted by the phosphor during the progress of the reaction. (Instantly during monitoring).
The phosphor that can be an indicator of the progress of the reaction can be appropriately selected according to the type of reaction to be generated in the reaction chamber. For example, when the reaction generated in the reaction chamber is PCR, a fluorescent substance whose fluorescence characteristics such as fluorescence intensity and fluorescence wavelength are changed depending on the amount of nucleic acid (eg, DNA) in the reaction solution can be used. Specifically, a fluorescent dye that changes properties such as fluorescence intensity and fluorescence wavelength by intercalating into double-stranded DNA can be used. From the viewpoint of easy measurement, a fluorescent dye having a property of increasing the fluorescence intensity by intercalation is preferable. Specific examples of such a fluorescent dye include ethidium bromide (EtBr), SYBR Green I, Pico Green, thiazole orange, oxazole yellow and the like. For example, ethidium bromide intercalated into DNA emits fluorescence when excited by energy transfer of ultraviolet rays (260 nm) absorbed by DNA or light absorbed by itself. SYBR GreenI intercalated into DNA emits green fluorescence when excited by ultraviolet light of about 260 nm or visible light of about 470 nm. Since the fluorescence intensity emitted from these fluorescent dyes is proportional to the amount of double-stranded DNA, measuring the fluorescence intensity of the fluorescent dye allows the progress of PCR in the reaction chamber (for example, the presence or absence of amplification of target nucleic acid by PCR, PCR The amount of the amplification product can be monitored in real time (ie, immediately during the progress of the PCR reaction).
In addition, as a fluorescent substance that can be an indicator of the progress of PCR, a fluorescent substance in which two types of fluorescent dyes of a reporter and a quencher are bound to an oligonucleotide probe complementary to an intermediate portion of a target sequence can be used. The reporter is a molecule that emits fluorescence when irradiated with excitation light, but in the case of an oligonucleotide probe having a quencher near the reporter, the energy absorbed by the reporter is absorbed by the quencher, and the reporter is not excited, No fluorescence that would otherwise occur is produced (quenching). When the quenched oligonucleotide probe is added to the PCR reaction solution contained in the reaction chamber, it binds to the target sequence. Thereafter, an extended chain is synthesized from the 3 'end of the primer by Tag polymerase. When the probe collides with the probe on the way, the probe which has been annealed is decomposed by the 5' → 3 'endonuclease activity, and is adjacent to the probe. The reporter and the quencher are separated, and the reporter that has been suppressed by the quencher emits fluorescence. Since this reaction occurs almost in proportion to the PCR cycle, the progress of PCR in the reaction chamber can be monitored in real time by measuring the fluorescence intensity of the reporter.
In addition, as a fluorescent substance that can be an indicator of the progress of PCR, one obtained by binding a fluorescent dye to two types of oligonucleotide probes that hybridize adjacent to a target nucleic acid can be used. A donor dye is bound to the 3 ′ end of the 5 ′ probe, and an acceptor dye is bound to the 5 ′ end of the 3 ′ probe. When two types of probes hybridize adjacent to the target nucleic acid, the donor dye The dye fluoresces when excited by an external light source, and the light is absorbed by the acceptor dye, which then emits light of a different wavelength. As the amount of the probe that hybridizes to the target nucleic acid increases as the PCR amplification product increases, the progress of PCR in the reaction chamber can be monitored in real time by measuring the fluorescence intensity.
(3) In the second aspect of the reaction container of the present invention, a part or the whole of the contact surface of the lid member is a flat surface.
In the reaction vessel according to this aspect, irradiation of the reaction solution with excitation light or detection of fluorescence emitted from the reaction solution can be performed through a part or the whole of the contact surface of the lid material that is a flat surface. This facilitates setting of the irradiation condition of the excitation light and the reception condition of the fluorescence.
(4) In the third aspect of the reaction vessel of the present invention, the contact surface of the lid member is a surface of a wall portion of the lid member having a substantially uniform thickness.
In the reaction vessel according to this aspect, the temperature of the reaction solution accommodated in the reaction chamber can be controlled via a substantially uniform-thickness wall (for example, a plate) having a contact surface of the lid member. Thus, the temperature of the reaction solution can be quickly and efficiently controlled. Further, setting of the temperature control condition at that time is also easy. Further, irradiation of excitation light to the reaction solution accommodated in the reaction chamber or detection of fluorescence from the reaction solution can be performed through a substantially uniform wall portion having a contact surface of the lid material, whereby It is easy to set the irradiation condition of the excitation light and the light receiving condition of the fluorescence. In particular, when a part or the whole of the contact surface of the lid member is a flat surface, the wall portion has a flat plate shape, and the contact surface of the lid member on the wall portion and the surface on the opposite side thereof are substantially parallel. It becomes easier to set the irradiation condition of the excitation light and the light receiving condition of the fluorescence.
(5) In the fourth embodiment of the reaction vessel of the present invention, a part or the whole of the contact surface of the reaction chamber is a flat surface.
In the reaction vessel according to this aspect, irradiation of the reaction solution with excitation light or detection of fluorescence from the reaction solution can be performed through a part or the whole of the contact surface of the reaction chamber which is a flat surface, whereby It is easy to set the irradiation condition of the excitation light and the light receiving condition of the fluorescence.
(6) In the fifth aspect of the reaction container of the present invention, the contact surface of the reaction chamber is a surface of a wall portion having a substantially uniform thickness which constitutes the reaction container main body.
In the reaction vessel according to this aspect, the temperature of the reaction solution contained in the reaction chamber can be controlled through a substantially uniform wall (for example, a plate) having a contact surface of the reaction chamber. Thus, the temperature of the reaction solution can be quickly and efficiently controlled. Further, setting of the temperature control condition at that time is also easy. Further, irradiation of excitation light to the reaction solution contained in the reaction chamber or detection of fluorescence from the reaction solution can be performed through a substantially uniform wall having a contact surface of the reaction chamber. It is easy to set the irradiation condition of the excitation light and the light receiving condition of the fluorescence. In particular, when a part or the whole of the contact surface of the reaction chamber is a flat surface, the wall is flat, and the contact surface of the reaction chamber and the surface on the opposite side of the wall are substantially parallel. It becomes easier to set the irradiation condition of the excitation light and the light receiving condition of the fluorescence.
(7) In the sixth aspect of the reaction container of the present invention, when the lid is attached to the reaction container main body, a closed space is formed by the contact surface of the lid and the contact surface of the reaction chamber. A part or all of the reaction solution is accommodated in the closed space.
In the reaction container according to this aspect, when the lid is attached to the reaction container main body, the end of the contact surface of the lid and the end of the contact surface of the reaction chamber are in close contact with each other to form a closed space. . Whether part or all of the reaction solution is accommodated in the closed space depends on the volume of the reaction solution accommodated in the reaction chamber, the volume of the sealed space formed, and the like.
In the reaction vessel according to this aspect, the entire outer surface (or almost the entirety) of the reaction solution accommodated in the closed space is a contact surface with the lid member and the reaction chamber, and therefore, the contact surface of the lid member and / or The temperature of the reaction solution can be quickly controlled through the contact surface of the reaction chamber.
Further, since gas such as air does not exist (or hardly exists) in the closed space, it is possible to suppress evaporation of the reaction solution into gas when a target reaction proceeds in the closed space. The reaction can proceed even if the amount of the reaction solution contained in the chamber is very small.
(8) In the seventh aspect of the reaction container of the present invention, when the lid is attached to the reaction container main body, a reaction liquid residual portion capable of storing a residual portion of the reaction liquid that cannot be stored in the closed space. A housing is formed in the reaction chamber.
In the reaction container according to the present embodiment, a larger volume of the reaction solution than can be accommodated in the closed space is accommodated in the reaction chamber, and the reaction solution is pressed by a lid member (for example, a convex portion provided on the lid member). As a result, air in the reaction chamber, bubbles in the reaction solution, and the like are pushed out to the reaction solution remaining storage section together with the reaction solution that cannot be contained in the closed space, thereby causing bubbles in the reaction solution contained in the closed space. And air can be prevented from entering. In addition, since a certain amount of the reaction solution is contained in the closed space, the reaction can proceed for a certain amount of the reaction solution regardless of the volume of the reaction solution contained in the reaction chamber. The labor required to accurately measure and accommodate the reaction in the reaction chamber can be reduced.
In the reaction vessel according to the present embodiment, the reaction solution remaining portion accommodating portion is formed in the reaction chamber as follows, for example. When the lid material is attached to the reaction vessel body, the outer peripheral surface of the convex portion of the lid material does not completely adhere to the inner peripheral surface of the reaction chamber. When a space is formed between itself and the peripheral surface, this space becomes the reaction liquid remaining portion storage portion.
(9) In an eighth aspect of the reaction container of the present invention, the reaction chamber has a facing surface facing a contact surface of the lid member, and the lid member is attached to the reaction container body. Then, a part or the whole of the reaction solution accommodated in the reaction chamber is thinly accommodated between the contact surface of the lid member and the opposing surface of the reaction chamber.
In the present invention, among the contact surfaces of the reaction chamber, a surface facing the contact surface of the lid member is referred to as a “facing surface of the reaction chamber”. The facing surface of the reaction chamber is preferably, for example, a flat surface facing the lid material if the contact surface is flat, and a curved surface facing the contact surface of the lid material if the contact surface is a curved surface. .
In the reaction vessel according to the present aspect, the reaction in the reaction solution thinly accommodated between the contact surface of the lid member and the opposite surface of the reaction chamber is monitored. At this time, since the reaction solution existing in a thin state has a large ratio of the surface area to the volume of the reaction solution, the heat transfer through the contact surface of the reaction chamber and / or the contact surface of the lid member causes the temperature of the reaction solution to be reduced. Control can be performed quickly. Further, when the reaction liquid is thin, temperature control can be uniformly performed on the entire reaction liquid.
(10) In a ninth aspect of the reaction container of the present invention, the opposed surface of the reaction chamber is a surface of a wall portion having a substantially uniform thickness constituting the reaction container main body.
In the reaction vessel according to this aspect, the temperature of the reaction solution accommodated in the reaction chamber can be controlled through a substantially uniform thickness wall (for example, a plate) having an opposing surface of the reaction chamber. Thus, the temperature of the reaction solution can be quickly and efficiently controlled. Further, setting of the temperature control condition at that time is also easy. Further, the irradiation of the reaction solution contained in the reaction chamber with the excitation light or the detection of the fluorescence from the reaction solution can be performed through the substantially uniform thickness wall having the opposing surface of the reaction chamber. It is easy to set the irradiation condition of the excitation light and the light receiving condition of the fluorescence. In particular, when a part or the whole of the opposing surface of the reaction chamber is a flat surface, the wall is flat, and the opposing surface of the reaction chamber and the surface on the opposite side of the wall are substantially parallel. It becomes easier to set the irradiation condition of the excitation light and the light receiving condition of the fluorescence.
(11) In the tenth aspect of the reaction vessel of the present invention, the reaction solution contained in the reaction chamber may be supplied from the outside of the reaction vessel and / or from the reaction solution contained in the reaction chamber to the outside of the reaction vessel. The wall having the opposed surface of the reaction chamber is made of a light transmitting material so that light can be transmitted through the opposed surface of the reaction chamber.
In the reaction vessel according to this aspect, when light can be transmitted from the outside of the reaction vessel to the reaction solution contained in the reaction chamber via the opposing surface of the reaction chamber, the light is contained in the reaction chamber from outside the reaction vessel. The reaction solution can be irradiated with light through the opposite surface of the reaction chamber. In addition, when light can be transmitted from the reaction solution contained in the reaction chamber to the outside of the reaction vessel through the opposite surface of the reaction chamber, light emitted from the reaction solution contained in the reaction chamber (for example, fluorescence or chemical Luminescence) can be detected outside the reaction vessel via the opposing surface of the reaction chamber. Therefore, irradiation of light to the reaction solution and / or detection of light emitted from the reaction solution at the time of monitoring the reaction can be performed through the opposed surface of the reaction chamber.
(12) In the eleventh aspect of the reaction vessel of the present invention, the reaction vessel main body is in contact with the lid member to define a distance between a contact surface of the lid member and an opposing surface of the reaction chamber. Having a surface.
In the reaction container according to this aspect, the distance between the contact surface of the lid member and the opposing surface of the reaction chamber is kept constant, so that the reaction container is accommodated between the contact surface of the lid member and the opposing surface of the reaction chamber. Since the thickness of the reaction solution is kept constant, temperature control can be uniformly performed on the entire reaction solution. The thickness of the reaction solution can be adjusted by adjusting the distance between the contact surface of the lid member and the corresponding surface of the reaction chamber.
In the reaction container according to this aspect, the reaction container body may have a contact surface inside the reaction chamber or may have a contact surface outside the reaction chamber. For example, the contact surface can be provided along the inner peripheral surface of the reaction chamber. In this case, when the lid member is attached to the reaction vessel main body, the lid member is in close contact with the periphery of the inner peripheral surface of the reaction chamber, and the reaction chamber is sealed.
(13) In a twelfth aspect of the reaction container of the present invention, the reaction chamber has an enclosing surface for enclosing the reaction solution existing between the contact surface of the lid member and the opposing surface of the reaction chamber. When the lid is attached to the reaction vessel main body, a closed space is formed by the contact surface of the lid, the opposing surface of the reaction chamber, and the surrounding surface of the reaction chamber, and the reaction liquid A part or the whole is stored in the closed space in a thin shape.
In the present invention, among the contact surfaces of the reaction chamber, the surface surrounding the reaction liquid existing between the contact surface of the lid member and the opposing surface of the reaction chamber is the “enclosure surface of the reaction chamber”. The shape of the surrounding surface of the reaction chamber is determined by the shape of the contact surface of the lid material and the shape of the facing surface of the reaction chamber. For example, when the contact surface of the lid material and the facing surface of the reaction chamber are circular, the surrounding surface of the reaction chamber is Is cylindrical, and when the contact surface of the lid member and the opposing surface of the reaction chamber are rectangular, the surrounding surface of the reaction chamber is rectangular. The shape of the cross section of the surrounding surface can be any shape such as a circular shape, a rectangular shape (including both squares and rectangles), a semi-circular half-square shape, and a parallelogram shape.
In the reaction vessel according to this aspect, the end of the opposing surface of the reaction chamber and the end (usually the lower end) of the surrounding surface of the reaction chamber are continuous, and when the lid material is attached to the reaction vessel main body. The end of the contact surface of the lid material and the end (usually the upper end) of the surrounding surface of the reaction chamber are in close contact with each other. Thus, a closed space is formed by the contact surface of the lid member, the opposing surface of the reaction chamber, and the surrounding surface of the reaction chamber.
In the reaction container according to the present embodiment, the reaction in the reaction solution stored in a thin space in the closed space is monitored. At this time, since the reaction solution existing in a thin state has a large ratio of the surface area to the volume of the reaction solution, the heat transfer through the contact surface of the reaction chamber and / or the contact surface of the lid member causes the temperature of the reaction solution to be reduced. Control can be performed quickly. Further, when the reaction solution is thin, the thickness of the reaction solution is substantially uniform, so that the temperature control can be uniformly performed on the entire reaction solution. Further, since gas such as air does not exist (or hardly exists) in the closed space, it is possible to suppress evaporation of the reaction solution into gas when a target reaction proceeds in the closed space. The reaction can proceed even if the amount of the reaction solution contained in the chamber is very small.
(14) In the thirteenth aspect of the reaction container of the present invention, a part or the whole of the surrounding surface of the reaction chamber is a flat surface.
In the reaction container according to this aspect, irradiation of the reaction solution with excitation light or detection of fluorescence from the reaction solution can be performed through a part or the whole of the surrounding surface of the reaction chamber which is a flat surface. It is easy to set the irradiation condition of the excitation light and the light receiving condition of the fluorescence.
(15) In a fourteenth aspect of the reaction vessel of the present invention, the cross section of the surrounding surface of the reaction chamber is rectangular.
In the reaction vessel according to this aspect, the surrounding surface of the reaction chamber is constituted by four planes, and the two opposing surfaces are parallel. Therefore, by utilizing the characteristic that light travels straight, the entire reaction liquid is irradiated with light through one plane constituting the surrounding surface of the reaction chamber, and the light emitted from the entire reaction liquid is emitted. It is possible to detect. Further, it is easy to set the irradiation condition of the excitation light and the light receiving condition of the fluorescence at that time.
(16) In a fifteenth aspect of the reaction container of the present invention, the surrounding surface of the reaction chamber is a surface of a wall portion having a substantially uniform thickness that constitutes the reaction container main body.
In the reaction vessel according to this aspect, the temperature of the reaction solution contained in the reaction chamber can be controlled through a substantially uniform thickness wall (for example, a plate) having an enclosing surface of the reaction chamber. Thus, the temperature of the reaction solution can be quickly and efficiently controlled. Further, setting of the temperature control condition at that time is also easy. Further, the irradiation of the excitation light to the reaction solution contained in the reaction chamber or the detection of fluorescence from the reaction solution can be performed through the surface of the substantially uniform wall having the surrounding surface of the reaction chamber, This facilitates setting of the irradiation condition of the excitation light and the light receiving condition of the fluorescence. In particular, when a part or the whole of the surrounding surface of the reaction chamber is flat, the wall portion has a flat plate shape, and the surrounding surface of the reaction chamber in the wall portion and the surface on the opposite side thereof are substantially parallel. It becomes easier to set the irradiation condition of the excitation light and the light receiving condition of the fluorescence.
(17) In the sixteenth aspect of the reaction vessel of the present invention, the reaction vessel contained in the reaction chamber may be supplied from outside the reaction vessel to / from the reaction vessel contained in the reaction chamber. The wall having the surrounding surface of the reaction chamber is made of a light-transmitting material so that light can be transmitted to the outside through the surrounding surface of the reaction chamber.
In the reaction vessel according to the present embodiment, when light can be transmitted from the outside of the reaction vessel to the reaction solution contained in the reaction chamber via the surrounding surface of the reaction chamber, the reaction solution is contained in the reaction chamber from outside the reaction vessel. The reaction solution can be irradiated with light through the surrounding surface of the reaction chamber. Further, when light can be transmitted from the reaction solution contained in the reaction chamber to the outside of the reaction vessel through the surrounding surface of the reaction chamber, light emitted from the reaction solution contained in the reaction chamber (for example, fluorescence or chemical Luminescence) can be detected outside the reaction vessel via the surrounding surface of the reaction chamber. Therefore, irradiation of light to the reaction solution and / or detection of light emitted from the reaction solution at the time of monitoring the reaction can be performed through the surrounding surface of the reaction chamber.
In the reaction vessel according to the present embodiment, the temperature of the reaction solution accommodated in the reaction chamber is controlled by heat transfer through the contact surface of the lid member and / or the opposing surface of the reaction chamber, and when the reaction is monitored. Irradiation of the reaction solution with light and / or detection of light emitted from the reaction solution can be performed through the surrounding surface of the reaction chamber. As described above, by separately providing the surface used for controlling the temperature of the reaction solution and the surface used for monitoring the progress of the reaction, the temperature of the reaction solution can be quickly controlled, and The area for monitoring the progress of the process can be set freely. It is also possible to monitor the progress of the reaction in the entire reaction solution.
(18) In the seventeenth aspect of the reaction container of the present invention, a nozzle tip fitted to a nozzle capable of sucking and discharging a liquid may be fitted to the lid of the reaction vessel according to the sixth aspect. A mounting space is formed, and in a state where the lid member is attached to the reaction vessel main body, the nozzle chip is mounted in the nozzle chip mounting space so that the nozzle chip can be mounted in the nozzle chip mounting space. A nozzle tip fitting opening communicating therewith is formed, and in a state where the lid member is attached to the reaction container main body, the outside of the reaction container and the closed space are formed by a perforated needle provided outside the reaction container. A through-hole may be formed in the reaction vessel main body and the lid member to communicate the nozzle space with the nozzle tip fitting space.
When performing a target reaction using the reaction vessel according to the present embodiment, a part or the whole of the reaction solution is accommodated in a closed space formed by the contact surface of the lid member and the contact surface of the reaction chamber, and A reaction occurs.
Before or after the reaction, the nozzle tip mounted on the nozzle is fitted from the nozzle tip fitting opening into the nozzle chip fitting space of the reaction vessel in a state where the lid member is attached to the reaction vessel main body. The nozzle tip fitted in the nozzle tip fitting space is a mediating member capable of transmitting the suction force (decompression) and the discharge force (pressure) of the nozzle to the outside of the nozzle tip. For example, a nozzle mounting port is formed at one end. In this case, a nozzle tip having a suction / discharge port formed at the other end which communicates with the nozzle mounting port can be used. When such a nozzle tip is used, for example, the nozzle tip is fitted in the nozzle tip fitting space such that the suction / discharge port of the nozzle tip communicates with the nozzle tip fitting space. At this time, the position of the suction / ejection port of the nozzle chip in the nozzle chip fitting space is defined, for example, by the contact between the lid member and the contact portion of the nozzle chip.
Before or after the fitting of the nozzle tip into the nozzle tip fitting space, by means of a puncture needle provided outside the reaction vessel, the outside of the reaction vessel and the closed space containing the reaction solution, the nozzle tip fitting space, Are formed in the reaction vessel main body and the lid member. The piercing needle first pierced the reaction vessel body to form a through-hole communicating the outside of the reaction vessel with the sealed space containing the reaction solution, and then pierced the lid material to contain the reaction solution. A through hole is formed to allow the closed space and the nozzle chip fitting space to communicate with each other. In a reaction container in which the nozzle tip has been fitted and the hole has been pierced by the piercing needle, the outside of the reaction container and the closed space containing the reaction solution communicate with each other through a through-hole formed in the reaction container main body. And the nozzle chip fitting space communicates through a through hole formed in the lid material, and the nozzle chip fitting space communicates with the suction / discharge port of the nozzle chip. And the discharge force can be transmitted to the outside of the reaction vessel. Therefore, when the reaction container is immersed in the liquid, the through-hole formed in the reaction container body is immersed in the liquid, and the suction and discharge by the nozzle are started. And flows out of the closed space with the discharge by the nozzle. By repeating suction and discharge by the nozzle, the reaction liquid contained in the closed space of the reaction container is extracted into the liquid. With the extraction of the reaction solution, the reaction products contained in the reaction solution are also extracted into the liquid.
As described above, if the reaction container according to the present embodiment is used, the reaction is performed in a state where the lid member is attached to the reaction container body, and the reaction solution is contained in the reaction solution without detaching the lid member from the reaction container body. A reaction product can be obtained.
The nozzle chip may be fitted in the nozzle chip fitting space such that the suction / discharge port of the nozzle chip contacts and is sealed with the wall of the lid material forming the nozzle chip fitting space. However, it is necessary that the wall of the lid member that seals the suction / discharge port of the nozzle tip has a contact surface with the closed space containing the reaction liquid. In this case, before or after fitting of the nozzle tip into the nozzle tip fitting space, the outside of the reaction vessel and the closed space containing the reaction solution and the nozzle are perforated by a piercing needle provided outside the reaction vessel. A through-hole communicating with the suction / discharge port of the chip is formed in the reaction vessel main body and the lid. A through-hole that allows the closed space containing the reaction liquid to communicate with the suction / discharge port of the nozzle tip is formed in the wall of the lid member that seals the suction / discharge port of the nozzle tip. In a reaction container in which the nozzle tip has been fitted and the hole has been pierced by the piercing needle, the outside of the reaction container and the closed space containing the reaction solution communicate with each other through a through-hole formed in the reaction container main body. The suction space and the suction / discharge port of the nozzle tip communicate with each other through the through-hole formed in the lid member, so that the suction and discharge forces of the nozzle can be transmitted to the outside of the reaction vessel. . Therefore, as described above, the reaction product contained in the reaction solution is obtained by immersing the reaction container in the liquid and immersing the through-hole formed in the reaction container body in the liquid, and repeating the suction and discharge by the nozzle. Extracted into the liquid.
In the reaction vessel according to this aspect, the shape and size of the nozzle chip fitting space formed in the lid member and the nozzle chip fitting opening communicating with the nozzle chip fitting space are fitted in the nozzle chip fitting space. It is appropriately adjusted according to the shape and size of the nozzle. Further, the nozzle chip fitting port is formed at a position where the nozzle chip can be fitted into the nozzle chip fitting space from the nozzle chip fitting port in a state where the lid member is attached to the reaction vessel main body.
In the reaction vessel according to this aspect, the material of the reaction vessel body and the lid material is a material that is not corroded by the reaction solution, can withstand the reaction conditions (for example, reaction temperature), and is provided outside the reaction vessel. A material that can pierce the reaction vessel main body and the lid material with the piercing needle is selected. When the perforation needle is made of a metal such as stainless steel, for example, plastic, glass or the like can be selected as the material of the reaction vessel main body and the lid material.
In the reaction vessel according to the present aspect, the relative position between the sealed space containing the reaction solution and the nozzle chip fitting space is such that the reaction solution and the outside of the reaction container are contained by a perforated needle provided outside the reaction container. It is adjusted so that a through hole for communicating the closed space and the nozzle chip fitting space can be formed in the reaction vessel main body and the lid member.
The shape of the piercing needle provided outside the reaction vessel according to the present embodiment is not particularly limited as long as it can pierce the lid member and the reaction vessel main body, and the shape of the piercing needle is, for example, a pointed tip. , Specifically, a conical shape, a pyramid shape, a needle shape, and the like. Here, the term "pointed shape" means a shape that becomes thinner toward the tip, and in addition to a shape with a sharp tip, a shape with a rounded tip, a shape with a flat tip, etc. Is also included. The material of the puncture needle is appropriately determined according to these materials so as to pierce the lid member and the reaction vessel main body, but is usually a metal such as stainless steel. The number of perforation needles used for perforation is not particularly limited. The length of the puncture needle is appropriately adjusted so that a desired through-hole can be formed.
(19) In an eighteenth aspect of the reaction container of the present invention, a nozzle tip capable of fitting a nozzle tip attached to a nozzle capable of sucking and discharging a liquid into a lid of the reaction container according to the twelfth aspect. The nozzle chip fitting space is formed so that the fitting space is formed and the nozzle tip can be fitted in the nozzle chip fitting space in a state where the lid member is attached to the reaction vessel main body. A nozzle tip fitting opening communicating with the reaction vessel is formed, and in a state where the lid member is attached to the reaction vessel main body, the outside of the reaction vessel is pre-sealed with a perforation needle provided outside the reaction vessel. A through-hole may be formed in the reaction vessel main body and the lid member for communicating the space with the nozzle chip fitting space.
When a target reaction is performed using the reaction vessel according to the present embodiment, a part or the whole of the reaction solution is sealed in a closed space formed by the contact surface of the lid member, the facing surface of the reaction chamber, and the surrounding surface of the reaction chamber. To produce the desired reaction.
When the reaction vessel according to this aspect is used, similarly to the reaction vessel according to the seventeenth aspect, the reaction is performed in a state where the lid is attached to the reaction vessel main body, and then the lid is detached from the reaction vessel main body. The reaction product contained in the reaction solution can be obtained without the need.
(20) In the nineteenth aspect of the reaction vessel of the present invention, the nozzle chip fitting space is formed such that the nozzle chip fitting space is sealed when the nozzle chip fitting opening is sealed. ing.
In the reaction vessel according to this aspect, since the nozzle chip fitting space is sealed by fitting the nozzle chip into the nozzle chip fitting space, the suction force and the discharge force of the nozzle are efficiently applied to the nozzle chip fitting space. Can communicate well. Here, “sealing” means a state in which there are no gaps, pores, and the like that hinder the transmission of the suction force (pressure reduction) and the discharge force (pressure) by the nozzle to the nozzle chip fitting space. The state in which the nozzle chip fitting space communicates with the suction / discharge port of the nozzle chip is included in “sealing”. In addition, “sealing” includes a state in which there are gaps and pores that do not impede transmission of suction force (pressure reduction) and discharge force (pressure) by the nozzle to the nozzle chip fitting space. It is.
(21) In the twentieth aspect of the reaction container of the present invention, the wall of the lid forming the nozzle chip fitting space has an inner peripheral surface that can be in close contact with the outer peripheral surface of the nozzle tip.
In the reaction vessel according to the present aspect, when the nozzle tip is fitted into the nozzle tip fitting space, the inner peripheral surface of the wall portion of the lid member forming the nozzle chip fitting space and the outer peripheral surface of the nozzle tip are in close contact. Thus, the nozzle chip fitting space is sealed.
(22) In the twenty-first aspect of the reaction container of the present invention, a concave portion provided on an outer peripheral surface of the nozzle tip is provided on an inner peripheral surface of a wall portion of the lid member which can be in close contact with an outer peripheral surface of the nozzle tip. And / or a convex portion and / or a concave portion capable of fitting with the convex portion are provided.
In the reaction vessel according to this aspect, the fitting state of the nozzle tip into the nozzle tip fitting space becomes strong, and the nozzle tip fitted into the nozzle tip fitting space is fitted into the nozzle tip fitting space. Even when a force in the opposite direction is applied, the nozzle tip does not detach from the nozzle chip fitting space. Therefore, it is possible to transfer the reaction vessel in a state in which the lid member is attached to the reaction vessel main body by transferring the nozzle having the nozzle tip fitted in the nozzle tip fitting space.
(23) In a twenty-second aspect of the reaction container of the present invention, the contact surface of the lid is a wall surface of the lid forming the nozzle tip fitting space.
In the reaction vessel according to the present aspect, the closed space containing the reaction liquid and the nozzle are formed on the wall of the lid forming the nozzle chip fitting space and having the contact surface with the reaction liquid. A through-hole that communicates with the chip fitting space can be formed. In addition, since the wall of the lid opposes any part of the wall of the reaction vessel main body forming a closed space containing the reaction solution, one piercing needle provided outside the reaction vessel is provided. Thereby, a through-hole that allows the outside of the reaction vessel to communicate with the sealed space containing the reaction solution and the nozzle chip fitting space can be formed in the reaction vessel main body and the lid member.
(24) In a twenty-third aspect of the reaction vessel of the present invention, the contact surface of the lid is a wall surface of the lid forming a deepest portion of the nozzle tip fitting space.
Here, the “deepest part of the nozzle chip fitting space” means a part of the nozzle chip fitting space that is most separated from the nozzle chip fitting opening, and in the reaction vessel according to this embodiment, the nozzle tip is a nozzle. The nozzle is fitted from the tip fitting port to the deepest part of the nozzle tip fitting space.
(25) In a twenty-fourth aspect of the reaction container of the present invention, the wall portion of the lid member forming the deepest portion of the nozzle chip fitting space forms the deepest portion of the closed space. Is provided so as to face the wall portion of the slab.
Here, “the deepest part of the closed space” means a portion of the closed space in which the reaction solution is stored, which is closest to the surface on which the reaction vessel is placed. In the reaction vessel according to this aspect, the outside of the reaction vessel, the closed space containing the skin solution, and the nozzle chip fitting space are provided by a piercing needle provided perpendicularly or substantially perpendicularly to the surface on which the reaction vessel is placed. Can be formed in the reaction vessel main body and the lid member.
(26) In the twenty-fifth aspect of the reaction vessel of the present invention, the fitting direction of the nozzle tip into the nozzle tip fitting space is perpendicular or substantially perpendicular to a surface on which the reaction vessel is mounted. Thus, the nozzle chip fitting space is formed.
In the reaction vessel according to this aspect, the force received by the reaction vessel from the nozzle tip when fitting the nozzle tip into the nozzle tip fitting space is perpendicular or substantially perpendicular to the surface on which the reaction vessel is placed. Power. Therefore, when the nozzle tip is fitted into the nozzle chip fitting space, the reaction container does not shift, and the nozzle tip can be easily fitted into the nozzle chip fitting space.
(27) In a twenty-sixth aspect of the reaction container of the present invention, the lid member has an outer peripheral surface that can be in close contact with the inner peripheral surface of the reaction chamber.
In the reaction vessel according to this aspect, the outer peripheral surface of the lid member and the inner peripheral surface of the reaction chamber are in close contact with each other, so that the hermeticity of the closed space containing the reaction liquid is more reliable. Therefore, the suction force and the discharge force by the nozzle can be efficiently transmitted to the outside of the reaction container.
(28) In the twenty-seventh aspect of the reaction container of the present invention, a concave portion and / or a convex portion are provided on an inner peripheral surface of the reaction chamber, and an inner peripheral surface of the lid is provided on an inner peripheral surface of the reaction chamber. Protrusions and / or recesses that can be fitted with recesses and / or protrusions provided on the peripheral surface are provided.
In the reaction container according to the present embodiment, the state in which the lid member is adhered to the reaction container body becomes strong, and even when the reaction container in which the lid member is adhered to the reaction container body is transferred (for example, Even if the lid is held and transported without supporting the reaction vessel main body), the lid does not detach from the reaction vessel main body. Therefore, it is possible to transfer the reaction vessel in a state in which the lid member is attached to the reaction vessel main body by transferring the nozzle having the nozzle tip fitted in the nozzle tip fitting space.
(29) In a twenty-eighth aspect of the reaction container of the present invention, the reaction container is a PCR reaction container.
In the reaction vessel according to this aspect, the reaction generated in the reaction chamber is PCR, and the reaction solution contained in the reaction chamber is a PCR reaction solution. The reaction solution for PCR includes, for example, H₂O, buffer, MgCl₂, DNTP mix, primers, template DNA, Taq polymerase, etc., and the PCR reaction solution after the reaction contains a PCR amplified fragment (eg, DNA fragment) as a reaction product.
In PCR, it is necessary to control the temperature of the reaction solution over time or periodically. However, the reaction container of the present invention can rapidly control the temperature of the reaction solution. By using it as a container, the time required for PCR can be reduced. In addition, PCR is a technique for amplifying a very small amount of template DNA, so contamination of other DNA becomes a serious problem.However, the reaction container of the present invention can prevent contamination to a reaction solution. Since the reaction container of the present invention can be used as a reaction container for PCR, the intended PCR can be performed accurately. Furthermore, since the reaction container of the present invention can suppress evaporation of the reaction solution contained in the reaction chamber, by using the reaction container of the present invention as a reaction container for PCR, a very small amount of the reaction solution for PCR can be obtained. However, it is possible to proceed with PCR. Furthermore, by using the reaction container of the present invention as a PCR reaction container, the progress of PCR can be monitored in real time.
When the reaction container according to this aspect is used, preparation of a sample containing a target nucleic acid (for example, extraction of a nucleic acid from a cell), amplification of a target nucleic acid by PCR, progress of PCR (for example, presence or absence of amplification of a target nucleic acid) , The amount of PCR amplification product, etc.) (for example, detection, measurement, qualitative analysis, quantitative analysis, etc.), and a series of operations such as acquisition of PCR amplification product can be automated.
(30) In order to achieve the above object, a first reaction apparatus of the present invention is a reaction apparatus including the reaction vessel according to the first aspect, a temperature control device, a light source, and a fluorescence detector, wherein The lid member and / or the reaction vessel body so that a control device can control the temperature of the reaction solution accommodated in the reaction chamber via the contact surface of the lid member and / or the contact surface of the reaction chamber. The light source is provided so as to be able to irradiate the reaction liquid contained in the reaction chamber with light through the contact surface of the lid material and / or the contact surface of the reaction chamber, The fluorescence detector is provided so as to be able to detect fluorescence emitted from a reaction solution contained in the reaction chamber via a contact surface of the lid member and / or a contact surface of the reaction chamber. And
In the first reaction device of the present invention, the temperature control device is attached to the lid and / or the reaction vessel main body, and the reaction liquid is moved by heat transfer through the contact surface of the lid and / or the contact surface of the reaction chamber. Temperature control can be performed quickly. The temperature control device may be directly attached to the lid member and / or the reaction vessel main body, or may be attached via another member. For example, when the reaction container of the present invention is provided with a heat conductive metal block or a heat conductive metal plate so as to be in contact with the reaction container main body and / or the lid member, the heat conductive metal block or the heat conductive metal plate may be used. The temperature control device can be attached to the lid member and / or the reaction vessel main body via the conductive metal plate. In the first reaction apparatus of the present invention, the temperature control device may be attached to only one of the lid member and the reaction vessel main body, but from the viewpoint of quickly controlling the temperature of the reaction solution, the lid member and the reaction vessel Preferably, it is attached to both of the main bodies.
In the first reaction apparatus of the present invention, the light source can irradiate the reaction liquid contained in the reaction chamber with excitation light via the contact surface of the lid member and / or the contact surface of the reaction chamber.
Further, in the first reaction device of the present invention, the fluorescence detector may detect the fluorescence emitted from the reaction solution contained in the reaction chamber via the contact surface of the lid member and / or the contact surface of the reaction chamber. it can.
In the first reaction device of the present invention, the reaction surface can be irradiated with excitation light and the fluorescence emitted from the reaction solution can be detected by arbitrarily combining the contact surface of the lid member and the contact surface of the reaction chamber. . That is, both the irradiation of the excitation light to the reaction solution and the detection of the fluorescence emitted from the reaction solution may be performed through the contact surface of the lid material, or both may be performed through the contact surface of the reaction chamber. May be performed through the contact surface of the lid member and the contact surface of the reaction chamber, respectively, or may be performed through the contact surface of the reaction chamber and the contact surface of the lid member, respectively.
In the first reaction apparatus of the present invention, the reaction solution can be irradiated with excitation light while the reaction solution is housed in the reaction chamber, and fluorescence emitted from the reaction solution can be detected. Therefore, if a phosphor that can be an indicator of the progress of the reaction is added to the reaction solution, the progress of the reaction can be monitored by detecting the fluorescence emitted by the phosphor during the progress of the reaction. In particular, in the first reaction apparatus of the present invention, the progress of the reaction occurring in the reaction chamber is monitored in real time (that is, Monitoring (on the fly).
(31) In the first aspect of the first reaction device of the present invention, the temperature control device is a wall portion having a substantially uniform thickness constituting the lid member and having a contact surface with the lid member. And / or is attached to a wall having a substantially uniform thickness constituting the reaction vessel main body and having a contact surface with the reaction chamber.
In the first reaction apparatus according to the present aspect, the temperature of the reaction solution accommodated in the reaction chamber is controlled by controlling the thickness of the wall having the contact surface of the lid material and the thickness of the wall having a substantially uniform thickness and / or the contact surface of the reaction chamber. Can be performed through a substantially uniform wall portion, whereby temperature control of the reaction solution can be performed quickly and efficiently. Further, setting of the temperature control condition at that time is also easy.
Irradiation of the excitation light to the reaction solution accommodated in the reaction chamber or detection of fluorescence from the reaction solution is performed by using a wall portion having a contact surface of a lid material and a substantially uniform thickness and / or a contact surface of the reaction chamber. This can be performed through a wall portion having a substantially uniform thickness, which facilitates setting of irradiation conditions of excitation light and light reception conditions of fluorescence. In particular, when a part or the whole of the contact surface of the lid material or the contact surface of the reaction chamber is a flat surface, the wall portion has a flat plate shape, and the contact surface of the lid material on the wall portion and the surface on the opposite side thereof, Alternatively, since the contact surface of the reaction chamber and the surface on the opposite side are substantially parallel, setting of the irradiation condition of the excitation light and the reception condition of the fluorescence becomes easier.
In the first reaction device according to this aspect, the temperature control device may be directly attached to the wall, or may be attached via another member. For example, when a heat conductive metal block or a heat conductive metal plate is provided so as to be in contact with the wall, the temperature control device is attached via the heat conductive metal block or the heat conductive metal plate. Can be.
(32) In the second aspect of the first reaction apparatus of the present invention, the reaction vessel is the reaction vessel according to the twelfth aspect, and the temperature control device is configured such that the contact surface of the lid material and / or the reaction The lid and / or the reaction vessel main body are attached to the reaction chamber so that the temperature of the reaction solution accommodated in the reaction chamber can be controlled through the facing surface of the chamber, and the light source is accommodated in the reaction chamber. The reaction solution is provided so as to be able to irradiate light through the surrounding surface of the reaction chamber, and the fluorescence detector emits fluorescence emitted from the reaction solution accommodated in the reaction chamber to the reaction chamber. It is provided so that it can be detected through the surrounding surface.
In the first reaction apparatus according to this aspect, the temperature of the reaction solution contained in the reaction chamber is rapidly controlled by heat transfer through the contact surface of the lid member and / or the opposing surface of the reaction chamber. By irradiating the reaction solution with the excitation light through the surrounding surface and detecting the fluorescence emitted from the reaction solution, the progress of the reaction occurring in the reaction chamber can be monitored in real time. In particular, in the first reaction apparatus according to this embodiment, the surface used for controlling the temperature of the reaction solution (the contact surface of the lid member and / or the surface facing the reaction chamber) and the surface used for monitoring the progress of the reaction ( Since the reaction chamber is separated from the reaction chamber, the area for monitoring the progress of the reaction can be freely set, and the progress of the reaction in the entire reaction solution can be monitored.
(33) In the third aspect of the first reaction device of the present invention, the temperature control device may be a wall having a substantially uniform thickness constituting the lid and having a contact surface with the lid. And / or is attached to a wall having a substantially uniform thickness constituting the reaction vessel main body and having a wall facing the reaction chamber.
In the first reaction apparatus according to this aspect, the temperature of the reaction solution accommodated in the reaction chamber is controlled by controlling the thickness of the wall having a contact surface of the lid member and having a substantially uniform thickness and / or the opposing surface of the reaction chamber. Can be performed through a substantially uniform wall portion, whereby temperature control of the reaction solution can be performed quickly and efficiently. Further, setting of the temperature control condition at that time is also easy.
Irradiation of the excitation light to the reaction solution accommodated in the reaction chamber or detection of fluorescence from the reaction solution is performed by using a wall portion having a contact surface of a lid material and having a substantially uniform thickness and / or a facing surface of the reaction chamber. This can be performed through a wall portion having a substantially uniform thickness, which facilitates setting of irradiation conditions of excitation light and light reception conditions of fluorescence. In particular, when a part or the whole of the contact surface of the lid material or the opposing surface of the reaction chamber is a flat surface, the wall portion has a flat plate shape, and the contact surface of the lid material on the wall portion and the surface on the opposite side thereof, Alternatively, since the opposing surface of the reaction chamber and the surface on the opposite side are substantially parallel, setting of the irradiation condition of the excitation light and the light receiving condition of the fluorescence becomes easier.
In the first reaction device according to this aspect, the temperature control device may be directly attached to the wall, or may be attached via another member. For example, when a heat conductive metal block or a heat conductive metal plate is provided so as to be in contact with the wall, the temperature control device is attached via the heat conductive metal block or the heat conductive metal plate. Can be.
(34) In a fourth aspect of the first reaction apparatus of the present invention, the reaction apparatus further includes a plurality of optical fibers arranged around a surrounding surface of the reaction chamber, and irradiates the reaction solution with light from the light source and / or Alternatively, detection of fluorescence emitted from the reaction solution is performed using the optical fiber.
In the first reaction apparatus according to this aspect, the optical fiber is disposed, for example, around a surface of the wall that forms the lid member and has the surrounding surface of the reaction chamber, on a surface opposite to the surrounding surface of the reaction chamber. When a part or the whole of the surrounding of the reaction chamber is flat and the thickness of the wall having the surrounding surface of the reaction chamber is substantially uniform, the wall has a flat plate shape, and the reaction chamber in the wall has Since the surrounding surface and the surface on the opposite side are almost parallel, by arranging the optical fiber perpendicular to the wall having the surrounding surface of the reaction chamber, it is easy to set the irradiation condition of the excitation light and the reception condition of the fluorescence. It becomes.
When multiple optical fibers are used to irradiate the reaction liquid and detect fluorescence from the reaction liquid, the irradiation area of each optical fiber is small. The area to be excited becomes smaller, and the fluorescence intensity emitted from that area becomes weaker. Therefore, when irradiating the reaction liquid existing in a thin state between the contact surface of the lid member and the facing surface of the reaction chamber with excitation light via the contact surface of the lid member and / or the facing surface of the reaction chamber. When optical fibers are used, the detection sensitivity of each optical fiber is low.
On the other hand, when the excitation light is applied to the reaction liquid existing in a thin state between the contact surface of the lid member and the opposite surface of the reaction chamber through the surrounding surface of the reaction chamber, the irradiation of each optical fiber is performed. Even though the area is small, the distance through which the irradiation light passes through the reaction solution is long, so that the region excited by each optical fiber is large, and the intensity of the fluorescence emitted from that region is high. Therefore, the detection sensitivity of each optical fiber is high.
Therefore, in the reaction vessel according to this aspect, the progress of the reaction in the reaction solution present in a thin state between the contact surface of the lid member and the facing surface of the reaction chamber is monitored with high sensitivity using an optical fiber. be able to.
In the first reaction device according to the present embodiment, one optical fiber may perform both irradiation of light from the light source to the reaction solution and detection of fluorescence emitted from the reaction solution, or may perform only one of them. . Further, an optical fiber used for irradiating the reaction solution with light from a light source and an optical fiber used for detecting fluorescence emitted from the reaction solution can be arbitrarily arranged around the surrounding surface of the reaction chamber. The type of irradiation light and the type of detected fluorescence may be the same for all optical fibers, or may be different for each optical fiber or each optical fiber group.
In the first reaction apparatus according to the present embodiment, a plurality of different fluorescent dyes are contained in a reaction solution accommodated in a reaction chamber, and irradiation of excitation light corresponding to each fluorescent dye and fluorescence emitted from each fluorescent dye are performed. Is performed for each optical fiber or each optical fiber group, whereby different PCRs are allowed to proceed simultaneously, and the progress of each reaction (the presence or absence of amplification of the target nucleic acid by each PCR, the amount of each PCR amplification product, etc.) is determined. It can be monitored in real time. When the same excitation light is irradiated by multiple optical fibers and the same fluorescence is detected, the progress of the reaction in the entire reaction solution is monitored by arranging the optical fibers around the entire surrounding surface of the reaction chamber. can do.
(35) In order to achieve the above object, a second reaction apparatus of the present invention includes a reaction vessel installation section in which the reaction vessel according to the seventeenth aspect or the eighteenth aspect is installed, and a first temperature control device. And a second temperature control device, a light source, and a fluorescence detector, wherein the first temperature control device is configured to seal the reaction container installed in the reaction container installation portion. The temperature of the reaction solution contained in the space is provided so as to be controllable through the contact surface of the reaction chamber, and the second temperature control device is attached to and detached from the nozzle chip fitting space of the lid member. Is mounted so as to be able to control the temperature of the reaction solution contained in the closed space of the reaction vessel installed in the reaction vessel installation section through the contact surface of the lid material, The light source is provided in the reaction vessel installed in the reaction vessel installation section. The reaction solution accommodated in the space is provided so as to be able to irradiate light through the contact surface of the lid material and / or the contact surface of the reaction chamber, and the fluorescence detector is provided in the reaction container installation section. Provided so that fluorescence emitted from a reaction solution contained in the closed space of the reaction vessel installed in the reaction vessel can be detected through the contact surface of the lid member and / or the contact surface of the reaction chamber. It is characterized by the following.
In the second reaction apparatus of the present invention, the reaction vessel before the reaction is installed in the reaction vessel installation portion, the temperature of the reaction solution contained in the closed space in the reaction vessel, the first and second temperature control device Controlled by. The first and second temperature control units include, for example, a heat conductive metal block or a heat conductive metal plate provided so as to be in contact with the reaction vessel main body or the lid material, and the first temperature control device The temperature of the reaction solution is controlled via the contact surface of the chamber, and the second temperature control device controls the temperature of the reaction solution via the contact surface of the lid. The second temperature control device is adapted to be attached to and detached from the nozzle chip fitting space of the lid member, is attached to the nozzle chip fitting space at the time of reaction, and is detached from the nozzle chip fitting space after the reaction. Is done.
In the second reaction apparatus of the present invention, the excitation light can be radiated from the light source to the reaction solution via the contact surface of the lid and / or the contact surface of the reaction chamber, and the fluorescence emitted from the reaction solution is brought into contact with the lid. It can be detected by a fluorescence detector via the surface and / or the contact surface of the reaction chamber. Thus, the progress of the reaction occurring in the reaction solution can be monitored in real time (that is, immediately during the progress of the reaction) while controlling the temperature of the reaction solution to progress the target reaction.
(36) In the first aspect of the second reaction apparatus of the present invention, the reaction vessel is the reaction vessel according to the eighteenth aspect, and the first temperature control device is installed in the reaction vessel installation section. The temperature of the reaction solution accommodated in the closed space of the reaction vessel is provided so as to be controllable through the opposed surface of the reaction chamber, and the light source is installed in the reaction vessel installation section. The reaction solution accommodated in the closed space of the reaction container is provided so as to be able to irradiate light through the surrounding surface of the reaction chamber, the fluorescence detector is installed in the reaction container installation part The fluorescent light emitted from the reaction solution contained in the closed space of the reaction container is provided so as to be detectable through the surrounding surface of the reaction chamber.
In the second reaction apparatus according to the present embodiment, the temperature of the reaction solution contained in the reaction chamber is rapidly controlled by heat transfer through the contact surface of the lid member and the opposing surface of the reaction chamber, thereby enclosing the reaction chamber. By irradiating the reaction solution with the excitation light through the surface and detecting the fluorescence emitted from the reaction solution, the progress of the reaction occurring in the reaction chamber can be monitored in real time. In particular, in the second reaction apparatus according to this embodiment, the surface used for controlling the temperature of the reaction solution (the contact surface of the lid member and the surface facing the reaction chamber) and the surface used for monitoring the progress of the reaction (the reaction chamber) ), The region for monitoring the progress of the reaction can be freely set, and the progress of the reaction in the entire reaction solution can be monitored.
(37) In the second embodiment of the second reaction apparatus of the present invention, the reaction apparatus further includes a plurality of optical fibers arranged around the surrounding surface of the reaction chamber, and the light source irradiates the reaction solution with light and / or Alternatively, detection of fluorescence emitted from the reaction solution is performed using the optical fiber.
In the second reaction device according to the present embodiment, the detection of fluorescence using an optical fiber can be performed in the same manner as in the first reaction device according to the fourth embodiment, and similar to the first reaction device according to the fourth embodiment. The effect of can be obtained.
(38) In the third aspect of the second reaction apparatus of the present invention, the temperature control section further includes a temperature control section attaching / detaching section for attaching and detaching the second temperature control section to / from the nozzle chip fitting space. The attaching / detaching unit performs an operation of attaching the second temperature control device to the nozzle chip fitting space before the reaction, and an operation of detaching the second temperature control device from the nozzle chip fitting space after the reaction. .
(39) In a fourth embodiment of the second reaction apparatus of the present invention, the apparatus further comprises a perforation container installation section in which a perforation container is installed, a nozzle capable of sucking and discharging a liquid, and a nozzle transfer section, The perforation container includes a liquid storage space capable of storing a liquid, an opening communicating with the liquid storage space, and a piercing needle, and the liquid storage space extends from the opening to the liquid storage space. A reaction container is formed, the perforation needle is provided so as to protrude from the wall of the perforation container forming the liquid storage space into the liquid storage space, and the nozzle transfer unit is provided. However, the operation of fitting the nozzle tip mounted on the nozzle into the nozzle chip fitting space of the reaction vessel installed in the reaction vessel installation part, and the reaction vessel fitted with the nozzle tip , Said The operation of transferring to the container container for hole, and the reaction container is accommodated in the liquid storage space of the container for perforation installed in the container installation unit for perforation, and the perforation needle provided in the container for perforation, Performing an operation of forming a through-hole in the lid member and the reaction vessel main body, which communicates the liquid storage space of the perforation container, the sealed space of the reaction vessel, and the nozzle chip fitting space, and performs the nozzle, An operation of extracting the reaction liquid contained in the closed space of the reaction container into the liquid is performed by sucking and discharging the liquid contained in the liquid containing space of the perforating container through the through hole. .
In the second reaction device according to this aspect, the nozzle is transferred by the nozzle transfer unit, and the nozzle tip mounted on the nozzle is inserted into the nozzle chip fitting space of the reaction container after the reaction, which is installed in the reaction container installation unit. Is fitted. After fitting the nozzle tip into the nozzle tip fitting space, the nozzle is transferred by the nozzle transfer unit, and the reaction container is transferred from the reaction container installation unit to the perforated container installation unit. Next, the nozzle is transferred by the nozzle transfer unit, and the reaction container is accommodated in the liquid accommodating space of the perforation container installed in the perforation container installation unit. At this time, the reaction vessel is pressed against a perforation needle provided in the perforation container, and the perforation needle is used to pass through the target through-hole (that is, the liquid storage space of the perforation container, the closed space containing the reaction solution, and the nozzle). A through-hole communicating with the chip fitting space is formed in the lid member and the reaction vessel main body. Next, suction and discharge by the nozzle are started, and the liquid stored in the liquid storage space of the container for perforation is suctioned and discharged through the through hole. By repeating suction and discharge by the nozzle, the reaction liquid contained in the closed space of the reaction container is extracted into the liquid. With the extraction of the reaction solution, the reaction products contained in the reaction solution are also extracted into the liquid.
As described above, if the second reaction apparatus according to the present embodiment is used, the reaction is performed in a state where the lid member is attached to the reaction container main body, and then the reaction container is removed without detaching the lid member from the reaction container main body. The reaction product contained in the reaction solution inside can be obtained.
In the second reaction device according to the present embodiment, the nozzle may have any structure as long as it can suck and discharge the liquid, for example, the same structure as the nozzle used in a known dispensing device. Can be used. Further, the nozzle transfer section may have any structure as long as it performs a predetermined operation.
The operation of fitting the nozzle chip into the nozzle chip fitting space is performed after the operation of detaching the second temperature control device from the nozzle chip fitting space. Further, the operation of fitting the nozzle chip into the nozzle chip fitting space and the operation of attaching and detaching the second temperature control device from the nozzle chip fitting space are controlled so as not to interfere with each other.
(40) In the fifth embodiment of the first and second reactors of the present invention, the reactor is a PCR reactor.
In the reaction device according to this aspect, the reaction generated in the reaction chamber is PCR, and the reaction solution accommodated in the reaction chamber is a reaction solution for PCR. In the reaction apparatus according to the present embodiment, the PCR is performed in a short time by rapidly controlling the temperature of the reaction solution for PCR, and the progress of the PCR (for example, whether or not the target nucleic acid is amplified by the PCR, the amount of the PCR amplification product). Etc.) in real time.
The reaction apparatus according to this aspect includes preparation of a sample containing a target nucleic acid (for example, extraction of a nucleic acid from a cell), amplification of a target nucleic acid by PCR, progress of PCR (for example, presence or absence of amplification of a target nucleic acid, PCR amplification). It enables the automation of a series of tasks such as monitoring of the amount of product (eg, detection, measurement, qualitative analysis, quantitative analysis, etc.).
(41) In order to achieve the above object, the method of the present invention comprises a step (a) of bringing a reaction solution contained in a reaction chamber into contact with a contact member, and a step of contacting the reaction solution with the reaction chamber and / or Or (b) controlling the temperature of the reaction solution via a contact surface between the reaction solution and the contact member, and a contact surface between the reaction solution and the reaction chamber and / or the reaction solution and the contact member. (C) irradiating the reaction solution with light via a contact surface with the reaction solution, and / or via a contact surface between the reaction solution and the reaction chamber and / or a contact surface between the reaction solution and the contact member. (D) detecting the fluorescence emitted from the reaction solution.
In the method of the present invention, step (b) is preferably performed after step (a). Thus, the temperature of the reaction solution can be quickly controlled through the contact surface between the reaction solution and the reaction chamber and the contact surface between the reaction solution and the contact member. The temperature control of the reaction solution via the contact surface between the reaction solution and the reaction chamber in the step (b) can be performed before the step (a) and simultaneously with the step (a).
In the method of the present invention, step (c) and step (d) are preferably performed after step (a). Thus, the progress of the reaction can be monitored while the reaction proceeds while rapidly controlling the temperature of the reaction solution. The reaction solution is irradiated with light through the contact surface between the reaction solution and the reaction chamber in step (c), and the fluorescence emitted from the reaction solution through the contact surface between the reaction solution and the reaction chamber in step (d). Can be detected before step (a) and simultaneously with step (a).
Further, in the method of the present invention, preferably, step (b), step (c) and step (d) are performed simultaneously. Thus, the progress of the reaction can be monitored in real time while the temperature of the reaction solution is quickly controlled to progress the reaction.
The method of the present invention can be carried out, for example, using the reaction vessel of the present invention or the reactor of the present invention.
(42) In the first embodiment of the method of the present invention, a contact surface of the reaction chamber used for controlling the temperature of the reaction solution, and a contact surface of the reaction chamber used for irradiating the reaction solution with light. Or, the contact surface of the reaction chamber used for detection of fluorescence from the reaction solution is different from the contact surface.
In the reaction according to this embodiment, of the surfaces where the reaction chamber comes into contact with the reaction solution (the contact surfaces of the reaction chambers), the surface used for controlling the temperature of the reaction solution (the surface used in step (b)) and the reaction By making the surface used for monitoring the progress (the surface used in step (c) and / or step (d)) separate, the temperature of the reaction solution can be quickly controlled and the reaction can be performed quickly. The area for monitoring the progress of the process can be set freely. This makes it possible to monitor the progress of the reaction in the entire reaction solution.
(43) In the second aspect of the method of the present invention, the front contact member is formed with a nozzle chip fitting space in which a nozzle chip mounted on a nozzle capable of sucking and discharging a liquid can be fitted. After the reaction in the reaction chamber is completed, a step of forming a through hole for communicating the outside of the reaction chamber, the inside of the reaction chamber, and the nozzle chip fitting space with a perforation needle provided outside the reaction chamber ( e), a step (f) of fitting the nozzle tip mounted on the nozzle into the nozzle chip fitting space, a step (g) of bringing the outside of the reaction chamber into contact with a liquid, and operating the nozzle. And (h) extracting the reaction liquid contained in the reaction chamber into the liquid by sucking and discharging the liquid through the through-hole.
In the method according to this embodiment, steps (e), (f) and (g) can be performed in any order. Step (e) is performed after completion of the reaction in the reaction chamber. Steps (f) and (g) are performed before the completion of the reaction in the reaction chamber (including both before the start of the reaction and in the reaction progress stage). The reaction may be performed, or may be performed after completion of the reaction. When using the second reactor according to the fourth aspect, of the steps (e), (f) and (g), the step (f) is performed first, and then the steps (e) and (g) are performed. ) In any order. Step (h) is performed after performing steps (e), (f) and (g).
(44) In the third aspect of the method of the present invention, the reaction occurring in the reaction chamber is PCR.
In the method according to this aspect, the reaction solution accommodated in the reaction chamber is a reaction solution for PCR. In the method according to the present embodiment, the progress of the PCR (for example, whether or not the target nucleic acid is amplified by PCR, the amount of ) Can be monitored in real time.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
(First embodiment)
FIG. 1 is a sectional view showing a first embodiment of a reaction vessel according to the present invention, FIG. 2 is a top view of a reaction vessel body of the reaction vessel according to the first embodiment, and FIG. FIG. 4 is a cross-sectional view showing a state where the lid member is attached to the reaction container body in the reaction vessel according to the first embodiment, and FIG. 5 is a first embodiment of the reaction apparatus according to the present invention. FIGS. 6 (i) to (iii) are schematic partial cross-sectional views showing the configuration, and are explanatory diagrams showing examples of the arrangement of optical fibers.
As shown in FIGS. 1 and 4, the reaction vessel 1a according to the present embodiment includes a reaction vessel main body 2a and a lid 3a.
As shown in FIGS. 1 and 2, the reaction vessel main body 2a has a rectangular bottom plate portion 22a in plan view, and a rectangular tubular first member standing upright from the periphery of the bottom plate portion 22a while maintaining the same diameter. The side plate portion 23a, a rectangular cylindrical second side plate portion 24a that is erected so as to gradually increase in diameter upward from the upper end portion of the first side plate portion 23a, and the same diameter upward from the upper end portion of the second side plate portion 24a. The third side plate 25a has a rectangular tubular shape and stands upright while maintaining the above-mentioned condition, and a protruding edge 26a provided at the upper end of the third side plate 25a.
As shown in FIG. 2, the cross section of the first side plate portion 23a, the second side plate portion 24a, and the third side plate portion 25a is rectangular, and the inner peripheral surface 213a of the first side plate portion 23a, the second side plate portion The inner peripheral surface 214a of 24a and the inner peripheral surface 215a of the third side plate portion 25a are each formed by four planes. The rectangular shape includes a square as well as a rectangle.
As shown in FIG. 1, a concave portion 261a is provided in the protruding edge portion 26a of the reaction vessel main body 2a, and when the lid member 3a is attached to the reaction vessel main body 2a, as shown in FIG. The concave portion 261a of the main body 2a and the convex portion 37a of the lid 3a are fitted to fix the lid 3a to the reaction vessel main body 2a.
As shown in FIG. 1, the reaction vessel main body 2a includes a reaction chamber 21a having an opening 211a at an upper end and capable of storing a reaction solution 4a. As shown in FIG. 1, the reaction chamber 21a is formed in the reaction vessel main body 2a as a recess having an opening 211a at the upper end. The reaction chamber 21a is a recess formed by the bottom plate 22a, the first side plate 23a, the second side plate 24a, and the third side plate 25a, and the upper surface 212a of the bottom plate 22a corresponds to the bottom of the reaction chamber 21a. The inner peripheral surface 213a of the first side plate portion 23a, the inner peripheral surface 214a of the second side plate portion 24a, and the inner peripheral surface 215a of the third side plate portion 25a correspond to the inner peripheral surface of the reaction chamber 21a.
As shown in FIG. 1, the opening area of the opening 211a of the reaction chamber 21a is slightly larger than the area of the bottom surface of the reaction chamber 21a, and the reaction liquid 4a added from the opening 211a remains unchanged (the reaction liquid 4a). (Without applying any force other than gravity downward). Depending on the manner in which the reaction solution 4a is added, the reaction solution 4a may adhere to the inner peripheral surface of the reaction chamber 21a. In such a case, the reaction container body 2a is vibrated using a vortex mixer or the like. Thereby, the reaction liquid 4a can reach the bottom of the reaction chamber 21a.
As shown in FIG. 1, the bottom plate 22a, the first side plate 23a, the second side plate 24a, and the third side plate 25a have a substantially uniform thickness. The “substantially uniform thickness” includes a case where the thickness is uniform. The thickness of each plate portion can be appropriately changed, but the bottom plate portion 22a is preferably a thin plate from the viewpoint of quick temperature control of the reaction solution 4a accommodated in the reaction chamber 21a. Further, the first side plate portion 23a is preferably a thin plate from the viewpoint that the conditions for irradiating the reaction solution 4a contained in the reaction chamber 21a with the excitation light and the conditions for detecting the fluorescence emitted from the reaction solution 4a are easily set. . The thickness of the thin plate can be appropriately determined depending on the material constituting the thin plate and the like. For example, in the case of plastic, the thickness is preferably about 0.1 to 0.5 mm.
As shown in FIG. 1, the number of the reaction chambers 21a provided in the reaction vessel main body 2a is one, but the number and the position of the reaction chambers provided in the reaction vessel main body can be appropriately changed. For example, the reaction vessel main body may include eight reaction chambers arranged in a line, or may include a total of 96 reaction chambers of eight columns × 12 rows. When the number of reaction chambers provided in the reaction vessel main body is plural, sample processing can be performed efficiently. For example, since a sample dispensing device equipped with an eight-nozzle unit is commercially available, such a sample dispensing device is used when the reaction vessel body has eight reaction chambers in one row. By doing so, the dispensing of the reaction solution into the reaction chamber can be automated.
As shown in FIG. 1, before the lid member 3a is attached to the reaction vessel main body 2a, the reaction solution 4a contained in the reaction chamber 21a contacts the bottom surface and the inner peripheral surface of the reaction chamber 21a. ing.
As shown in FIG. 1, the lid member 3a has a convex portion 31a projecting downward, and a flat plate portion 36a provided at an upper end portion of the convex portion 31a.
As shown in FIG. 1, a protruding edge protruding downward is provided at the peripheral edge of the flat plate portion 36a, and a protruding portion 37a protruding in the direction of the protruding portion 31 is provided at the lower end of the protruding edge. Have been. When the lid member 3a is attached to the reaction container main body 2, as shown in FIG. 4, the concave portion 261a of the reaction container main body 2a and the convex portion 37a of the lid member 3a are fitted, and the lid member 3a is fitted to the reaction container main body 2a. It is fixed.
As shown in FIGS. 1 and 3, the convex portion 31 a has a pressing portion 32 a formed of a flat plate having a rectangular shape in a plan view, and a rectangular cylindrical shape that is erected so as to gradually increase in diameter upward from the periphery of the pressing portion 32 a. A first side plate portion 33a, a second side plate portion 34a of a rectangular tubular shape erected so as to gradually increase in diameter upward from an upper end portion of the first side plate portion 33a, and upward from an upper end portion of the second side plate portion 34a. The third side plate portion 35a is a rectangular tubular member that stands upright while maintaining the same diameter, and is continuous with the flat plate portion 36a at the upper end of the third side plate portion 35a.
As shown in FIG. 3, the cross section of the first side plate portion 33a, the second side plate portion 34a and the third side plate portion 35a is rectangular, and the outer peripheral surface 313a of the first side plate portion 33a and the second side plate portion 34 are formed. And the outer peripheral surface 315a of the third side plate portion 35a are each formed by four planes. The rectangular shape includes a square as well as a rectangle.
As shown in FIG. 1, the pressing portion 32a, the first side plate portion 33a, the second side plate portion 34a, and the third side plate portion 35a have a substantially uniform thickness. The “substantially uniform thickness” includes a case where the thickness is uniform. Although the thickness of each plate portion can be appropriately changed, the pressing portion 32a is preferably a thin plate in terms of quick temperature control of the reaction solution 4a accommodated in the reaction chamber 21a. The thickness of the thin plate can be appropriately determined depending on the material constituting the thin plate and the like. For example, in the case of plastic, the thickness is preferably about 0.1 to 0.5 mm.
As shown in FIGS. 1 and 3, the number of the convex portions 31 a included in the lid member 3 a is one, and the number and the position of the convex portions in the lid member are determined by the number and the position of the reaction chambers provided in the reaction container body. Can be changed as appropriate.
The convex portion 31a is provided on the lid member 3a so as to fit with the reaction chamber 21a formed as a concave portion on the reaction container main body 2a. When the lid member 3a is attached to the reaction container main body 2a, As shown in FIG. 4, the opening 211a of the reaction chamber 21a is sealed by the lid 3a.
The convex portion 31a is provided such that the lower surface 312a of the pressing portion 32a of the convex portion 31a does not contact the bottom surface of the reaction chamber 21a when the lid 3a is attached to the reaction vessel main body 2a. Therefore, when the lid member 3a is attached to the reaction vessel main body 2a, as shown in FIG. 4, a gap is formed between the lower surface 312a of the pressing portion 32a of the convex portion 31a and the bottom surface of the reaction chamber 21a (sealed space S1a). Is formed.
The pressing portion 32a of the convex portion 31a is provided so as to face the bottom plate portion 22a of the reaction container main body 2a when the lid 3a is attached to the reaction container main body 2a. 4, the lower surface 312 (contact surface of the lid member) of the pressing portion 32a of the convex portion 31a and the upper surface 212a of the bottom plate portion 22a of the reaction vessel main body 2a (opposing surface of the reaction chamber) as shown in FIG. ), The reaction liquid 4a is accommodated in a thin shape.
At this time, the lower surface 312a (the contact surface of the lid member) of the pressing portion 32a of the convex portion 31a and the upper surface 212a of the bottom plate portion 22a of the reaction vessel main body 2a (the facing surface of the reaction chamber) are present in a thin shape. The reaction solution 4a is in a state of being surrounded by the inner peripheral surface 213a (surrounding surface of the reaction chamber) of the first side plate 23a of the reaction vessel main body 2a. That is, when the lid member 3a is attached to the reaction container body 2a, as shown in FIG. 4, the lower surface 312a (contact surface of the lid member) of the pressing portion 32a of the convex portion 31a and the bottom plate portion 22a of the reaction container body 2a. And the inner peripheral surface 213a (surrounding surface of the reaction chamber) of the first side plate portion 23a of the reaction vessel body 2a, form a closed space S1a, and a part of the reaction solution 4a The thin space is accommodated in the closed space S1a.
As shown in FIG. 4, the reaction liquid 4a contained in the closed space S1a is applied to the lower surface 312a (the contact surface of the lid member) of the pressing portion 32a of the convex portion 31a and the upper surface 212a of the bottom plate portion 22a of the reaction container main body 2a (the reaction surface). And the inner peripheral surface 213a (surrounding surface of the reaction chamber) of the first side plate 23a of the reaction vessel main body 2a.
The pressing portion 32a of the convex portion 31a is provided so as to press the reaction solution 4a contained in the reaction chamber 21a in the process of attaching the lid member 3a to the reaction container body 2a. Thereby, the reaction liquid 4a is gradually extruded to the upper part of the reaction chamber 21a. Then, when the lid 3a is attached to the reaction vessel main body 2a, as shown in FIG. 4, the outer peripheral surface 313a of the first side plate 33a of the lid 3a and the second side plate 24a of the reaction vessel main body 2a are formed. The inner peripheral surface 214a comes into contact with the inner peripheral surface 214a. As a result, the downward movement of the pressing portion 32a is restricted, and the lower surface 312a (the contact surface of the lid member) of the pressing portion 32a of the convex portion 31a and the upper surface 212a of the bottom plate portion 22a of the reaction vessel main body 2a (the opposing surface of the reaction chamber). ) Is constant. In the present embodiment, the surface (contact surface) where the lid member 3a and the reaction vessel main body 2a come into contact with each other is tapered, but, for example, with respect to the direction in which the lid material is attached to the reaction vessel main body. A vertical surface may be used as the contact surface.
When the lid 3a is attached to the reaction vessel main body 2a, as shown in FIG. 4, the outer surface 313a of the first side plate 33a of the lid 3a and the inner peripheral surface of the second side plate 24a of the reaction vessel main body 2a. 214a and the outer surface 315a of the third side plate 35a of the lid 3a and the inner peripheral surface 215a of the third side plate 25a of the reaction vessel body 2a. . Thus, the hermeticity of the sealed space S1a can be maintained, and contamination of the reaction solution 4a contained in the sealed space S1a can be prevented.
On the other hand, even if the lid 3a is attached to the reaction vessel main body 2a, as shown in FIG. 4, the second side plate 34a of the lid 3a and the second side plate 24a and the third side plate 25a of the reaction vessel main body 2a. And do not adhere. That is, between the second side plate portion 34a of the lid member 3a and the second side plate portion 24a and the third side plate portion 25a of the reaction vessel main body 2a, the remaining portion of the reaction liquid 4a that cannot be accommodated in the closed space S1a is accommodated. A closed space S2a (reaction liquid remaining portion accommodating portion) is formed.
When the reaction liquid 4a is pressed by the pressing portion 32a, the air in the reaction chamber 21a and the bubbles in the reaction liquid 4a are pushed out to the upper part of the reaction chamber 21a together with the reaction liquid 4a, and are stored in the closed space S2a. , A part of which is discharged to the outside of the reaction chamber 21a, it is possible to prevent air from entering the sealed space S1a and air bubbles from entering the reaction solution 4a accommodated in the sealed space S1a. .
The convex portion 31a has a lower surface 312a (contact surface of the lid member) of the pressing portion 32a of the convex portion 31a and an upper surface 212a of the bottom plate portion 22a of the reaction container main body 2a when the lid member 3a is attached to the reaction container main body 2a. The reaction solution 4a existing between the reaction solution 4a and the reaction solution 4a is provided to be thin. That is, when the lid 3a is attached to the reaction vessel body 2a, the projection 31a is formed between the lower surface 312a (contact surface of the lid) of the pressing portion 32a of the projection 31a and the bottom plate 22a of the reaction vessel body 2a. It is provided so that the distance to the upper surface 212a (the opposing surface of the reaction chamber) is reduced.
The distance between the lower surface 312a of the pressing portion 32a of the convex portion 31a (the contact surface of the lid member) and the upper surface 212a of the bottom plate 22a of the reaction vessel main body 2a (the surface facing the reaction chamber) (that is, the thickness of the thin reaction solution 4a). Is preferably 0.1 to 0.5 mm. Further, the distance between the lower surface 312a of the pressing portion 32a of the convex portion 31a (the contact surface of the lid member) and the upper surface 212a of the bottom plate portion 22a of the reaction vessel main body 2a (the surface facing the reaction chamber) (that is, the thin reaction solution 4). Is preferably uniform at any position.
The material of the reaction vessel main body 2a and the lid material 3a is a material that is not corroded by the reaction solution 4a, can withstand the reaction conditions (for example, reaction temperature) generated in the reaction chamber 21a, and has a light transmitting property. is there.
Since the whole reaction vessel body 2a is made of a light transmitting material, the reaction vessel 4a contained in the closed space S1a and the reaction solution 4a contained in the closed space S1a from the outside of the reaction vessel body 2a. Light can be transmitted to the outside of the reaction vessel main body 2a through the bottom plate 22a and the first side plate 23a of the reaction vessel main body 2a.
Further, since the entire lid 3a is also made of a light-transmitting material, the reaction solution 4a contained in the sealed space S1a and the reaction solution 4a contained in the sealed space S1a from outside the reaction vessel main body 2a. Light can be transmitted to the outside of the reaction vessel main body 2a through the pressing portion 32a of the lid 3a.
However, the reaction vessel body 2a and the lid 3a do not need to be entirely made of a light-transmitting material, and need to transmit light in order to monitor the progress of the reaction occurring in the reaction chamber 21a. It is sufficient if a certain portion is made of a light transmitting material.
For example, when irradiating the reaction solution 4a with excitation light and detecting fluorescence emitted from the reaction solution 4a via the first side plate portion 23a of the reaction container body 2a, the first side plate portion 23a of the reaction container body 2a is used. Is sufficient if it is composed of a light transmitting material. When irradiating the reaction solution 4a with excitation light and detecting fluorescence emitted from the reaction solution 4a via the bottom plate portion 22a of the reaction container body 2a, the bottom plate portion 22a of the reaction container body 2a is not transparent. It is sufficient if it is composed of a material. When the excitation light is applied to the reaction solution 4a and the fluorescence emitted from the reaction solution 4a is detected via the pressing portion 32a of the lid 3a, the pressing portion 32a of the lid 3a is made of a light-transmitting material. It is enough if it is configured.
Alternatively, only one of the reaction vessel main body 2a and the lid 3a may be made of a light-transmissive material, and the other may be made of a light-impermeable material. For example, when irradiating the reaction solution 4a with excitation light and detecting fluorescence emitted from the reaction solution 4a via the first side plate portion 23a of the reaction container body 2a, the lid 3a is made of a light-impermeable material. You may.
Examples of the material of the reaction container main body 2a and the lid material 3a include transparent or translucent thermoplastic resin and glass. If a thermoplastic resin is selected as the material for the reaction vessel main body 2a and the lid 3a, the reaction vessel main body 2a and the lid 3a can be easily formed by a conventional method such as injection molding. When the reaction temperature reaches a high temperature (for example, 90 to 100 ° C.), it is preferable to use a material having excellent heat resistance, for example, an engineering plastic.
As shown in FIG. 5, a reaction device 10a according to the present embodiment includes a reaction container 1a held by a pedestal 53a, a temperature control device 6a having thermoelectric semiconductor elements 61a and 62a, a light source 7a, and a fluorescence detector. 8a and a plurality of optical fibers 9a.
As shown in FIG. 5, the thermoelectric semiconductor element 61a included in the temperature control device 6a is attached to the pressing portion 32a (the upper surface of the pressing portion 32a) of the lid member 3a via the heat conductive metal plate 51a. The semiconductor element 62a is attached to the bottom plate portion 22a (the lower surface of the bottom plate portion 22a) of the reaction vessel main body 2a via a heat conductive metal plate 51a. The thermoelectric semiconductor element can be used as a cooling element (cooling) and / or used as a heating element (heating), and is, for example, a Peltier element.
The temperature control device 6a is configured to be able to control cooling and heating by the thermoelectric semiconductor devices 61a and 62a, and the thermoelectric semiconductor devices 61a and 62a are electrically connected to the temperature control device 6a. Further, as shown in FIG. 5, a heat radiating portion 52a having a cooling fin is mounted on the thermoelectric semiconductor elements 61a and 62a, so that the thermoelectric semiconductor elements 61a and 62a can be forcibly cooled. According to the temperature control device 6a, the reaction liquid accommodated in the closed space S1a of the reaction container 1a by the heat transfer through the pressing portion 32a of the lid 3a and the heat transfer through the bottom plate portion 22a of the reaction container main body 2a. 4a can be quickly controlled.
The reaction solution 4a is a reaction solution for PCR, and the amount of the reaction solution 4a accommodated in the closed space S1a is preferably about 2 to 50 μl. When the temperature of the reaction solution 4a is controlled by the temperature controller 6a, the PCR proceeds. At this time, since the reaction solution 4a is accommodated in the closed space S1a in a thin shape, the ratio of the surface area to the volume is large, and most of the surface area is the upper and lower surfaces of the thin layer, that is, the cover material 3a. It is occupied by the lower surface 312a of the pressing portion 32a (the contact surface of the lid member) and the upper surface 212a of the bottom plate portion 22a of the reaction vessel main body 2a (the surface facing the reaction chamber). Therefore, the temperature of the reaction solution 4a accommodated in the closed space S1a can be quickly controlled by the heat transfer through the pressing portion 32a of the lid member 3a and the heat transfer through the bottom plate portion 22a of the reaction vessel main body 2a. Thus, the time required for PCR can be reduced.
The light source 7a is a device that can emit excitation light that excites a fluorescent dye contained in the reaction solution 4a. As shown in FIG. 5, a plurality of optical fibers 9a are mounted on the light source 7a, and the excitation light emitted from the light source 7a is radiated through the optical fiber 9a. As shown in FIGS. 5 and 6 (ii), the optical fiber 9a is disposed around the first side plate portion 23a of the reaction vessel main body 2a (the outer peripheral surface of the first side plate portion 23a). The excitation light emitted through the reaction vessel 4a is applied to the reaction solution 4a contained in the closed space S1a via the first side plate 23a of the reaction vessel main body 2a.
The reaction solution 4a contains H₂O, buffer, MgCl₂, DNTP mix, primers, template DNA, Taq polymerase, etc., and a fluorescent dye which serves as an indicator of the progress of PCR (amplification of target nucleic acid, amount of PCR amplification product, etc.) such as ethidium bromide, SYBR Green I, Pico Green Etc. are contained. Therefore, when the reaction liquid 4a contained in the closed space S1a is irradiated with the excitation light, the fluorescent dye emits fluorescence. The fluorescent light emitted from the reaction solution 4a contained in the closed space S1a is transmitted to the outside of the reaction vessel main body 2a through the first side plate 23a of the reaction vessel main body 2a.
The fluorescence detector 8a is a device that can detect fluorescence emitted from the reaction solution 4a. As shown in FIG. 5, a plurality of optical fibers 9a are mounted on the fluorescence detector 8a, emitted from the reaction solution 4 contained in the closed space S1a, and passed through the first side plate 23a of the reaction container body 2a. The fluorescence transmitted to the outside of the reaction vessel main body 2a is received through the optical fiber 9a and is detected by the fluorescence detector 8a.
The configurations of the light source 7a and the fluorescence detector 8a are not particularly limited, and a general device including a filter, a reflecting mirror, a lens, and the like can be used.
Since the fluorescence intensity emitted from the reaction solution 4a is proportional to the amount of DNA contained in the reaction solution 4a, by detecting the fluorescence intensity, the progress of PCR (for example, the presence or absence of amplification of the target nucleic acid by PCR, the PCR amplification product Amount, etc.) can be monitored in real time (ie, immediately while the PCR reaction is in progress).
One end of the optical fiber 9a is attached to the light source 7a or the fluorescence detector 8a, and the other end is disposed around the first side plate portion 23a of the reaction vessel main body 2a (the outer peripheral surface of the first side plate portion 23a). I have.
FIG. 6 shows an example of the arrangement of the optical fibers 9a. FIG. 6 is a view corresponding to the AA cross-sectional view of FIG.
In FIG. 6 (i), one end of a plurality of optical fibers 9a is arranged on one surface of the outer peripheral surface of the first side plate portion 23a having a rectangular cross section, and the excitation light to the reaction liquid 4a is generated by each optical fiber 9a. And the reception of the fluorescence emitted from the reaction solution 4a.
In FIG. 6 (ii), one end of a plurality of optical fibers 9a is arranged on two opposing outer peripheral surfaces of a first side plate portion 23a having a rectangular cross section, and the reaction solution is formed by the optical fibers 9a arranged on one surface. The excitation light is radiated to 4a, and the optical fiber 9a arranged on the other surface can receive the fluorescence emitted from the reaction solution 4a.
In FIG. 6 (iii), one end of the plurality of optical fibers 9 is arranged on two orthogonal surfaces of the outer surface of the first side plate portion 23 having a rectangular cross section, and the reaction liquid is formed by the optical fibers 9 arranged on one surface. 4 is irradiated with excitation light, and the fluorescence emitted from the reaction liquid 4 can be received by the optical fiber 9 arranged on the other surface.
In the examples shown in FIGS. 6 (i) to (iii), the optical fibers are arranged so as to be orthogonal to the outer peripheral surface of the first side plate portion 23a, which facilitates setting of irradiation conditions and light receiving conditions. .
In the reaction device 10a, a portion (the pressing portion 32a of the lid 3a and the bottom plate 22a of the reaction container 2a) used for controlling the temperature of the reaction solution 4a, of the reaction container main body 2a and the lid 3a, Since the part used for monitoring the situation (the first side plate 23a of the reaction vessel body 2a) is separate, the temperature of the reaction solution 4a can be quickly controlled and the progress of the reaction is monitored. The area can be set freely.
In the reaction device 10a, the type of the excitation light to be irradiated and the type of the detected fluorescence may be the same for each optical fiber, or may be different for each optical fiber or each optical fiber group. A plurality of different fluorescent dyes are contained in the reaction solution 4a, and irradiation of excitation light corresponding to each fluorescent dye and detection of fluorescence emitted from each fluorescent dye are performed for each optical fiber or each optical fiber group, Different PCRs can proceed simultaneously, and the progress of each reaction (such as the presence or absence of amplification of the target nucleic acid by each PCR, the amount of each PCR amplification product, etc.) can be monitored in real time. In the case where the same excitation light is irradiated by a plurality of optical fibers and the same fluorescence is detected, the plurality of optical fibers are applied to the entire outer peripheral surface of the first side plate portion 23a having a rectangular cross section. By arranging, the entire reaction solution 4a can be irradiated with excitation light, fluorescence emitted from the entire reaction solution 4a can be detected, and the progress of the reaction in the entire reaction solution 4a can be monitored.
The first embodiment described above is described to facilitate understanding of the present invention, and does not limit the present invention in any way. Therefore, each element disclosed in the first embodiment is intended to include all design changes and equivalents belonging to the technical scope of the present invention.
For example, the optical fiber 9a can be arranged on the upper surface of the pressing portion 32a of the lid 3a. In this case, it is possible to irradiate the reaction solution 4a with excitation light and / or detect fluorescence emitted from the reaction solution 4a via the pressing portion 32a of the lid 3a.
Further, the optical fiber 9a can be arranged on the lower surface of the bottom plate 22a of the reaction vessel main body 2a. In this case, irradiation of the reaction liquid 4a with excitation light and / or detection of fluorescence emitted from the reaction liquid 4a can be performed via the bottom plate portion 22a of the reaction container main body 2a.
Further, the irradiation of the reaction solution 4a with the excitation light and the detection of the fluorescence emitted from the reaction solution 4a can be performed not by using an optical fiber but by using a lens or the like. By using the lens, it is possible to easily irradiate the entire reaction solution 4a with excitation light and detect fluorescence emitted from the entire reaction solution 4a.
(Second embodiment)
FIG. 7 is a cross-sectional view showing a second embodiment of the reaction vessel according to the present invention, and FIG. 8 (i) shows a state in which a lid member is attached to the reaction vessel body in the reaction vessel according to the second embodiment. FIG. 8 (ii) is a cross-sectional view showing a state in which a nozzle tip is fitted to a lid member attached to a reaction vessel main body in the reaction vessel according to the second embodiment, and FIG. FIG. 10 (i) is a partial cross-sectional view showing a second embodiment of such a reaction device, and FIG. 10 (i) is an exploded view showing the structure of a first temperature control unit and a second temperature control unit provided in the reaction device according to the second embodiment. FIG. 10 (ii) is a perspective view showing a state of the first temperature control unit and the second temperature control unit at the time of reaction, and FIG. 11 is a reaction vessel at the time of reaction in the reaction apparatus according to the second embodiment. FIG. 12 is a cross-sectional view showing the vicinity, and FIG. It is a partial sectional view illustrating the operation.
As shown in FIGS. 7 and 8, the reaction container 1b according to the present embodiment includes a reaction container main body 2b and a lid 3b.
As shown in FIG. 7, the reaction vessel main body 2b includes a disc-shaped bottom plate portion 22b, and a cylindrical first side plate portion 23b standing upright from the periphery of the bottom plate portion 22b so as to maintain the same diameter. The tapered second side plate 24b erected so as to gradually increase in diameter upward from the upper end of the first side plate 23b, and maintain the same diameter upward from the upper end of the second side plate 24b. It has an upright cylindrical third side plate portion 25b and a flange portion 26b provided on an upper peripheral portion of the third side plate portion 25b.
The bottom plate portion 22b and the first side plate portion 23b to the third side plate portion 25b of the reaction vessel main body 2b are made of a material that is not corroded by the reaction liquid and can withstand the reaction conditions (for example, reaction temperature) generated in the reaction chamber. It is made of a thin plate made of a material having light transmissivity (for example, a transparent or translucent thermoplastic resin, glass, or the like), and the thickness of the thin plate is preferably about 0.1 to 0.5 mm.
As shown in FIG. 7, a reaction chamber 20b surrounded by a bottom plate 22b and a first side plate 23b to a third side plate 25b is formed in the reaction vessel main body 2b. An opening 21b communicating with the reaction chamber 20b is formed.
The reaction chamber 20b can accommodate a reaction solution from the opening 21b. Further, since the reaction chamber 20b does not communicate with an opening other than the opening 21b, the opening 21b is sealed when the opening 21b is sealed (see FIG. 8 (i)).
The inner diameter of the third side plate portion 25b of the reaction container body 2b is substantially the same as the outer diameter of the second side plate portion 34b of the lid member 3b, and when the lid member 3b is attached to the reaction container main body 2, a reaction occurs. The inner peripheral surface of the third side plate portion 25b of the container main body 2b and the outer peripheral surface of the second side plate portion 34b of the lid member 3b are in close contact with each other (see FIG. 8 (i)).
As shown in FIG. 7, a convex portion 27b is provided on the inner peripheral surface of the third side plate portion 25b of the reaction vessel main body 2b, and the convex portion 27b is provided on the outer peripheral surface of the second side plate portion 34b of the lid member 3b. (See FIG. 8 (i)).
As shown in FIG. 7, a contact surface 28b is provided at an upper end portion of the first side plate portion 23b of the reaction vessel main body 2b, and the contact surface 28b is attached to the lid 3b by the reaction vessel main body 2b. When this is done, it comes into contact with the bottom plate 32b of the lid 3b (see FIG. 8 (i)).
As shown in FIG. 7, the lid member 3b includes a disc-shaped bottom plate portion 32b, a tapered first side plate portion 33b that is erected so as to gradually increase in diameter upward from the periphery of the bottom plate portion 32b, It has a cylindrical second side plate portion 34b erected so as to maintain the same diameter upward from the upper end portion of the one side plate portion 33b, and a flange portion 35b provided at an upper peripheral edge of the second side plate portion 34b. .
The bottom plate portion 32b, the first side plate portion 33b, and the second side plate portion 34b of the lid member 3b are made of a material that is not corroded by the reaction liquid and can withstand a reaction condition (for example, a reaction temperature) generated in the reaction chamber, and It is made of a thin plate made of a material having light transmittance (for example, a transparent or translucent thermoplastic resin, glass, or the like), and the thickness of the thin plate is preferably about 0.1 to 0.5 mm.
As shown in FIG. 7, a nozzle chip fitting space 30b surrounded by a bottom plate portion 32b, a first side plate portion 33b, and a second side plate portion 34b is formed in the lid member 3b, and is provided at an upper end of the lid member 3b. Is formed with a nozzle tip fitting opening 31b communicating with the nozzle tip fitting space 30b.
The nozzle tip fitting space 30b is formed so that the nozzle tip 4b can be fitted from the nozzle tip fitting opening 31b (see FIG. 8 (ii)). Further, since the nozzle chip fitting space 30b does not communicate with an opening other than the nozzle chip fitting port 31b, the nozzle chip fitting port 31b is sealed when the nozzle chip fitting port 31b is sealed (FIG. 8 ( ii))).
Since the nozzle tip fitting port 31b is formed in a portion of the lid 3b other than the portion that seals the opening 21b of the reaction vessel main body 2b, the lid 3b is attached to the reaction vessel main body 2b. Also in the state, the nozzle tip 4 can be fitted into the nozzle tip fitting space 30b from the nozzle tip fitting opening 31b (see FIG. 8 (ii)).
The deepest part of the nozzle chip fitting space 30b (the part of the nozzle chip fitting space 30b furthest away from the nozzle chip fitting opening 31b) is formed by the bottom plate portion 32b of the lid 3b, and the nozzle chip 4b is The tip is fitted from the tip fitting opening 31b toward the deepest part of the nozzle tip fitting space 30b (see FIG. 8 (ii)).
The fitting direction of the nozzle tip 4b into the nozzle tip fitting space 30b is formed to be perpendicular or substantially perpendicular to the surface on which the reaction vessel 1b is placed (the lower face of the bottom plate 22b of the reaction vessel 1b). (See FIG. 8 (ii)), the force of the reaction vessel 1b receiving the force from the nozzle tip 4b when fitting the nozzle tip 4b is perpendicular or substantially perpendicular to the surface on which the reaction vessel 1b is placed. The force in the vertical direction. Therefore, the position of the reaction vessel 1b does not shift when the nozzle tip 4b is fitted, and the nozzle tip 4b can be easily fitted into the nozzle chip fitting space 30b.
The outer diameter of the second side plate portion 34b of the lid member 3b is substantially the same as the inner diameter of the third side plate portion 25b of the reaction container main body 2b, and when the lid member 3b is attached to the reaction container main body 2b, a reaction occurs. The inner peripheral surface of the third side plate portion 25b of the container main body 2b and the outer peripheral surface of the second side plate portion 34b of the lid member 3b are in close contact with each other (see FIG. 8 (i)).
As shown in FIG. 7, a concave portion 36b is provided on the outer peripheral surface of the second side plate portion 34b of the lid member 3b, and the concave portion 36b is provided on the inner peripheral surface of the third side plate portion 25b of the reaction vessel main body 2b. The protrusion 27b can be fitted (see FIG. 8 (i)).
As shown in FIG. 7, a convex portion 37b is provided on the inner peripheral surface of the second side plate portion 34b of the lid member 3b, and the convex portion 37b is provided on the outer peripheral surface of the second side plate portion 44b of the nozzle tip 4b. The recess 49b provided can be fitted (see FIG. 8 (ii)).
As shown in FIG. 7, a contact surface 38b is provided at an upper end portion of the second side plate portion 34b of the lid member 3b, and the contact surface 38b allows the nozzle tip 4b to be fitted into the nozzle chip fitting space 30b. When mounted, it comes into contact with the third side plate portion 46b of the nozzle tip 4b. (See FIG. 8 (ii)).
When the lid 3b is attached to the reaction vessel main body 2b, as shown in FIG. 8 (i), the inner peripheral surface of the third side plate 25b of the reaction vessel main body 2b and the second side plate 34b of the lid 3b. The opening 21b of the reaction container body 2b is sealed by the bottom plate portion 32b, the first side plate portion 33b, and the second side plate portion 34b of the lid member 3b, and the reaction chamber 20b of the reaction container body 2b is sealed. Is done. At this time, as shown in FIG. 8 (i), the convex portion 27b provided on the third side plate portion 25b of the reaction vessel main body 2b and the concave portion 36b provided on the second side plate portion 34b of the lid 3b are fitted. Accordingly, the lid 3b is fixed to the reaction vessel main body 2b, and the state of attachment of the lid 3b to the reaction vessel main body 2b becomes strong.
When the lid member 3b is attached to the reaction container main body 2b, as shown in FIG. 8 (i), the contact surface 28b provided on the upper end of the first side plate 23b of the reaction container main body 2b and the lid The position of the bottom plate 32b of the lid 3b in the reaction chamber 20b is defined by the contact of the bottom plate 32b of the member 3b (in the present embodiment, the bottom plate 32b of the lid 3b is attached to the bottom plate of the reaction vessel body 2b. The closed space S1b is formed between the bottom plate portion 22b of the reaction vessel main body 2b and the bottom plate portion 32b of the lid 3b. That is, the lower surface of the bottom plate portion 32b of the lid member 3b (contact surface of the lid material), the upper surface of the bottom plate portion 22b of the reaction vessel main body 2b (opposing surface of the reaction chamber), and the first side plate 23b of the reaction vessel main body 2b. A sealed space S1b is formed by the inner peripheral surface (the surrounding surface of the reaction chamber), and a part of the reaction solution is thinly accommodated in the sealed space S1b. The reaction liquid contained in the closed space S1b is supplied to the lower surface of the bottom plate 32b of the lid 3b (contact surface of the lid), the upper surface of the bottom plate 22b of the reaction vessel main body 2b (the surface facing the reaction chamber), and the reaction liquid. It is in contact with any surface of the inner peripheral surface (the surrounding surface of the reaction chamber) of the first side plate portion 23b of the container body 2b.
When the lid 3b is attached to the reaction container main body 2b, as shown in FIG. 8 (i), the second side plate 24b and the third side plate 25b of the reaction container main body 2b and the first side of the lid 3b. A sealed space S2b is formed between the side plate 33b. The reaction liquid is accommodated in the closed space S1b formed in the reaction chamber 20b by the attachment of the lid member 3b, and the remainder of the reaction liquid that cannot be accommodated in the closed space S1b is accommodated in the closed space S2b. At this time, the reaction solution is pressed by the bottom plate portion 32b of the lid member 3b, and the air in the reaction chamber 20b and bubbles in the reaction solution are pushed out to the upper part of the reaction chamber 20b together with the reaction solution, and stored in the closed space S2b. At the same time, a part thereof is discharged from the opening 21b to the outside of the reaction chamber 20b. This prevents air from being mixed into the closed space S1b and air bubbles from being mixed into the reaction solution contained in the closed space S1b.
As shown in FIG. 7, the nozzle tip 4b according to the present embodiment has a disk-shaped tip plate 43b constituting the tip of the nozzle tip 4b, and has a diameter gradually increasing upward from the periphery of the tip plate 43b. A first side plate portion 44b having a tapered shape, a cylindrical second side plate portion 45b standing upright from the upper end of the first side plate portion 44b so as to maintain the same diameter, and a second side plate. A tapered third side plate portion 46b erected so as to gradually increase in diameter from the upper end portion of the portion 45b, and a cylindrical shape erected upward from the upper end portion of the third side plate portion 46b to maintain the same diameter. Of the fourth side plate portion 47b, and a flange portion 48b provided on a peripheral edge of an upper end of the fourth side plate portion 47b.
As shown in FIG. 7, the nozzle tip 4b is formed with an inner space 40b surrounded by the end plate 43b and the first to fourth side plates 44b to 47b. A nozzle mounting port 41b communicating with the internal space 40b is formed at the upper end of the nozzle tip 4b, and a suction / discharge port 42b communicating with the nozzle mounting port 41b via the internal space 40b is formed at the tip plate 43b of the nozzle chip 4b. Is formed.
The nozzle tip 4b is configured such that the nozzle 16b can be attached to the internal space 40b from the nozzle tip attachment port 41b (see FIG. 12). Through the nozzle tip 4b.
As shown in FIG. 7, a filter 6b is provided in the internal space 40b of the nozzle tip 4b. As shown in FIG. 7, the filter 6b is provided so as to be located in the vicinity of the suction / discharge port 42b, and prevents liquid droplets from entering the internal space 40b from the suction / discharge port 42b. It is designed to prevent contamination.
The outer diameter of the second side plate portion 45b of the nozzle tip 4b is substantially the same as the inner diameter of the second side plate portion 34b of the lid 3b, and the nozzle tip 4b is fitted in the nozzle chip fitting space 30b of the lid 3b. At this time, the outer peripheral surface of the second side plate portion 45b of the nozzle tip 4b and the inner peripheral surface of the second side plate portion 34b of the lid member 3b are in close contact with each other (see FIG. 8 (ii)).
As shown in FIG. 7, a concave portion 49b is provided on the outer peripheral surface of the second side plate portion 45b of the nozzle tip 4b, and the concave portion 49b is provided on the inner peripheral surface of the second side plate portion 34b of the lid member 3b. The projection 37b can be fitted (see FIG. 8 (ii)).
When the nozzle tip 4b is fitted in the nozzle tip fitting space 30b of the lid 3b, as shown in FIG. 8 (ii), the inner peripheral surface of the second side plate portion 34b of the lid 3b and the nozzle tip 4b of the nozzle tip 4b. The outer peripheral surface of the second side plate portion 45b is in close contact with the outer peripheral surface of the second side plate portion 45b, and the tip end portion 43b, the first side plate portion 43b, and the second side plate portion 45b of the nozzle tip 4b seal the nozzle chip fitting opening 31b, and the nozzle chip fitting space 30b Is sealed. Here, “sealing” means a state in which there is no gap, pore, or the like that hinders transmission of the suction force and the discharge force by the nozzle 16b to the nozzle chip fitting space 30b. The state in which it is connected to the suction / ejection port 42b is included in “sealing”.
Further, when the nozzle tip 4b is fitted into the nozzle tip fitting space 30b of the lid 3b, as shown in FIG. 8 (ii), the projection 37b provided on the second side plate portion 34b of the lid 3b The concave portion 49b provided in the second side plate portion 45b of the nozzle tip 4b is fitted, the nozzle tip 4b is fixed to the cover 3b, and the fitting state of the nozzle tip 4b into the nozzle tip fitting space 30b is changed. It will be strong.
Further, when the nozzle tip 4b is fitted into the nozzle tip fitting space 30b of the lid 3b, as shown in FIG. 8 (ii), the nozzle tip 4b is provided at the upper end of the second side plate 34b of the lid 3b. When the contact surface 38b and the third side plate portion 46b of the nozzle tip 4b abut, the position of the tip plate portion 43b of the nozzle tip 4b in the nozzle tip fitting space 30b is defined (in the present embodiment, the nozzle tip 4b The tip plate 43b of the lid 3b is not in contact with the bottom plate 32b of the lid 3b, so that the suction / discharge port 42b of the nozzle tip 4b is not sealed.) A sealed space S3b communicating with the suction / discharge port 42b of the nozzle tip 4b is formed between the front end plate 43b and the distal end plate 43b. Since the closed space S3b has no opening other than the suction / discharge port 42b of the nozzle tip 4b, the suction force and discharge force by the nozzle 6b can be efficiently transmitted from the suction / discharge port 42b of the nozzle chip 4b to the closed space S3b. Has become.
When the lid 3b is attached to the reaction vessel main body 2b and the nozzle tip 4b is fitted on the lid 3b, the closed space S1b is, as shown in FIG. 8 (ii), a bottom plate of the reaction vessel main body 2b. It has a contact surface with the portion 22b and a contact surface with the bottom plate portion 32b of the lid member 3b. As shown in FIG. 8 (ii), the closed space S3b has a contact surface with the bottom plate 32b of the lid 3b. Therefore, by forming through holes in the bottom plate portion 22b of the reaction container body 2b and the bottom plate portion 32b of the lid member with a perforated needle provided outside the reaction container 1b, the outside of the reaction container 1b and the closed space S1b are sealed. The space S3b can be communicated (see FIG. 12 (iii)). At this time, since the bottom plate portion 32b of the lid member 3b faces the deepest portion of the closed space S1b (the bottom plate portion 22b of the reaction container body 2b constituting the surface on which the reaction container 1b is placed), the reaction container 1b The reaction vessel 1b is provided by a piercing needle provided perpendicularly or substantially perpendicularly to the surface on which is mounted (the lower surface of the bottom plate portion 22b of the reaction vessel main body 2b) (for example, by a piercing needle 51b provided in the piercing vessel 5b). Can be formed in the bottom plate portion 22b of the reaction vessel main body 2b and the bottom plate portion 32b of the lid member (see FIG. 12 (iii)).
As shown in FIG. 9, the perforating container 5b according to the present embodiment includes a main body 50b and a perforating needle 51b. The main body 50b has a rectangular bottom plate in a plan view, a rectangular tubular side plate erected from the periphery of the bottom plate, and a flange provided on the upper edge of the side plate. A liquid storage space 501b surrounded by a bottom plate and a side plate is formed in the main body 50b, and an opening 502b communicating with the liquid storage space 501b is formed at the upper end of the main body 50b.
In the liquid storage space 501b of the perforation container 5b, a liquid can be stored from the opening 502b, and the reaction container 1b can be stored (see FIG. 12 (iii)).
As shown in FIG. 9, the perforation needle 51 b is projected from the bottom plate of the main body 50 b into the liquid storage space 501 b and on the surface on which the reaction vessel 1 b is placed (the upper surface of the bottom plate of the main body 50 b). When the reaction container 1b in which the lid 3b is attached to the reaction container main body 2b is accommodated in the liquid accommodation space 501b, the bottom plate portion of the reaction container main body 2b is provided. A through hole can be formed in the bottom plate portion 32b of the cover member 22b and the lid member 3b (see FIG. 12 (iii)).
As shown in FIG. 9, the tip of the puncture needle 51b has a pointed shape, and the material of the puncture needle 51b is a material such as stainless steel or the like that can pierce the plastic, glass, or the like constituting the reaction vessel main body 2b and the lid member 3b. Metal.
As shown in FIG. 9, the reaction apparatus 10b according to the present embodiment includes a reaction vessel installation section 17b in which the reaction vessel 1b is installed, a perforation vessel installation section 18b in which the perforation vessel 5b is installed, and a liquid suction device. And a nozzle 16b capable of discharging the nozzle 16b, a nozzle transfer unit 15b for transferring the nozzle 16b in a predetermined direction, a first temperature control unit 11b provided in the reaction container installation unit 17b, a second temperature control unit 13b, and a second A temperature control unit attaching / detaching unit 14b for transferring the temperature control unit 13b in a predetermined direction.
As shown in FIG. 9, the reaction vessel setting section 17b and the perforating vessel setting section 18b are provided on a base 100b. Above the base 100b, a second temperature control section 13b and a nozzle 16b are arranged vertically and horizontally. There is a space that can be moved.
As shown in FIG. 9, a first temperature control unit 11b is provided in the reaction container setting unit 17b, and the reaction container 1b is set in the first temperature control unit 11b.
As shown in FIGS. 9 to 11, the first temperature control unit 11b includes a heat insulating ring 110b, a heat transfer body 111b, a heat insulating housing 112b, a thermoelectric semiconductor element 113b, and a heat sink 114b.
As shown in FIG. 9 to FIG. 11, a space is formed at the approximate center of the heat insulating ring 110b so that the reaction vessel main body 2b can be accommodated from the upper end opening and the projection of the heat transfer body 111b can be fitted from the lower end opening. The reaction vessel main body 2b accommodated in the space is supported by a protrusion of the heat transfer body 111b fitted in the space. The heat insulating ring 110b is made of a heat insulating material such as ceramics, so that heat transfer between the heat transfer body 111b and the reaction vessel main body 2b is efficiently performed.
As shown in FIGS. 9 to 11, the heat insulating ring 110b is provided with an optical fiber fitting hole 115b communicating with the space in which the reaction vessel main body 2b is accommodated, and the optical fiber is fitted into the optical fiber fitting hole 115b. By doing so, the optical fiber can be arranged around the first side plate 23b of the reaction vessel body 2b supported by the protrusion of the heat transfer body 111b (the outer peripheral surface of the first side plate 23b). A plurality of optical fiber fitting holes 115b are provided in the heat insulating ring 110b, and the plurality of optical fiber fitting holes 115b are connected to an optical fiber connected to a light source (not shown) and a fluorescence detector (not shown). The optical fiber is fitted therein, and the excitation light emitted from the light source through the optical fiber can be applied to the reaction solution contained in the closed space S1b via the first side plate 23b of the reaction vessel main body 2b, and the closed space S1b Fluorescence emitted from the reaction solution contained in the reaction vessel 2b and transmitted through the first side plate 23b of the reaction vessel main body 2b to the outside of the reaction vessel main body 2b is received through an optical fiber and can be detected by a fluorescence detector. ing.
As shown in FIGS. 9 to 11, the heat transfer body 111 b includes a disk portion and a protrusion, and the protrusion is fitted on the heat insulating ring 110 b, and the disk portion is provided on the heat sink 114 b. The upper surface of the thermoelectric semiconductor element 113b. The heat transfer body 111b is made of a metal such as copper, so that heat generated by the thermoelectric semiconductor element 113b can be efficiently transmitted to the reaction vessel main body 2b.
The thermoelectric semiconductor element 113b can be used as a cooling element (cooling) and / or can be used as a heating element (heating), for example, a Peltier element. The thermoelectric semiconductor element 113b is connected to a power supply (not shown), so that when power is supplied from the power supply, the heat transfer body 111b can be cooled and / or heated. As shown in FIGS. 9 to 11, the lower surface of the thermoelectric semiconductor element 113b is in contact with a heat sink 114b having cooling fins, and the thermoelectric semiconductor element 113b is forcibly cooled by the heat sink 114.
As shown in FIGS. 9 to 11, the heat transfer body 111b and the thermoelectric semiconductor element 113b are housed inside a heat insulating casing 112b made of a heat insulating material such as ceramics. The cooling and / or heating of 111b is performed efficiently.
The first temperature control unit 11b transfers the heat applied to the heat transfer member 111b by the thermoelectric semiconductor element 113b to the reaction container body 2b via the contact surface between the lower surface of the reaction container body 2b and the protrusion of the heat transfer member 111b. The temperature of the reaction solution accommodated in the closed space S1b can be controlled by the transmission and the heat transfer through the bottom plate portion 22b of the reaction vessel main body 2b.
As shown in FIGS. 9 to 11, the second temperature control unit 13b includes a heat insulating ring 130b, a heat transfer body 131b, a heat insulating housing 132b, a thermoelectric semiconductor element 133b, a heat sink 134b, and a telescopic arm of the temperature control unit attaching / detaching unit 14b. An arm mounting portion 135b to which 142b is mounted is provided.
As shown in FIGS. 9 to 11, a space is formed at substantially the center of the heat insulating ring 130 b so that the protrusion of the heat transfer body 131 b can be inserted from the upper end opening and the lid 3 b can be inserted from the lower end opening. I have. The heat insulating ring 130b is made of a heat insulating material such as ceramics, so that heat transfer between the heat transfer body 131b and the lid member 3b is efficiently performed.
As shown in FIGS. 9 to 11, the heat transfer body 131 b includes a disk portion and a protrusion, and the protrusion is inserted into the heat insulating ring 130 b, and the disk portion is connected to the lower surface of the thermoelectric semiconductor element 133 b. In contact. The protrusion of the heat transfer body 131b is formed so as to be mounted in the nozzle chip fitting space 30b of the lid 3b, and the protrusion of the heat transfer body 131b mounted in the nozzle chip fitting space 30b is formed of a lid material. 3b, the bottom plate 32b, the first side plate 33b, and the second side plate 34b are in contact with each other. The outer diameter of the protrusion of the heat transfer body 131b is smaller than the inner diameter of the heat insulation ring 130b, and when the protrusion of the heat transfer body 131b is inserted into the heat insulation ring 130b, the outer circumference of the protrusion of the heat transfer body 131b. A gap leading to the lower end opening of the heat insulating ring 130b is formed between the surface and the inner peripheral surface of the heat insulating ring 130b. The cover 3b can be inserted into the gap, and even when the protrusion of the heat transfer body 131b is inserted into the heat insulating ring 130b, the protrusion of the heat transfer body 131b can be inserted into the nozzle tip of the cover 3b. It can be mounted in the fitting space 30b. The heat conductor 131b is made of a metal such as copper, so that heat generated by the thermoelectric semiconductor element 133b can be efficiently transmitted to the lid 3b.
The thermoelectric semiconductor element 133b can be used as a cooling element (cooling) and / or can be used as a heating element (heating), for example, a Peltier element. The thermoelectric semiconductor element 133b is connected to a power supply (not shown), so that when power is supplied from the power supply, the heat conductor 131b can be cooled and / or heated. As shown in FIGS. 9 to 11, the upper surface of the thermoelectric semiconductor element 133b is in contact with a heat sink 134b having cooling fins, and the thermoelectric semiconductor element 131b is forcibly cooled by the heat sink 134b.
As shown in FIGS. 9 to 11, the heat transfer body 131b and the thermoelectric semiconductor element 133b are housed inside a heat insulating casing 132b made of a heat insulating material such as ceramics. The cooling and / or heating of 131b can be performed efficiently.
When the protrusion of the heat transfer member 131b is mounted in the nozzle chip fitting space 30b of the cover member 3b, the second temperature control section 13b transfers the heat applied to the heat transfer member 131b by the thermoelectric semiconductor element 133b to the cover member 3b. The heat is transmitted to the lid 3b via the contact surface between the bottom plate portion 32b, the first side plate portion 33b, and the second side plate portion 34b and the protrusion of the heat transfer body 111b, and the heat generated through the bottom plate portion 32b of the lid material 3b. By the movement, the temperature of the reaction solution accommodated in the closed space S1b can be controlled.
As shown in FIG. 9, the temperature control unit attaching / detaching unit 14b is provided on the rail 140b provided substantially vertically on the upper surface of the base 100b, the movable unit 141b movable along the rail 140b, and the movable unit 141b. And a telescopic arm 142b.
As shown in FIG. 9, the telescopic arm 142b is provided on the movable part 141b so as to be able to expand and contract horizontally with respect to the upper surface of the base 100b. As shown in FIG. 9, a second temperature control unit 13b is attached to a distal end of the telescopic arm 142b via an arm mounting unit 135b. It is transported in the horizontal direction with respect to the upper surface of the base 100b, and is also transported in the vertical direction with respect to the upper surface of the base 100b by the movement of the movable portion 141b.
The temperature control unit attaching / detaching unit 14b transfers the protrusion of the heat transfer body 131b to the first temperature control unit 11b by transferring the second temperature control unit 13b in the horizontal direction and the vertical direction with respect to the upper surface of the base 100b. It can be mounted in the nozzle chip fitting space 30b of the lid 3b of the installed reaction vessel 1b, and can be detached from the nozzle chip fitting space 30b.
The nozzle 16b is connected to a liquid suction / discharge device (not shown), and can suck and discharge the liquid through the suction / discharge hole 160b (see FIG. 12). Further, the suction / discharge hole 160b communicates with the tip of the nozzle 16b, so that the suction force and the discharge force can be transmitted to the nozzle tip 4b mounted on the tip of the nozzle 16b via an O-ring or the like. .
As shown in FIG. 9, the nozzle transfer unit 15b is provided on a rail 150b provided in a horizontal direction with respect to the upper surface of the base 100b, a movable unit 151b movable along the rail 150b, and a movable unit 151b. And a telescopic arm 152b.
As shown in FIG. 9, the telescopic arm 152b is provided on the movable part 151b so as to be able to expand and contract in the vertical direction with respect to the upper surface of the base 100b. As shown in FIG. 9, the nozzle 16b attached to the distal end of the telescopic arm 152b is moved vertically by the expansion and contraction of the telescopic arm 152b with respect to the upper surface of the base 100b, and is moved by the movement of the movable part 151b. Are transported in the horizontal direction with respect to the upper surface of the base 100b.
The nozzle transfer unit 15b transfers the nozzle tip 4b mounted on the nozzle 16b to the reaction set in the first temperature control unit 11b by transferring the nozzle 16b in the horizontal direction and the vertical direction with respect to the upper surface of the base 100b. The nozzle tip fitting space 30b of the lid 3b of the container 1b can be fitted. Further, the nozzle transfer section 15b transfers the reaction vessel 1b, in which the nozzle tip 4b is fitted, to the perforating vessel setting section 18b, and the liquid accommodating space of the perforating vessel 5b installed in the perforating vessel setting section 18b. A through-hole can be formed in the bottom plate portion 22b of the reaction vessel main body 2b and the bottom plate portion 32b of the lid member 3b by a piercing needle 51b provided in the opening container 502b and provided in the piercing container 5b.
The operation of the temperature control unit attaching / detaching unit 14b and the operation of the nozzle transfer unit 15b are controlled so as not to interfere with each other.
The operation of the reaction device 10b will be described by taking as an example a case where PCR is carried out while a PCR reaction solution is accommodated in the reaction container 1b.
After accommodating the PCR reaction solution through the opening 21b in the reaction chamber 20b of the reaction vessel body 2b, the lid 3b is attached to the reaction vessel body 2b. At this time, the convex portion 27b of the reaction vessel main body 2b and the concave portion 36b of the lid 3b are fitted, and the lid 3b is fixed to the reaction vessel main body 2b (see FIG. 8 (i)). In addition, the closed space S1b and the closed space S2b are formed in the reaction chamber 20b by the attachment of the lid member 3b, and the PCR reaction solution is accommodated in the closed space S1b and the PCR reaction solution that cannot be accommodated in the closed space S1b. The remainder is accommodated in the closed space S2b (see FIG. 8 (i)). The reaction vessel 1b in this state is installed in the first temperature control section 11b provided in the reaction vessel installation section 17b (see FIGS. 9 and 11).
The reaction device 10b transfers the second temperature control unit 13b to the reaction vessel 1b installed in the first temperature control unit 11b by the temperature control unit attachment / detachment unit 14b, and the protrusion of the heat transfer body 131b of the second temperature control unit 13b. The part is mounted on the nozzle chip fitting space 30b of the lid member 3b (see FIGS. 9 and 11).
The reaction device 10b mounts the protrusion of the heat transfer body 131b in the nozzle chip fitting space 30b, and then controls the first temperature control unit 11b and the second temperature control unit 13b to use the PCR reaction solution contained in the closed space S1b. An operation for controlling the temperature is performed. Thereby, PCR proceeds in the PCR reaction solution accommodated in the closed space S1b, and a PCR amplified fragment 7b is generated as a reaction product in the PCR reaction solution (see FIG. 12 (i)).
At this time, since the PCR reaction solution is contained in the closed space S1b in a thin shape, the ratio of the surface area to the volume is large, and most of the surface area is the upper and lower surfaces of the thin layer, that is, the lid material 3b. It is occupied by the lower surface of the bottom plate portion 32b (the contact surface of the lid member) and the upper surface of the bottom plate portion 22b of the reaction vessel main body 2b (the surface facing the reaction chamber). Therefore, the temperature of the PCR reaction solution accommodated in the closed space S1b can be quickly controlled by the heat transfer through the bottom plate portion 32b of the lid member 3b and the heat transfer through the bottom plate portion 22b of the reaction vessel main body 2b. Thus, the time required for PCR can be reduced.
In addition, the excitation light emitted from the light source is irradiated to the PCR reaction solution contained in the closed space S1b through the optical fiber fitted in the optical fiber fitting hole 115b of the heat insulating ring 110b, and the PCR reaction contained in the closed space S1b. By receiving the fluorescence emitted from the solution and detecting it with a fluorescence detector, the progress of PCR (for example, whether or not the target nucleic acid is amplified by PCR, the amount of PCR amplification product, etc.) can be monitored in real time.
After completion of the PCR, the reaction device 10b transfers the second temperature control unit 13b by the temperature control unit attaching / detaching unit 14b, and fits the protrusion of the heat transfer body 131b of the second temperature control unit 13b with the nozzle tip of the lid 3b. An operation of detaching from the space 30b is performed (see FIG. 9).
After detaching the projection of the heat transfer member 131b from the nozzle chip fitting space 30b, the reaction device 10b moves the nozzle 16b by the nozzle transfer unit 15b to above the reaction vessel 1b installed in the first temperature control unit 11b. Then, the nozzle tip 4b mounted on the nozzle 16b is fitted into the nozzle tip fitting space 30b from the nozzle tip fitting opening 31b (see FIGS. 12 (i) and (ii)). At this time, the convex portion 37b of the lid 3b and the concave portion 49b of the nozzle tip 4b are fitted, and the nozzle tip 4b is fixed to the lid 3b. Further, by fitting the nozzle tip 4b into the nozzle tip fitting space 30b, a closed space S3b communicating with the suction / discharge port 42b of the nozzle tip 4b is formed in the nozzle tip fitting space 30b.
After fitting the nozzle tip 4b attached to the nozzle 16b into the nozzle tip fitting space 30b, the reaction device 10b transfers the nozzle 16b by the nozzle transfer unit 15b, and the nozzle tip 4b attached to the nozzle 16b is fitted. An operation of transporting the reaction vessel 1b, which has been performed, to above the perforated vessel installation section 18b is performed (see FIG. 9). Since the lid member 3b is fixed to the reaction container main body 2b, and the nozzle tip 4b is fixed to the lid member 3b, the lid member 3b does not detach from the reaction container main body 2b during transfer. Does not detach from the lid 3b.
After transferring the reaction vessel 1b above the perforating vessel setting section 18b, the reaction device 10b extends the telescopic arm 152b, and moves the reaction vessel 1b to the liquid storage space of the perforating vessel 5b installed on the perforating vessel setting section 18b. 501b is received from the opening 502b (at this time, the lower surface of the bottom plate 22b of the reaction vessel body 2b is pressed against the piercing needle 51b provided in the piercing vessel 5b), and the piercing needle 51b provided in the piercing vessel 5b. Thereby, a through hole for communicating the liquid storage space 501b of the perforation container 5b, the sealed space S1b of the reaction container 1b, and the nozzle chip fitting space 30b is formed in the bottom plate portion 22b of the reaction container body 2b and the bottom plate portion 32b of the lid member 3b. (See FIG. 12 (c)). At this time, the piercing needle 51b first pierces the bottom plate portion 22b of the reaction vessel main body 2b to form a through hole for communicating the liquid storage space 501b of the piercing vessel 5b with the closed space S1b of the reaction vessel 1b, and then The bottom plate portion 32b of the lid member 3b is perforated to form a through hole for communicating the closed space S1b of the reaction vessel 1b with the closed space S3b in the nozzle chip fitting space 30b.
In the reaction vessel 1b pierced by the piercing needle 51b, the liquid storage space 501b of the piercing vessel 5b and the closed space S1b of the reaction vessel 1b communicate with each other through a through hole formed in the bottom plate 22b of the reaction vessel main body 2b. The closed space S1b of the reaction vessel 1b and the closed space S3b in the nozzle chip fitting space 30b communicate with each other through a through hole formed in the bottom plate portion 32b of the lid member 3b. Since the space S3b communicates with the suction / discharge port 42b of the nozzle tip 4b, the suction force and the discharge force of the nozzle 16b can be transmitted to the liquid storage space 501b of the perforation container 5b.
After piercing with the piercing needle 51b, the reaction device 10b starts suction and discharge by the nozzle 16b, and removes the extract 8b (eg, buffer solution) stored in the liquid storage space 501b of the piercing container 5b through the through hole. By performing suction and discharge, the operation of extracting the PCR amplified fragment 7b contained in the PCR reaction solution in the closed space S1b of the reaction container 1b into the extraction solution 8b is performed (see FIG. 12 (iii)). At this time, when the suction and discharge by the nozzle 16b are started, the extract 8b stored in the liquid storage space 501b of the perforation container 5b flows into the closed space S1b with the suction by the nozzle 16b, and is discharged by the nozzle 16b. Flows out of the closed space S1b. By repeating the suction and discharge by the nozzle 16b, the flow of the extract 8b into and out of the closed space S1b is repeated, and the PCR amplified fragment 7b contained in the PCR reaction solution in the closed space S1b of the reaction vessel 1b is repeated. Is extracted into the extraction liquid 8b stored in the liquid storage space 501b of the perforation container 5b.
After the PCR is performed with the lid 3b attached to the reaction vessel main body 2b in this manner, the PCR is contained in the PCR reaction solution in the closed space S1b of the reaction vessel 1b without removing the lid 3b from the reaction vessel main body 2b. PCR amplified fragment 7b can be obtained.
The second embodiment described above is described to facilitate understanding of the present invention, and does not limit the present invention in any way. Therefore, each element disclosed in the second embodiment is intended to include all design changes and equivalents belonging to the technical scope of the present invention.
When the lid 3b is attached to the reaction vessel body 2b to seal the reaction chamber 20b, the inner peripheral surface of the third side plate 25b of the reaction vessel main body 2b and the outer peripheral surface of the second side plate 34b of the lid 3b. Can be brought into close contact with each other through a member capable of maintaining airtightness such as an O-ring. Similarly, when the nozzle tip 4b is fitted into the nozzle tip fitting space 30b of the lid 3b to seal the nozzle tip fitting space 30b, the inner peripheral surface of the second side plate portion 34b of the lid 3b and the nozzle tip Instead of directly contacting the outer peripheral surface of the second side plate portion 45b of the second side plate 4b, the second side plate portion 45b can be brought into close contact with a member such as an O-ring that can maintain airtightness. At this time, a gap, a pore, and the like are formed in a member such as an O-ring so as to communicate the inside and the outside of the reaction chamber 20b or the nozzle tip fitting space 30b, and the lid member 3b is attached to the reaction vessel main body 2b. When or when fitting the nozzle tip 4b into the nozzle tip fitting space 30b, the air inside the reaction chamber 20b or the nozzle tip fitting space 30b can be discharged to the outside. Also, on the inner peripheral surface of the third side plate portion 25b of the reaction vessel main body 2b or the outer peripheral surface of the second side plate portion 34b of the lid member 3b, or on the inner peripheral surface of the second side plate portion 34b of the lid member 3b or the nozzle tip 4b. On the outer peripheral surface of the second side plate portion 45b, gaps, pores, and the like that allow the inside and the outside of the reaction chamber 20b or the nozzle chip fitting space 30b to communicate with each other may be formed.
Industrial potential
According to the present invention, it is possible to automate the reaction without the necessity of centrifugation when accommodating the reaction solution in the reaction chamber, and to quickly control the temperature of the reaction solution accommodated in the reaction chamber, A reaction vessel that allows the reaction to proceed even if the amount of the reaction solution contained in the reaction chamber is very small, and that can monitor the reaction occurring in the reaction chamber in real time (ie, immediately during the reaction). , A reaction apparatus and a method are provided.
Further, according to the present invention, after performing a reaction in a state where the lid member is attached to the reaction container body, the reaction product contained in the reaction solution in the reaction container can be removed without detaching the lid member from the reaction container body. A reaction vessel, a reaction apparatus and a method that can be obtained are provided.
According to the reaction vessel, reaction apparatus and method of the present invention, preparation of a sample containing a target nucleic acid (for example, extraction of a nucleic acid from a cell), amplification of a target nucleic acid by PCR, and progress of PCR (for example, Monitoring (eg, detection, measurement, quantification, etc.) of the presence or absence of amplification, the amount of PCR amplification products, etc. (eg, detection, measurement, quantification, etc.), and automation of a series of operations such as the acquisition of PCR amplified fragments can be performed. It becomes possible.
[Brief description of the drawings]
FIG. 1 is a sectional view showing a first embodiment of the reaction vessel according to the present invention.
FIG. 2 is a top view of the reaction vessel main body of the reaction vessel according to the first embodiment.
FIG. 3 is a bottom view of the lid member according to the first embodiment.
FIG. 4 is a cross-sectional view showing a state in which the lid member is attached to the reaction container main body in the reaction container according to the first embodiment.
FIG. 5 is a schematic partial sectional view showing a first embodiment of the reaction apparatus according to the present invention.
FIGS. 6 (i) to (iii) are explanatory diagrams showing examples of the arrangement of optical fibers (FIG. 6 (ii) corresponds to a cross-sectional view taken along line AA of FIG. 5).
FIG. 7 is a sectional view showing a second embodiment of the reaction vessel according to the present invention.
FIG. 8 (i) is a cross-sectional view showing a state in which a lid member is attached to a reaction vessel main body in the reaction vessel according to the second embodiment, and FIG. 8 (ii) is a reaction vessel according to the second embodiment. FIG. 3 is a cross-sectional view showing a state in which a nozzle tip is fitted to a lid material attached to a reaction container body.
FIG. 9 is a partial sectional view showing a second embodiment of the reactor according to the present invention.
FIG. 10 (i) is an exploded perspective view showing the structure of the first temperature control unit and the second temperature control unit provided in the reaction device according to the second embodiment, and FIG. It is a perspective view showing the state of the 1st temperature control part and the 2nd temperature control part.
FIG. 11 is a cross-sectional view showing a state near a reaction vessel during a reaction in the reaction device according to the second embodiment.
FIG. 12 is a partial cross-sectional view showing the operation of the reaction device according to the second embodiment up to the extraction of the reaction product.

Claims (44)

A reaction vessel including a reaction vessel body having an opening and having a reaction chamber capable of containing a reaction solution, and a lid member capable of sealing the opening of the reaction chamber,
The lid member and the reaction chamber each have a contact surface that comes into contact with the reaction solution contained in the reaction chamber in a state where the lid member is attached to the reaction container main body.
The lid is made of a light-transmitting material so that light can pass through the contact surface of the lid from the reaction solution contained in the reaction chamber to the outside of the reaction vessel, or The reaction vessel main body is made of a light-transmitting material so that light can pass through the contact surface of the reaction chamber from the reaction solution contained in the reaction chamber to the outside of the reaction vessel. Characteristic reaction vessel. The lid is made of a light-transmitting material so that light can pass through the contact surface of the lid from the outside of the reaction vessel to the reaction solution contained in the reaction chamber, or The reaction container main body is made of a light-transmitting material so that light can pass through the contact surface of the reaction chamber from the outside of the reaction container to a reaction solution contained in the reaction chamber. The reaction vessel according to claim 1, wherein The reaction container according to claim 1, wherein a part or the whole of a contact surface of the lid member is a flat surface. The reaction container according to claim 1, wherein the contact surface of the lid member is a wall surface having a substantially uniform thickness that forms the lid member. The reaction vessel according to claim 1 or 2, wherein a part or the whole of a contact surface of the reaction chamber is a flat surface. 3. The reaction container according to claim 1, wherein the contact surface of the reaction chamber is a surface of a wall portion having a substantially uniform thickness that forms the reaction container body. When the lid is attached to the reaction vessel body, a closed space is formed by the contact surface of the lid and the contact surface of the reaction chamber, and a part or all of the reaction solution is contained in the closed space. 3. The reaction vessel according to 1 or 2, wherein When the lid member is attached to the reaction container main body, a reaction solution remaining portion accommodating portion capable of accommodating a remaining portion of the reaction solution that cannot be accommodated in the closed space is formed in the reaction chamber. The reaction container according to claim 7, wherein The reaction chamber has an opposing surface opposing a contact surface of the lid material, and when the lid material is attached to the reaction container main body, a part of the reaction solution contained in the reaction chamber or The reaction vessel according to claim 1, wherein the entirety is housed in a thin shape between a contact surface of the lid member and an opposing surface of the reaction chamber. 10. The reaction container according to claim 9, wherein the opposed surface of the reaction chamber is a surface of a wall having a substantially uniform thickness constituting the reaction container body. Light is transmitted from the outside of the reaction vessel to the reaction solution contained in the reaction chamber and / or from the reaction solution contained in the reaction chamber to the outside of the reaction vessel via the opposing surface of the reaction chamber. 11. The reaction vessel according to claim 10, wherein the wall having an opposing surface of the reaction chamber is made of a light-transmitting material so as to obtain the reaction chamber. 10. The reaction vessel according to claim 9, wherein the reaction vessel main body has a contact surface that comes into contact with the lid member and defines a distance between a contact surface of the lid member and an opposing surface of the reaction chamber. The reaction chamber has a surrounding surface surrounding the reaction solution existing between the contact surface of the lid member and the facing surface of the reaction chamber,
When the lid is attached to the reaction container body, a closed space is formed by the contact surface of the lid, the opposing surface of the reaction chamber, and the surrounding surface of the reaction chamber, and a part of the reaction solution or 10. The reaction vessel according to claim 9, wherein the whole is accommodated in the closed space in a thin shape. 14. The reaction vessel according to claim 13, wherein a part or the entire surrounding surface of the reaction chamber is flat. The reaction vessel according to claim 14, wherein the cross section of the surrounding surface of the reaction chamber is rectangular. 14. The reaction container according to claim 13, wherein the surrounding surface of the reaction chamber is a surface of a wall portion having a substantially uniform thickness that constitutes the reaction container main body. Light is transmitted from the outside of the reaction vessel to the reaction solution contained in the reaction chamber and / or from the reaction solution contained in the reaction chamber to the outside of the reaction vessel via the surrounding surface of the reaction chamber. 17. The reaction vessel according to claim 16, wherein the wall having the surrounding surface of the reaction chamber is made of a light-transmitting material so as to obtain the reaction chamber. A state in which a nozzle chip fitting space in which a nozzle chip mounted on a nozzle capable of sucking and discharging a liquid is fitted is formed in the lid member, and the lid member is attached to the reaction container body. A nozzle tip fitting opening communicating with the nozzle tip fitting space is formed so that the nozzle tip can be fitted into the nozzle tip fitting space,
In a state where the lid member is attached to the reaction vessel main body, a penetrating needle provided outside the reaction vessel communicates the outside of the reaction vessel with the closed space and the nozzle chip fitting space. The reaction vessel according to claim 7, wherein a hole can be formed in the reaction vessel body and the lid member. A state in which a nozzle chip fitting space in which a nozzle chip mounted on a nozzle capable of sucking and discharging a liquid is fitted is formed in the lid member, and the lid member is attached to the reaction container body. A nozzle tip fitting opening communicating with the nozzle tip fitting space is formed so that the nozzle tip can be fitted into the nozzle tip fitting space,
In a state where the lid member is attached to the reaction container main body, a penetrating needle provided outside the reaction container allows the outside of the reaction container to communicate with the front sealed space and the nozzle chip fitting space. 14. The reaction vessel according to claim 13, wherein a hole can be formed in the reaction vessel body and the lid. 20. The reaction vessel according to claim 18, wherein the nozzle chip fitting space is formed so that the nozzle chip fitting space is sealed when the nozzle chip fitting opening is sealed. . 21. The reaction vessel according to claim 20, wherein a wall portion of the lid forming the nozzle chip fitting space has an inner peripheral surface that can be in close contact with an outer peripheral surface of the nozzle tip. On the inner peripheral surface of the wall portion of the lid member that can be in close contact with the outer peripheral surface of the nozzle tip, a convex portion and / or concave portion that can be fitted with a concave portion and / or a convex portion provided on the outer peripheral surface of the nozzle tip. 22. The reaction container according to claim 21, wherein the reaction container is provided. 20. The reaction vessel according to claim 18, wherein the contact surface of the lid is a surface of a wall of the lid forming the nozzle chip fitting space. 20. The reaction vessel according to claim 18, wherein the contact surface of the lid is a surface of a wall of the lid forming a deepest portion of the nozzle chip fitting space. The wall portion of the lid member forming the deepest portion of the nozzle chip fitting space is provided so as to face the wall portion of the reaction vessel main body forming the deepest portion of the closed space. The reaction container according to claim 24. The nozzle chip fitting space is formed such that a fitting direction of the nozzle tip into the nozzle chip fitting space is perpendicular or substantially perpendicular to a surface on which the reaction vessel is mounted. The reaction vessel according to claim 18 or 19, wherein: 20. The reaction container according to claim 18, wherein the lid has an outer peripheral surface that can be in close contact with an inner peripheral surface of the reaction chamber. A concave portion and / or a convex portion are provided on an inner peripheral surface of the reaction chamber,
28. A convex portion and / or a concave portion which can be fitted to a concave portion and / or a convex portion provided on an inner peripheral surface of the reaction chamber on an outer peripheral surface of the lid member. Reaction vessel. 3. The reaction container according to claim 1, which is a reaction container for PCR. A reaction device comprising the reaction container according to claim 2, a temperature control device, a light source, and a fluorescence detector,
The lid and / or the reaction so that the temperature controller can control the temperature of the reaction solution contained in the reaction chamber via the contact surface of the lid and / or the contact surface of the reaction chamber. It is attached to the container body,
The light source is provided so as to be able to irradiate the reaction solution contained in the reaction chamber with light through the contact surface of the lid member and / or the contact surface of the reaction chamber,
The fluorescence detector is provided so that fluorescence emitted from a reaction solution contained in the reaction chamber can be detected through a contact surface of the lid member and / or a contact surface of the reaction chamber. And the reactor. The temperature control device may be a wall portion having a substantially uniform thickness that forms the lid member, the wall portion having a contact surface with the lid member, and / or a wall portion having a substantially uniform thickness that forms the reaction vessel body. 31. The reactor of claim 30, wherein the reactor is attached to a wall having a contact surface of the reaction chamber. The reaction vessel is the reaction vessel according to claim 13,
The lid and / or the reaction so that the temperature controller can control the temperature of the reaction solution contained in the reaction chamber via the contact surface of the lid and / or the opposing surface of the reaction chamber. It is attached to the container body,
The light source is provided so as to be able to irradiate the reaction solution contained in the reaction chamber with light through the surrounding surface of the reaction chamber,
31. The reaction apparatus according to claim 30, wherein the fluorescence detector is provided so as to be able to detect fluorescence emitted from a reaction solution contained in the reaction chamber via an enclosing surface of the reaction chamber. . The temperature control device may be a wall portion having a substantially uniform thickness that forms the lid member, the wall portion having a contact surface with the lid member, and / or a wall portion having a substantially uniform thickness that forms the reaction vessel body. 33. The reaction apparatus according to claim 32, wherein the reaction apparatus is attached to a wall having an opposing surface of the reaction chamber. Further comprising a plurality of optical fibers disposed around the surrounding surface of the reaction chamber,
33. The reaction apparatus according to claim 32, wherein irradiation of light from the light source to the reaction solution and / or detection of fluorescence emitted from the reaction solution are performed using the optical fiber. 20. A reaction device comprising a reaction container installation part in which the reaction container according to claim 18 or 19 is installed, a first temperature control device, a second temperature control device, a light source, and a fluorescence detector. ,
The first temperature control device is provided so that the temperature of the reaction solution contained in the closed space of the reaction vessel installed in the reaction vessel installation section can be controlled via the contact surface of the reaction chamber. Has been
The second temperature control device is detachably attached to the nozzle chip fitting space of the lid member, and adjusts the temperature of the reaction solution contained in the closed space of the reaction vessel installed in the reaction vessel installation section. , Is provided so that it can be controlled via the contact surface of the lid material,
The light source irradiates the reaction liquid contained in the closed space of the reaction vessel installed in the reaction vessel installation section with light through the contact surface of the lid member and / or the contact surface of the reaction chamber. Is provided to obtain
The fluorescence detector emits fluorescence emitted from a reaction solution contained in the closed space of the reaction vessel installed in the reaction vessel installation section, by using a contact surface of the lid member and / or a contact surface of the reaction chamber. A reaction device, which is provided so as to be detectable through a reactor. The reaction container is the reaction container according to claim 19,
The first temperature control device is provided so as to be able to control the temperature of the reaction solution accommodated in the closed space of the reaction vessel installed in the reaction vessel installation section via the facing surface of the reaction chamber. Has been
The light source is provided so as to be able to irradiate the reaction liquid contained in the closed space of the reaction vessel installed in the reaction vessel installation section with light through the surrounding surface of the reaction chamber,
The fluorescence detector is provided so as to be able to detect fluorescence emitted from a reaction solution contained in the closed space of the reaction vessel installed in the reaction vessel installation section via the surrounding surface of the reaction chamber. The reaction device according to claim 35, wherein: Further comprising a plurality of optical fibers disposed around the surrounding surface of the reaction chamber,
37. The reaction apparatus according to claim 36, wherein irradiation of light from the light source to the reaction solution and / or detection of fluorescence emitted from the reaction solution is performed using the optical fiber. The apparatus further includes a temperature control unit attaching / detaching unit for attaching and detaching the second temperature control unit to / from the nozzle chip fitting space,
An operation of attaching the second temperature control device to the nozzle chip fitting space before the reaction, and attaching and detaching the second temperature control device from the nozzle chip fitting space after the reaction. 36. The reaction product apparatus according to claim 35, wherein an operation of causing the reaction product is performed. A perforation container installation unit in which a perforation container is installed, a nozzle capable of sucking and discharging a liquid, and a nozzle transfer unit,
The perforation container includes a liquid storage space capable of storing a liquid, an opening communicating with the liquid storage space, and a perforation needle.
The liquid storage space is formed so that the reaction container can be stored in the liquid storage space from the opening,
The perforation needle is provided to protrude from the wall of the perforation container forming the liquid storage space to the liquid storage space,
An operation of fitting the nozzle tip mounted on the nozzle to the nozzle tip fitting space of the reaction vessel installed in the reaction vessel installation part, wherein the nozzle tip is fitted; The operation of transferring the reaction vessel to the perforation vessel installation section, and accommodating the reaction vessel in the liquid accommodation space of the perforation vessel installed in the perforation vessel installation section, and provided in the perforation vessel. The operation of forming a through-hole in the lid member and the reaction container main body by the perforation needle to communicate the liquid storage space of the perforation container, the closed space of the reaction container, and the nozzle chip fitting space of the reaction container. Do,
The nozzle extracts and extracts the reaction liquid contained in the closed space of the reaction container into the liquid by sucking and discharging the liquid contained in the liquid storage space of the perforation container through the through hole. The reaction device according to claim 35, wherein the reaction is performed. The reaction device according to claim 30 or 35, which is a reaction device for PCR. (A) contacting the reaction solution accommodated in the reaction chamber with the contact member; and the reaction via the contact surface between the reaction solution and the reaction chamber and / or the contact surface between the reaction solution and the contact member. Controlling the temperature of the solution (b), and irradiating the reaction solution with light through a contact surface between the reaction solution and the reaction chamber and / or a contact surface between the reaction solution and the contact member ( c) and detecting (d) fluorescence emitted from the reaction solution via a contact surface between the reaction solution and the reaction chamber and / or a contact surface between the reaction solution and the contact member. . A contact surface of the reaction chamber used for controlling the temperature of the reaction solution; and a reaction surface used for detecting fluorescence from the contact surface of the reaction chamber and / or the reaction solution used for irradiating the reaction solution with light. 42. The method of claim 41, wherein the contact surface of the chamber is a different surface. In the front contact member, a nozzle chip fitting space in which a nozzle tip mounted on a nozzle capable of sucking and discharging liquid is fitted is formed,
After the reaction in the reaction chamber is completed, a step of forming a through hole for communicating the outside of the reaction chamber, the inside of the reaction chamber, and the nozzle chip fitting space with a perforated needle provided outside the reaction chamber. (E), a step (f) of fitting the nozzle tip mounted on the nozzle into the nozzle chip fitting space, a step (g) of bringing the outside of the reaction chamber into contact with a liquid, and 42. The method according to claim 41, further comprising the step of: (h) extracting the reaction liquid contained in the reaction chamber into the liquid by operating to suck and discharge the liquid through the through-hole. . The method according to claim 41, wherein the reaction occurring in the reaction chamber is PCR.

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