WO2008004695A1

WO2008004695A1 - Reaction container and reaction device

Info

Publication number: WO2008004695A1
Application number: PCT/JP2007/063695
Authority: WO
Inventors: Hideji Tajima
Original assignee: Universal Bio Research Co., Ltd.
Priority date: 2006-07-07
Filing date: 2007-07-09
Publication date: 2008-01-10
Also published as: JPWO2008004695A1

Abstract

A reaction container and a reaction device capable of quickly controlling the temperature of reaction liquid and detecting a reaction result produced in the reaction liquid at a desired point in time. The reaction container comprises a liquid storing unit formed by first and second main walls facing each other and a sub-wall continuous to the first and second main walls and having at one end an opening to be able to store liquid in a thin layer form, and a reaction container body provided with a tubular unit having a first opening at one end and a second opening at the other end, wherein the end on the opening side of the liquid storing unit and the end on the second opening side of the tubular unit are continuous so as to allow the inner space of the liquid storing unit to communicate with the inner space of the tubular unit, and the first main wall and/or the second main wall have/has thermal conductivity and optical transparency.

Description

Reaction vessel and reactor

Technical field

The present invention relates to a reaction vessel and a reaction apparatus that can quickly control the temperature of a reaction solution and detect a reaction result generated in the reaction solution at a desired time.

Background art

[0002] 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 its temperature. Genetic engineering and biological test methods Widely used in fields such as detection methods!

[0003] The principle of PCR is that a double-stranded DNA containing a target DNA sequence is maintained at a temperature at which it is dissociated into single strands, and forward and reverse primers are annealed to the dissociated single-stranded DNA. According to the thermal profile (temperature increase / decrease) set in the third stage, which is maintained at the temperature at which to ring, and the third stage in which DNA polymerase maintains the temperature at which the DNA strand complementary to the single-stranded DNA is synthesized. The target DNA is amplified exponentially by repeating the cycle many times.

[0004] For example, a reaction solution containing a double-stranded DNA containing a target DNA sequence, an excess of a pair of primers, and a thermostable polymerase is treated at 95 ° C for 30 seconds, 65 ° C for 30 seconds, 72 ° PCR can proceed by reacting with C for 30 to 40 cycles, with 1 minute as one cycle. At 95 ° C, double-stranded DNA dissociates into single-stranded DNA. Next, when it is cooled to an appropriate temperature according to the base sequence of the primer (65 ° C in the above example), the primer and the single-stranded DNA ring. Next, when the temperature is raised to the polymerase reaction temperature (72 ° C in the above example), the DNA synthesis reaction by polymerase proceeds.

[0005] As described above, since temperature control of a reaction solution is important for PCR, PCR usually uses a thermostat that can be programmed with temperature control and a reaction vessel that can be used for the device. To be implemented.

[0006] 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 (2 A device that repeats the cycle of dissociation of double-stranded DNA, cooling (primer annealing), and heating (extension reaction with polymerase) is used. There are two types of metal block cooling methods, one using a compressor and one using a Peltier cooling method.

[0007] If the number of specimens is large, such as when PCR is performed for screening purposes, PCR of 96 specimens is performed using a PCR microtiter plate (96 wells) to process a large number of specimens at once. Devices that can be performed at once have also been developed.

[0008] Particularly recently, in order to efficiently process a large number of specimens in genetic diagnosis and genome projects, preparation of a sample containing the target nucleic acid (eg, extraction of nucleic acid from cells), amplification of the target nucleic acid by PCR, Monitor the progress of PCR (for example, whether target nucleic acid has been amplified, the amount of PCR amplification product, etc.) (for example, detection, measurement, qualitative analysis, quantitative analysis, etc.) There is a growing need for efficient parallel processing.

[0009] Under these circumstances, various PCR reaction vessels and PCR reactors have been developed that can reduce the time required for PCR by rapid temperature control of the reaction solution and can measure the progress of PCR in real time. (Patent Documents 1 to 5).

Patent Document 1: JP-A-8-196299

Patent Document 2: US Patent No. 5958349

Patent Document 3: US Patent 6565815

Patent Document 4: US Patent No. 6403037

Patent Document 5: US Pat. No. 6,660,228

Disclosure of the invention

Problems to be solved by the invention

[0010] An object of the present invention is to provide a reaction vessel and a reaction apparatus capable of quickly controlling the temperature of a reaction solution and detecting a reaction result generated in the reaction solution at a desired time. Means for solving the problem

[0011] (1) In order to solve the above-mentioned problem, the present invention is formed by the first and second main wall portions facing each other, and the sub-wall portion continuous with the first and second main wall portions. A liquid storage portion that has an opening at one end and can store liquid in a thin layer, and has a first opening at one end and the other end A reaction vessel main body comprising a cylindrical portion having a second opening in the first chamber, wherein the internal space of the liquid storage portion and the internal space of the cylindrical portion communicate with each other. An end portion on the opening side of the liquid storage portion and an end portion on the second opening portion side of the cylindrical portion are continuous, and the first and Z or the second main wall portions are thermally conductive and light. A permeable reaction vessel is provided.

[0012] According to the reaction container of the present invention, the first and second main wall portions having a large contact area with the reaction solution (for example, the CRC reagent) accommodated in a thin layer in the liquid accommodation portion. It can be heated or cooled through. Therefore, the temperature of the reaction solution can be quickly controlled. In addition, when light (for example, fluorescence, chemiluminescence, etc.), which also generates reaction force, is an indicator of the reaction result (for example, presence / absence of PCR amplified fragment, amount, etc.) generated in the reaction solution, By detecting the light emitted from the reaction solution through the wall, the reaction result generated in the reaction solution can be detected. When the light emitted from the reaction solution is fluorescent, irradiation with excitation light is required, but the reaction solution can be irradiated with excitation light through the first and second main walls.

[0013] (2) The reaction container of the present invention preferably includes a lid that can seal the first opening of the cylindrical portion. This is useful when the reaction needs to proceed in a sealed state, such as PCR.

(3) In the reaction container of the present invention, the tip end portion of the pipette tip attached to the nozzle of the dispensing device enters the first opening force of the cylindrical portion, and the cylindrical portion It is preferable that it can pass through the second opening and the opening of the liquid container to reach the bottom of the liquid container! /. It is possible to automate the dispensing of the reaction liquid into the reaction vessel using the dispensing device.

[0015] (4) In the reaction container of the present invention, it is preferable that the lid is attached to the reaction container main body so as to be freely opened and closed. It is possible to automate a series of operations from storing the reaction solution in the reaction vessel to collecting the reaction solution after the reaction, and further analyzing the collected reaction solution.

[0016] (5) In the reaction container of the present invention, the lid contacts the end portion on the first opening portion side of the cylindrical portion that does not fit into the first opening portion of the cylindrical portion. By doing so, it is preferable that the first opening of the cylindrical portion can be sealed. Since the lid attached to the reaction vessel body can be easily detached from the reaction vessel body, the reaction solution after the reaction is collected and the collected reaction solution It is possible to automate the analysis of

(6) In the reaction container of the present invention, the lid is provided with a magnet or a magnetic body, and the lid in the open state can be closed by magnetic force, and the lid in the closed state can be closed. It is preferable that the lid can be opened. It is possible to automate the opening and closing of the lid by magnetic force.

[0018] (7) In the reaction container of the present invention, the lid body is formed by a continuous wall portion, has an opening at one end, has a gas storage portion that can store gas, and the gas storage portion Can be deformed while maintaining the continuity of the wall so that the volume of gas accommodated in the gas accommodating portion is reduced, and the lid is attached to the reaction vessel main body. It is preferable that the opening of the gas accommodating portion communicates with the first opening of the cylindrical portion. The gas container is deformed while maintaining the continuity of the wall so that the volume of the gas accommodated in the gas container is reduced, so that the reaction chamber remains sealed while the internal space of the reaction vessel is maintained. The air pressure in the interior space of the container increases. When the atmospheric pressure in the internal space of the reaction vessel increases, the generation of bubbles due to heating of the reaction solution (eg, PCR reagent) contained in the reaction vessel can be effectively prevented.

[0019] (8) In order to solve the above-mentioned problems, the present invention provides a holding unit that holds the reaction container of the present invention, and a liquid storage unit of the reaction vessel that is held by the holding unit. When the passage capable of moving in the surface direction of the main wall portion of 2 and the liquid storage portion of the reaction vessel held by the holding portion are in a predetermined position of the passage, the first and Z or second of the reaction vessel A first to n-th temperature control unit provided at a predetermined position of the passage and a liquid storage unit of the reaction vessel held by the holding unit so that the main wall can be heated or cooled to a predetermined temperature. The first to n-th temperature control units can be relatively moved along the passage, and heating or cooling by the first to n-th temperature control units is performed in the first and second reaction vessels. The first to nth temperature control units are provided so as to be performed on the Z and the second main wall. Providing a reactor having e Bei and a moving portion that can control the speed of the liquid containing portion of the reaction vessel at the location.

[0020] According to the reaction apparatus of the present invention, the liquid container in the reaction vessel held by the holder is moved relative to the first to nth temperature control units, thereby forming a thin layer in the liquid container. Rapidly transfer the contained reaction solution (e.g., PCR reagent) through the 1st and Z or 2nd main walls. It can be heated or cooled.

“N” represents an integer of 1 or more. Further, “controlling the speed of the liquid container” includes stopping the liquid container, reducing the speed of the liquid container, and the like. The same applies hereinafter.

(9) Further, according to the present invention, the first and second main wall portions include a holding portion that holds the reaction vessel of the present invention, and a liquid storage portion of the reaction vessel that is held by the holding portion. When the passage capable of moving in the surface direction and the liquid storage portion of the reaction vessel held by the holding portion are at a predetermined position of the passage, the first and Z or second main wall portions of the reaction vessel are A first to nth temperature control unit provided at a predetermined position of the passage and a liquid storage unit of the reaction vessel held by the holding unit are provided in the passage so as to be heated or cooled to a predetermined temperature. A light receiving portion provided at a predetermined position of the passage and held by the holding portion so as to receive light emitted from the first and second main walls or the second main wall portion of the reaction vessel. The liquid container of the reaction vessel is connected to the first to nth temperature control units and the light receiving unit. The first to nth temperature control units can be heated or cooled, and the light receiving unit can receive light with respect to the first and Z or second main walls of the reaction vessel. A moving part capable of controlling the speed of the liquid container of the reaction container at the position where the first to nth temperature control parts and the light receiving part are provided, and the first and Z of the reaction container or There is provided a reaction device including a light detection unit that detects light emitted from a second main wall, and a light supply unit that supplies light from the light receiving unit to the light detection unit.

[0022] According to the reaction apparatus of the present invention, the liquid container in the reaction vessel held by the holding unit is moved relative to the first to nth temperature control units to form a thin layer in the liquid container. The contained reaction liquid can be quickly heated or cooled through the first and Z or second main walls. In addition, by moving the liquid storage part of the reaction container held by the holding part relative to the light receiving part, light emitted from the reaction liquid through the first and second main walls at a desired time (for example, , Chemiluminescence, etc.) can be detected, and thereby the reaction result (for example, the presence / absence of reaction, degree, etc.) generated in the reaction solution can be detected at a desired time. Reaction fluid Power is useful in cases where excitation light irradiation is required to generate light (for example, chemiluminescence). It is for.

[0023] (10) Further, according to the present invention, there are provided a holding part for holding the reaction container of the present invention, and a liquid storage part of the reaction container held by the holding part for the first and second main wall parts. When the passage movable in the surface direction and the liquid container of the reaction vessel held by the holding portion are at a predetermined position of the passage, the first and Z or the second main wall portions of the reaction vessel The first to nth temperature control units provided at predetermined positions of the passage and the liquid storage portion of the reaction container held by the holding portion are arranged at predetermined positions of the passage so as to be heated or cooled to a predetermined temperature. The first and Z or second main walls of the reaction vessel can be irradiated with light, and light emitted from the first and Z or second main walls of the reaction vessel can be received. As described above, the light irradiation 'light receiving portion provided at a predetermined position of the passage and the reaction held by the holding portion The liquid storage part of the container can be moved relative to the first to nth temperature control part and the light irradiation 'light receiving part along the passage, and the first to nth temperature control part Heating or cooling, and light irradiation 'The first to nth temperature control units and the light irradiation and light reception by the light receiving unit are performed on the first and Z or second main walls of the reaction vessel and A moving part capable of controlling the speed of the liquid storage part of the reaction container at a position where the light irradiation and light receiving part is provided; and an excitation light, and the first and Z or second main walls of the reaction container. A light detection unit that detects light emitted from the light source, and a light supply unit that supplies light from the light detection unit to the light irradiation / light receiving unit and from the light irradiation / light receiving unit to the light detection unit. Provided is a reactor equipped.

[0024] According to the reaction apparatus of the present invention, the liquid container in the reaction vessel held by the holder is moved relative to the first to n-th temperature control units to form a thin layer in the liquid container. The contained reaction solution (eg, PCR reagent) can be rapidly heated or cooled through the first and Z or second main walls. In addition, by moving the liquid storage part of the reaction vessel held by the holding part relative to the first to nth temperature control parts, the reaction can be performed through the first and second main wall parts at a desired time. By irradiating the liquid with excitation light, it is possible to detect light (for example, fluorescence, etc.) that also emits reaction liquid force through the first and second main walls. The presence / absence, amount, etc. of the PCR amplified fragment can be detected at a desired point in time. Reaction fluid When light is generated and excitation light irradiation is necessary (for example, For example, fluorescence).

[0025] (11) Furthermore, the present invention provides a holding unit for holding a plurality of reaction vessels of the present invention, and each reaction vessel held by the holding unit in the surface direction of the first and second main wall portions. When the movable passage and each reaction vessel held in the holding portion are in a predetermined position of the passage, the first and Z or second main wall portions of each reaction vessel can be heated or cooled to a predetermined temperature. In addition, when the first to n-th temperature control units provided at predetermined positions of the passage and the respective reaction vessels held by the holding portion are at the predetermined positions of the passages, The light receiving portion provided at a predetermined position of the passage and the liquid storage portion of each reaction vessel held by the holding portion so that the light emitted from Z or the second main wall portion can be received. It can be moved relative to the nth temperature control unit and the light receiving unit along the passage. The first to n-th temperature control units perform heating or cooling and the light-receiving unit receives light on the first and Z or second main walls of each reaction vessel. _A moving part that can control the speed of the liquid storage part of each reaction container at the position _where the temperature control part and the light receiving part are provided, and the first and Z or second main wall parts of each reaction container. A reaction device comprising a light detection unit for detecting light and a light supply unit for supplying light from the light receiving unit to the light detection unit, wherein the light supply unit is a first and a second of any reaction vessel. Decide whether to receive light emitted from Z or the second main wall, select a light receiving unit corresponding to the determined reaction vessel, and supply light received by the selected light receiving unit to the light detection unit The reactor is provided.

[0026] According to the reaction apparatus of the present invention, the liquid storage portions of the plurality of reaction vessels held in the holding portion are moved relative to the first to n-th temperature control portions, thereby being accommodated in each reaction vessel. The reaction solution thus obtained can be rapidly heated or cooled through the first and Z or second main walls. In addition, by moving the liquid storage parts of a plurality of reaction containers held in the holding part relative to the light receiving part, the light emitted from the reaction liquid stored in the desired reaction container is detected at a desired time. Thereby, the reaction result (for example, the presence or absence, degree of reaction, etc.) generated in a desired reaction vessel can be detected at a desired time. Multiple specimens can be processed at one time. This is useful when excitation light irradiation is not required because reaction fluid light is generated (for example, chemiluminescence). [0027] (12) Further, the present invention provides a holding portion for holding a plurality of reaction vessels of the present invention, and each reaction vessel held by the holding portion in the surface direction of the first and second main wall portions. When the movable passage and each reaction vessel held in the holding portion are in a predetermined position of the passage, the first and Z or second main wall portions of each reaction vessel can be heated or cooled to a predetermined temperature. In addition, when the first to n-th temperature control units provided at predetermined positions of the passage and the respective reaction vessels held by the holding portion are at the predetermined positions of the passages, Provided at a predetermined position of the passage so that light can be emitted to the Z or second main wall and light emitted from the first and Z or second main walls of each reaction vessel can be received. A light irradiating light receiving portion, and a liquid storage portion of each reaction vessel held by the holding portion, the first to nth temperature control portions; and The light irradiation 'can be moved relative to the light receiving portion along the passage, and is heated or cooled by the first to nth temperature control portions, and the light irradiation' light by the light receiving portion. At the position where the first to nth temperature control units and the light irradiation 'light receiving unit are provided so that irradiation and light reception are performed on the first and Z or second main walls of each reaction vessel. A moving part that can control the speed of the liquid container of each reaction container, and a light detection part that generates excitation light and detects light emitted from the first and Z or second main walls of each reaction container And a light supply unit that supplies light from the light detection unit to the light irradiation / light reception unit and from the light irradiation / light reception unit to the light detection unit, wherein the light supply unit comprises: Decide which reaction vessel to irradiate and receive light to the first and Z or second main walls. Select the light irradiation and light receiving unit corresponding to the specified reaction vessel, and supply the selected light irradiation 'excitation light to the light receiving unit and the light received by the selected light irradiation / light receiving unit to the light detection unit The reactor is provided.

[0028] According to the reaction apparatus of the present invention, the liquid storage portions of the plurality of reaction vessels held in the holding portion are moved relative to the first to n-th temperature control portions, thereby being accommodated in each reaction vessel. The reaction solution (for example, PCR reagent) can be rapidly heated or cooled through the first and Z or second main walls. In addition, by moving the liquid storage parts of a plurality of reaction containers held in the holding part relative to the light irradiation * light receiving part, excitation light is emitted to the reaction liquid stored in the desired reaction container at a desired time. It is possible to detect the light emitted from the reaction solution by irradiation, and thereby the reaction result generated in a desired reaction container (for example, PCR amplified fragment Presence, amount, etc.) can be detected at a desired time. Multiple specimens can be processed at one time. It is useful when excitation light irradiation is required to generate reaction fluid light (eg fluorescence).

[0029] (13) In the reaction apparatus of the present invention, the first to n-th temperature control units have a heating surface or a cooling surface, and the reaction vessel held by the holding unit is located at a predetermined position of the passage. In some cases, the heating surface or the cooling surface is located in the vicinity of the first and Z or second main wall portions of the reaction vessel or in contact with the first and Z or second main wall portions of the reaction vessel. Is preferred. Efficient heating or cooling through the first and Z or second main walls by the first to nth temperature control units is possible.

[0030] (14) The reaction apparatus of the present invention preferably includes a pressurizing unit that pressurizes the lid of the reaction vessel held by the holding unit against the reaction vessel main body. The sealed state of the reaction vessel held in the holding part can be held. This is useful when a sealed state is necessary for the progress of the reaction, such as PCR.

[0031] (15) In the reaction apparatus of the present invention, the reaction vessel held by the holding unit is the reaction vessel of (6), and the pressurizing unit has a magnet or magnetic body of a lid of the reaction vessel. And a pressing part moving part that moves the pressing part so that the lid body is in an open state force closed state or in an open state from a closed state. Preferred. By automating the opening and closing of the lid, it is possible to automate a series of operations from storing the reaction solution in the reaction vessel to collecting the reaction solution after the reaction and further analyzing the collected reaction solution.

[0032] (16) In the reaction apparatus of the present invention, the reaction container held in the holding part is the reaction container described in (3), a nozzle capable of sucking and discharging a liquid, and the nozzle It is preferable to include a nozzle moving part that moves the tip part of the pipette tip attached to the liquid container of the reaction container held by the holding part into and out of the liquid container. It is possible to automate the dispensing of the reaction liquid into the reaction vessel using the dispensing device.

[0033] (17) In the reaction apparatus of the present invention, after the nozzle moving section has caused the tip of the pipette tip to enter the liquid storage section of the reaction container, the first and second of the reaction container It is preferable to move in the surface direction of the main wall. As the pipette tip moves between the sub-walls of the liquid container, the reaction liquid is discharged into the liquid container, so that the pipette tip is contained in the liquid container. It is possible to prevent bubbles from being contained in the reaction solution to be stored.

[0034] (18) In the reaction apparatus of the present invention, the reaction container held in the holding part is the reaction container described in (7), and the volume of the gas stored in the gas storage part of the lid is It is preferable to include a pressing part that can press the gas accommodating part of the lid body and deform the gas accommodating part so as to decrease. When the pressing portion presses the gas accommodating portion, the gas accommodating portion is deformed while maintaining the continuity of the wall portion so that the volume of the gas accommodated in the gas accommodating portion is reduced. As a result, the air pressure in the internal space of the reaction vessel increases while maintaining the hermeticity of the internal space of the reaction vessel, and the generation of bubbles due to the heating of the reaction solution (eg, PCR reagent) contained in the reaction vessel is effective. Can be prevented.

The invention's effect

[0035] According to the present invention, there are provided a reaction vessel and a reaction apparatus capable of quickly controlling the temperature of a reaction solution and detecting a reaction result generated in the reaction solution at a desired time.

Brief Description of Drawings

FIG. 1 is a plan view showing an embodiment of a reaction apparatus of the present invention.

FIG. 2 is a partial cross-sectional side view showing an embodiment of the reaction apparatus of the present invention (before the lid is put on the reaction vessel body).

FIG. 3 (a) is a perspective view showing an embodiment of the reaction container (open state) of the present invention, and (b) is a perspective view showing an embodiment of the reaction container (closed state) of the present invention. (C) is a cross-sectional view taken along line AA in (b), and (d) is a cross-sectional view taken along line BB in (c).

[FIG. 4] (a) is a plan view of the temperature control unit, (b) is a side view of the temperature control unit, and (c) is a cross-sectional view showing the positional relationship between the temperature control unit and the reaction vessel. It is.

FIG. 5 is a perspective view of a light detection unit and a light supply unit.

FIG. 6 is a partial cross-sectional side view showing an embodiment of the reaction apparatus of the present invention (after attaching a lid to the reaction vessel main body).

FIG. 7 is a cross-sectional view of a single bead.

FIG. 8 is a cross-sectional view showing a modification of the reaction vessel of the present invention.

Explanation of symbols

[0037] la, lb, 1. · · · Reaction vessels 11 ··· Reaction vessel body

111 ··· Liquid container

111a, 111b ... Main wall

111c, 11 Id, llle… 畐 ij wall

12 ... Cover body

13 ... Magnet

10 ... Reactor

2 ··· Reaction vessel holder

4a, 4b, 4c—temperature controller

6 ... Light detector

7. Light supply section

91 ... Nozzle

P ... Pipette

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the reaction container and the reaction apparatus of the present invention will be described with reference to the drawings.

As shown in FIGS. 1 and 2, the reaction apparatus 10 includes a container group 200, a suction / discharge unit 9 that sucks and discharges liquid, and a suction / discharge unit 9 in the X-axis direction, the Y-axis direction, and the Z-axis direction. The suction / discharge part moving part 8 to be moved, the reaction container holding part 2 holding the three reaction containers la, lb and lc, and the lid of the reaction container la, lb and lc held in the reaction container holding part 2 Lid opening / closing part 3 for opening and closing 12 with respect to the reaction vessel body 11, and temperature control units 4a, 4b and 4c for controlling the temperatures of the reaction vessels 1a, lb and lc held in the reaction vessel holding part 2, respectively. And a light detection unit 6 that generates excitation light and detects fluorescence generated by the excitation light, and from the temperature control units 4a, 4b, and 4c to the light detection unit 6 or from the light detection unit 6 to the temperature control units 4a, 4b. And a light supply unit 7 for supplying light (excitation light, fluorescence) to 4c.

As shown in FIG. 1, a container group 200 is provided on the stage 100. Container group 200 contains predetermined PCR reagents (eg, buffer, MgCl, dNTP mix, primer, saddle type)

2

A container containing DNA, Taq polymerase, fluorescent dye, etc.), a container for containing the reaction solution after PCR, etc. are installed in place. [0040] As shown in FIG. 2, the suction / discharge unit 9 has a nozzle portion 91 to which the pipette tip P is attached, and the pipette tip P attached to the nozzle portion 91 by using a pump, a cylinder or the like. The inside can be depressurized or pressurized, and when the inside of the pipette tip P is depressurized by the suction / discharge section 9, the tip force of the pipette tip P is sucked into the pipette tip P while the suction When the inside of the pipette tip P is pressurized by the discharge section 9, the liquid inside the pipette tip P is discharged from the tip of the pipette tip P.

As shown in FIG. 1, the suction / discharge unit moving unit 8 includes two rails R2 and R3 installed in the Y-axis direction above the stage 100, and the Y-axis direction along the rails R2 and R3. Moving bodies 82 and 83 provided on rails R2 and R3 so that they can move, rail R1 with one end fixed to moving body 82 and the other end fixed to moving body 83, and X-axis direction along rail R1 And a moving body 81 provided on the rail R1 so that the suction arch I discharge section 9 is fixed to the moving body 81.

[0042] The moving bodies 82 and 83 can move in the Y-axis direction along the rails R2 and R3 using the driving force of the motor, etc., and the moving bodies 82 and 83 are moved along the rails R2 and R3. Then, when moving in the Y-axis direction, the moving body 81 provided on the rail Rl, the rail R1 and the suction / discharge unit 9 fixed to the moving body 81 can move in the Y-axis direction. In addition, the moving body 81 can move in the X-axis direction along the rail R1 using a driving force of a motor or the like. When the moving body 81 moves in the X-axis direction along the rail R1, the moving body 81 moves. The suction / discharge unit 9 fixed to the body 81 can move in the X-axis direction. The moving body 81 has a Z-axis moving mechanism including a motor, a ball screw, a nut screwed to the ball screw, etc., and the ball screw is rotated by the driving force of the motor, and the nut is moved along the ball screw. It can move in the axial direction, and when the nut moves in the Z-axis direction, the suction / discharge unit 9 fixed to the moving body 81 can move in the Z-axis direction. The X-axis direction is horizontal with respect to stage 100 (left and right in FIGS. 1 and 2), and the Y-axis direction is horizontal with respect to stage 100 (up and down in FIG. 1). Yes, the Z-axis direction is perpendicular to the stage 100 (the vertical direction in FIG. 2).

As shown in FIGS. 3 (a) to 3 (d), the reaction vessels la, lb and lc have a reaction vessel main body 11 and a lid 12 attached to the reaction vessel main body 11 so as to be freely opened and closed. [0044] As shown in Figs. 3 (a) to (d), the reaction vessel main body 11 includes a liquid storage portion 111, a cylindrical portion 112, and a flange portion 113 provided at the upper end portion of the cylindrical portion 112. Have

[0045] As shown in FIGS. 3 (c) and 3 (d), the liquid storage portion 111 includes the main wall portions 11la and 111b facing each other, and the sub wall portion 11 lc continuous with the main wall portions 11a and 11 lb. 1 l id and 1 l ie, and has an upper end opening 11 If and an inner space S111 leading to the upper end opening 11 If so that liquid can be stored in the inner space S111 in a thin layer. It is summer. The thickness of the liquid accommodated in the inner space S 111 in a thin layer (distance between the opposing inner wall surfaces 11 la and 11 lb) is usually 0.3 to 3. Omm, preferably 0.5 to 1. Omm.

[0046] As shown in FIGS. 3 (c) and 3 (d), the cylindrical portion 112 includes an upper end opening 112a, a lower end opening 112b, and an internal space S leading to the upper end opening 112a and the lower end opening 112b. 112. In the present embodiment, the cylindrical portion 112 has a cylindrical force, but is not limited to this, and may be a rectangular tube shape or the like. In this embodiment, the diameter of the cylindrical part 112 is substantially the same in any part, but a part of the diameter may be enlarged or reduced.

As shown in FIGS. 3 (c) and 3 (d), the upper end opening 11 of the liquid storage portion 111 and the end on the If side and the end on the lower end opening 112b side of the cylindrical portion 112 are liquid storage. The inner space S 111 of the portion 111 and the inner space S 112 of the cylindrical portion 112 are continuous so as to communicate with each other, and the tip portion force of the pipette tip P attached to the nozzle portion 91 of the suction / discharge portion 9 is cylindrical portion 112. From the upper end opening 112a of the tube, passes through the lower end opening 112b of the cylindrical portion 112 and the upper end opening 11 If of the liquid storage portion 111, and reaches the bottom (sub-wall portion 1 l ie) of the liquid storage portion 111. The main wall 11 la and 11 lb can be moved in the plane direction (the direction toward the subwall 11 Id from the subwall 11 lc or the direction from the subwall 11 Id to the subwall 11 lc). Become! /

[0048] The sub-wall portions ll lc, 111 (1 and 11 ^, and the cylindrical portion 112 are not corroded by the liquid stored in the liquid storage portion 111, but are supplied with the liquid stored in the liquid storage portion 111. It is made of a material that can withstand the reaction conditions (for example, temperature, pressure, etc.) In addition to the above properties, the main wall portions 11 la and 11 lb are materials having thermal conductivity and light transmittance (for example, The main wall 111a and 111b, the subwall ll lc, 111 (1 and 11 ^, and the cylindrical part 112 are made of a thin plate, The thickness of the thin plate is preferably 0.1 to 0.5 mm. As shown in FIG. 3 (a), the flange portion 113 provided at the upper end of the cylindrical portion 112 is provided with support portions 122 and 123 for rotatably supporting the shaft member 121. The shaft member 121 is fitted into the through hole of the lid body 12, and the lid body 12 can be rotated about the shaft member 121. The lid body 12 rotates and the lid body 12 and the flange portion 113 come into surface contact with each other, whereby the opening 112a of the cylindrical portion 112 is sealed (this state is a “closed state”). When the opening 112a of the cylindrical portion 112 is sealed with the lid 12, the internal space (internal spaces S111 and S112) of the reaction vessel main body 11 is sealed.

[0050] As shown in Figs. 3 (a) to (d), the lid 12 is provided with a magnet 13, and the lid 12 in the open state can be closed by magnetic force, and the lid 12 can be closed. A lid 12 can be opened.

[0051] As shown in FIG. 2, the reaction container holding part 2 includes a reaction container la 11, lb, and lc of the reaction container main body 11. The liquid storage part 111 and the cylindrical part 112 can pass through the cylindrical part. The flange portion 113 provided at the upper end of 112 has three through-holes that cannot pass through, and the reaction vessel holding portion 2 can hold the reaction vessels la, lc, and Id by supporting the flange portion 113. It ’s like that. In this embodiment, the number of reaction vessels held in the reaction vessel holding unit 2 is three, but the number of reaction vessels held in the reaction vessel holding unit 2 is not particularly limited.

[0052] As shown in FIGS. 1 and 2, the reaction vessels la, lb, and lc have the reaction vessel holding portion 2 so that the surface direction of the main walls 11 la and 11 lb and the X-axis direction match. Retained. Although not shown, the reaction vessel holding unit 2 can be moved on the stage 100 in the X-axis direction (the surface direction of the main wall portions 11 la and 11 lb) using a driving force such as a motor. Become! / The stage 100 has a reaction vessel holder 2 so that the reaction vessel holder 2 can move in the X-axis direction (main wall 111a and 11 lb surface direction) while holding the reaction vessels la, lb and lc. A through-hole of a size that prevents the movement of the reaction vessels la, lb and lc held in the chamber is provided! / When the reaction vessels la, lb, and lc are held in the reaction vessel holding part 2, the liquid storage portions 111 of the reaction vessels la, lb, and lc pass through this through hole and are provided below the stage 100. It is located in the passage 40 (see FIG. 4) of the temperature controllers 4a, 4b and 4c.

As shown in FIG. 1, the reaction container holding part 2 is provided with a lid opening / closing part 3. Lid open The closed portion 3 includes arm portions 31 and 32, a shaft 36 fitted in a through hole of the arm portion 31, support portions 34 and 35 that rotatably support the shaft 36, and a through hole provided in the arm portion 32. A shaft 39 fitted in the hole; support portions 37 and 38 for rotatably supporting the shaft 39; and a pressure portion 33 having one end fixed to the arm portion 31 and the other end fixed to the arm 32. The

[0054] As shown in FIG. 1, when the reaction vessels la, lb and lc held in the reaction vessel holding part 2 are in the open state, the pressurizing part 33 has a lid 12 for the reaction containers la, lb and lc. Can be placed. The pressurization unit 33 is provided with a magnet 34 at a position where the lid 12 is placed (see FIG. 6), and the magnet 13 of the lid 12 and the magnet 34 of the pressurization unit 33 act, The lid 12 is fixed to the pressure unit 33 by magnetic force. In this state, when the shafts 36 and 39 are rotated, the pressure unit 33 is rotated with the lid 12 fixed, and the lid 12 is changed from the open state to the closed state and from the closed state to the open state. Has become possible. After the lid 12 is closed, the pressurizing unit 33 continues to pressurize the lid 12 to the reaction vessel body 11 as it is, so that the sealed state of the reaction vessel 1 is maintained. .

[0055] As shown in FIG. 2, below the stage 100, temperature control units 4a and 4b are provided at positions corresponding to the positions of the reaction vessels la, lb and lc held in the reaction vessel holding unit 2. In addition, the liquid storage portions 111 of the reaction vessels la, lb, and lc are positioned in the passages 40 (see FIG. 4) of the temperature control portions 4a, 4b, and 4c.

[0056] As shown in FIGS. 4 (a) and (b), the temperature control units 4a, 4b, and 4c have the liquid storage portions 111 of the reaction vessels la, lb, and lc as the main wall portions 11 la and 11 lb, respectively. Passage 40 that can move in the surface direction (X-axis direction), and heating unit 41, cooling unit 42, heating unit 43, light irradiation / light receiving unit 44, heating unit 45, cooling provided along passage 40 Part 46, heating part 47, and light irradiation and light receiving light 48. The caloric heat ridges 41, 43, 45 and 47 have calo heat blocks 411, 431, 4 51 and 471, respectively, and the heating blocks 411 and 451 are provided so as to face each other. 471 is provided to face each other. The cooling units 42 and 46 have cooling blocks 421 and 461, respectively, and the cooling blocks 421 and 461 are provided to face each other. In addition, the light receiving portions 44 and 48 have mouth lenses 441 and 481, respectively, so that the rod lenses 441 and 481 are opposed to each other. It is Optical fibers 442 and 482 are connected to the rod lenses 441 and 481, respectively.

[0057] As shown in FIG. 4 (c), when the liquid storage portion 111 is positioned between the heating blocks 411 and 451, the heating surface force of the heating blocks 411 and 451 contacts the main wall portions 11 la and 11 lb, respectively. It comes to touch. When the liquid storage unit 111 is positioned between the heating blocks 431 and 471, the heating surfaces of the heating blocks 431 and 471 are in contact with the main wall portions 11la and 111b, respectively. In addition, when the liquid storage unit 111 is positioned between the cooling blocks 421 and 461, the cooling surfaces (cooling air blowing ports) of the cooling blocks 421 and 461 are located near the main wall portions 11 la and 11 lb, respectively. It comes to be located and speaks.

[0058] Each heating block and each cooling block is made of metal such as copper. A thermoelectric semiconductor element is connected to each heating block. When power is supplied to the power supply thermoelectric semiconductor element, each heating block is heated. The thermoelectric semiconductor element is an element that can be used (heated) as a heat generating element, for example, a Peltier element. Each cooling block is provided with a cooling air vent (see Fig. 4 (c)).

[0059] The calo heat blocks 411 and 451 are heated to a temperature at which the double-stranded DNA is dissociated (for example, 95 ° C), and the liquid container 111 is positioned between the heating blocks 411 and 451. When heating, the heating surfaces of the heating blocks 411 and 451 come into contact with the main wall 11 la and 11 lb, and the liquid (PCR reaction liquid) stored in the liquid storage 111 dissociates the double-stranded DNA. It is heated to a certain temperature.

[0060] The heating blocks 411 and 451 are heated to a temperature at which an extension reaction by the polymerase occurs (for example, 72 ° C). When the liquid container 111 is located between the heating blocks 431 and 471, the heating blocks 411 and 451 are heated. The heating surface of the blocks 431 and 471 and the main wall 11 la and 11 lb are in force S contact with each other, and the liquid (PCR reaction liquid) stored in the liquid storage 111 is heated to a temperature at which the elongation reaction by the polymerase occurs. It's like! /

[0061] When the liquid storage unit 111 is positioned between the cooling blocks 421 and 461, the cooling air generated by the blower (not shown) is blown through the ventilation ports of the cooling blocks 421 and 461. The primer is cooled to a temperature at which it anneals (eg 65 ° C). As shown in FIG. 2, the reaction container holding unit 2 moves in the X-axis direction to move the liquid storage unit 111 between the heating blocks 411 and 451, between the cooling blocks 421 and 461, and the heating block 431. And 471 are placed in a predetermined order and can be stopped for a predetermined time! When the liquid storage unit 111 stops between the heating blocks 411 and 451, the liquid storage unit 111 is heated to a predetermined temperature, and when the liquid storage unit 111 stops between the cooling blocks 421 and 461, the liquid storage unit 111 reaches a predetermined temperature. When the liquid container 111 is cooled and stopped between the heating blocks 431 and 471, the liquid container 111 is heated to a predetermined temperature.

[0063] As shown in Fig. 4 (a), the light irradiation of the temperature control units 4a, 4b and 4c * The light receiving units 44 and 48 respectively receive the excitation light generated by the light detection unit 6 in the temperature control unit 4a, Optical fibers 442 and 482 are connected to supply to 4b and 4c and the fluorescence generated by the temperature control units 4a, 4b and 4c to the light detection unit 6 is connected. When located between 44 and 48, the excitation light generated from the light source of the light detection unit 6 is applied to the main walls 11 la and 11 lb through the optical fibers 442 and 482, and also contains liquid. Fluorescence generated from the reaction liquid stored in the section 111 is supplied to the light detection section 6 through the optical fibers 442 and 482.

[0064] The light detection unit 6 includes a light source, a half mirror, a lens, a spectral filter, a fluorescence detection device, and the like, and can generate excitation light having a predetermined wavelength and detect fluorescence generated by the excitation light. I can do it!

As shown in FIG. 5, the light supply unit 7 includes a light path 71 connected to the light detection unit 6, a light path 72 connected to the light path 71 via the light reflection unit 74, and a light An optical path 73 connected to the optical path 72 via the reflection section 75; a rotating plate 76 connected to the optical path 73; and a support plate 77 that supports the optical fiber. The excitation light introduced into the light path 71 from the light source force is reflected by the light reflecting part 74 and introduced into the light path 72, reflected by the light reflecting part 75 and introduced into the light path 73 by the rotating body 76. It will be introduced into the light passage port 79 provided in.

As shown in FIG. 5, a shaft member 761 provided at the center of the rotating plate 76 is fitted into a through hole 771 provided at the center of the support plate 77, so that the rotating plate 76 can rotate. Supported by a support plate 77. [0067] As shown in FIG. 5, the support plate 77 is provided with three light passage openings 772, 773, and 774 force S on the rotation locus of the light passage opening 79 of the rotation plate 76, and the light passage is performed. Optical ports 78a, 78b, and 78c are connected to the ports 772, 773, and 774, respectively, and the optical fibers 78a, 78b, and 78c are respectively connected to the optical fibers 442 and 442 of the light irradiators 44 and 48, respectively. Connected to 482.

The light detection unit 6 can rotate the light path 71, and the rotation plate 76 can rotate about the shaft member 761 as the light path 71 rotates. The rotation axis of the optical path 71 and the rotation axis of the rotator 76 are adjusted so as to be located on the same straight line.

[0069] When the rotation plate 76 rotates and the light supply port 79 of the rotation plate communicates with the light supply port 772 of the support plate 77, the excitation light from the light source of the light detection unit 6 passes through the optical fiber 78a. The light irradiation of the temperature control unit 4a is supplied to the light receiving units 44 and 48, and the fluorescence from the light irradiation of the temperature control unit 4a and the light receiving units 44 and 48 is supplied to the light detection unit 6 through the optical fiber 78a. It has become. Further, when the rotating plate 76 rotates and the light supply port 79 of the rotating plate communicates with the light supply port 773 of the support plate 77, the excitation light from the light source of the light detection unit 6 is temperature controlled through the optical fiber 78b. The light irradiation of the unit 4b is supplied to the light receiving units 44 and 48, and the fluorescence from the light irradiation of the temperature control unit 4b and the light receiving units 44 and 48 is supplied to the light detecting unit 6 through the optical fiber 78b. ing. Further, when the rotating plate 76 rotates and the light supply port 79 of the rotating plate communicates with the light supply port 774 of the support plate 77, the excitation light having the light source power of the light detection unit 6 passes through the optical fiber 78c and the temperature control unit. 4c Light irradiation 'The light is supplied to the light receiving parts 44 and 48, and the light from the temperature control part 4c' The fluorescence from the light receiving parts 44 and 48 is supplied to the light detecting part 6 through the optical fiber 78c. ing. As a result, the optical detection unit 6 can optically and continuously detect the PCR results (for example, the amount of PCR amplified fragments) generated in the reaction vessels la, lb and lc! / RU

[0070] Hereinafter, the operation of the reactor 10 will be described.

Before operating the reaction apparatus 10, as shown in FIG. 1, the reaction containers la, lb and lc which are in an open state are installed in the reaction container holding part 2. At this time, each reaction vessel is held by the reaction vessel holding unit 2 so that the surface direction of the main walls 111a and 111b and the X-axis direction coincide with each other. The liquid container 111 of the reaction container is located in the passage 40 of the temperature controllers 4a, 4b and 4c. In addition, the lid 12 of each reaction container is placed on the pressurizing unit 33 of the lid opening / closing unit 3. Thereby, the magnet 34 of the pressurizing part 33 and the magnet 13 of the lid 12 act, and the lid 12 is fixed to the pressurizing part 33 by the magnetic force.

[0071] First, the reaction apparatus 10 moves the suction / discharge section 9 in the X-axis direction and the Y-axis direction by the suction / discharge section moving section 8, and the suction / discharge section 9 contains the PCR reagent in the container group 200. After moving the container above the predetermined container, the suction / discharge part 9 is moved in the Z-axis direction by the suction / discharge part moving part 8, and the tip of the pipette tip P attached to the nozzle part 91 of the suction / discharge part 9 is moved. Enter the container.

[0072] Next, in the reaction vessel 10, the inside of the pipette tip P is depressurized by the suction / discharge section 9, the PCR reagent is sucked into the pipette chip P, and then the suction / discharge section is moved by the suction / discharge section moving section 8. 9 is moved in the Z-axis direction, and the tip of the pipette tip P attached to the nozzle part 91 of the suction / discharge part 9 is retracted by a predetermined container force.

Next, the reaction apparatus 10 moves the suction / discharge part 9 in the X-axis direction and the Y-axis direction by the suction / discharge part moving part 8, and the reaction container la held in the reaction container holding part 2. , Lb and lc (open state), then move the suction / discharge part 9 in the Z-axis direction by the suction / discharge part moving part 8, and the pipette tip P attached to the nozzle part 91 of the suction / discharge part 9 The tip of is inserted into the reaction vessel la, lb and lc. At this time, the tip end portion of the pipette tip P enters from the upper end opening 112a of the cylindrical portion 112, passes through the lower end opening 112b of the cylindrical portion 112 and the upper end opening 11 If of the liquid storage portion 111, and becomes liquid. It reaches the bottom of the accommodating part 111 (sub-wall part 1 l ie).

[0074] Next, the reaction apparatus 10 pressurizes the inside of the pipette chip P by the suction / discharge section 9, and discharges the PCR reagent from the inside of the pipette chip P to the liquid storage section 111. At this time, the reaction apparatus 10 moves the suction / discharge part 9 in the X-axis direction by the suction / discharge part moving part 8, and the pipette tip P moves between the sub-wall parts 1lc and 11Id of the liquid storage part 111, Dispense the PCR reagent into the liquid container 1 11. This prevents bubbles from being contained in the PCR reagent stored in the liquid storage unit 111.

[0075] When the PCR reagent is completely discharged into the liquid storage unit 111, the PCR reagent is stored in the liquid storage unit 111. Housed in a thin layer.

Next, the reaction container 10 moves the suction / discharge part 9 in the Z-axis direction by the suction / discharge part moving part 8 to react the tip of the pipette tip P attached to the nozzle part 91 of the suction / discharge part 9. After leaving the containers la, lb and lc, the lid 12 is attached to the reaction vessel main body 11 by the lid opening / closing part 3. By attaching the lid 12 to the reaction vessel body 11, the opening 112a of the cylindrical portion 112 is sealed with the lid 12, and the internal space (internal spaces S111 and S112) of the reaction vessel body 11 is hermetically sealed. Is done. The lid opening / closing part 3 pressurizes the lid 12 against the reaction vessel main body 11 even after the lid 12 is attached to the reaction vessel main body 11. Thereby, the sealed state of the internal space of the reaction vessel main body 11 is maintained.

[0077] Next, the reaction apparatus 10 moves the liquid container 111 of the reaction containers la, lb, and lc along the passage 40 by moving the reaction container holder 2 in the X-axis direction. 1 11 is positioned between the heating blocks 411 and 451, and the liquid storage unit 111 is stopped at that position. When the liquid storage part 111 is located between the heating blocks 411 and 451, the heating surfaces of the calo heat blocks 411 and 451 are in contact with the main wall parts 11 la and 11 lb, respectively, and the main wall parts 11 la and 111b are , Heated to a temperature at which the double-stranded DNA is dissociated (eg, 95 ° C). As a result, the double-stranded DNA contained in the PCR reagent housed in the liquid container 111 is dissociated into a single-stranded DNA. The PCR reagent stored in the liquid storage unit 111 is thin and has a large contact area with the PCR reagent! PCR Since the PCR reagent is heated through the main walls 11 la and 11 lb, the double-stranded DNA contained in the PCR reagent dissociates rapidly. The liquid storage unit 111 is stopped between the heating blocks 411 and 451 until the double-stranded DNA contained in the PCR reagent is dissociated. The power stop time is usually 20 to 30 seconds. Although there is a possibility that bubbles in the PCR reagent force are generated when the PCR reagent is heated, the generation of bubbles in the PCR reagent force is prevented by the expansion pressure due to the heating of the air stored in the cylindrical portion 112.

[0078] Next, the reaction apparatus 10 moves the liquid container 111 of the reaction containers la, lb, and lc along the passage 40 by moving the reaction container holder 2 in the X-axis direction. 1 Place 11 between cooling blocks 421 and 461, and stop liquid storage unit 111 at that position. When the liquid storage unit 111 is positioned between the cooling blocks 421 and 461, cooling air is sent by a blower (not shown), and the main walls 11 la and 11 lb It is cooled to one ring temperature (eg 65 ° C). As a result, the primer anneals to the single-stranded DNA contained in the PCR reagent stored in the liquid storage unit 111. The PCR reagent stored in the liquid storage unit 111 is a thin layer, has a large contact area with the PCR reagent, and the PCR reagent is cooled through the main walls 11 la and 11 lb. Primer rapidly anneals to strand DNA. The liquid storage unit 111 normally stops between the cooling blocks 421 and 461 until the primer anneals to the single-stranded DNA contained in the PCR reagent.

[0079] Next, the reaction apparatus 10 moves the liquid container 111 of the reaction containers la, lb, and lc along the passage 40 by moving the reaction container holder 2 in the X-axis direction. 1 11 is positioned between the heating blocks 431 and 471, and the liquid container 111 is stopped at that position. When the liquid storage part 111 is located between the heating blocks 431 and 471, the heating surfaces of the calo heat blocks 4 31 and 471 are in contact with the main wall parts 11 la and 11 lb, respectively, and the main wall parts 11 la and 11 lb Is heated to a temperature at which an extension reaction by the polymerase occurs (eg, 72 ° C.). As a result, the extension reaction of the primer annealed to the single-stranded DNA contained in the PCR reagent contained in the liquid container 111 occurs. The PCR reagent stored in the liquid storage unit 111 is a thin layer and has a large contact area with the PCR reagent. P The PCR reagent is heated through the main walls 11 la and 11 lb. The extension reaction of the primer annealed to the double-stranded DNA occurs rapidly. The liquid container 111 has a force to stop between the heating blocks 431 and 471 until the primer extension annealed to the single-stranded DNA contained in the PCR reagent is sufficiently generated. The stop time is usually 30 to 60 seconds. . Although there is a risk that bubbles may be generated in the PCR reagent force when the PCR reagent is heated, the generation of bubbles from the PCR reagent is prevented by the expansion pressure due to the heating of the air stored in the cylindrical portion 112.

[0080] As described above, one cycle of PCR (eg, 30 seconds at 95 ° C, 30 seconds at 65 ° C, 1 minute at 72 ° C) is completed. In order to repeat this for 30 to 40 cycles, the reaction apparatus 10 reciprocates the reaction container holding part 2 in the X-axis direction to move the liquid container part 111 of the reaction containers la, lb and lc along the passage 40. The liquid container 111 is stopped at a predetermined position. [0081] After the completion of the predetermined PCR cycle, the reaction apparatus 10 moves the liquid container 111 of the reaction containers la, lb, and lc along the passage 40 by moving the reaction container holder 2 in the X-axis direction. Move the liquid storage unit 111 to light irradiation * position between the light receiving units 44 and 48, and stop the liquid storage unit 111 at that position. When the liquid storage unit 111 is positioned between the light irradiation / light reception units 44 and 48, the excitation light generated by the light source force of the light detection unit 6 passes through the light supply unit 7 to the main walls 11 la and 11 lb of the liquid storage unit 111. Irradiated. The fluorescent dye contained in the PCR reagent generates fluorescence by irradiation with excitation light, and the generated fluorescence is supplied to the light detection unit 6 through the light supply unit 7 and detected.

[0082] As the fluorescent dye, one that changes fluorescence characteristics such as fluorescence intensity and fluorescence wavelength depending on the amount of nucleic acid (for example, DNA) is used. For example, fluorescent dyes that change properties such as fluorescence intensity and fluorescence wavelength by inter-forced with double-stranded DNA can be used. From the viewpoint of ease of measurement, a fluorescent dye having a property that fluorescence intensity increases by intercalation is preferable. Specific examples of such fluorescent dyes include ethidium bromide (EtBr), SYBR Greenl, PicoGreen, thiazole orange, oxazole yellow and the like. For example, ethidium bromide inter-forced with DNA is excited by ultraviolet (260 nm) energy transfer absorbed by DNA or its own absorbed light, and emits fluorescence. In addition, SYBR Greenl inter-forced with DNA emits green fluorescence when excited by ultraviolet light around 26 Onm or visible light around 470 nm. Since the fluorescence intensity emitted by these fluorescent dyes is proportional to the amount of double-stranded DNA, the amount of PCR amplification product can be detected by measuring the fluorescence intensity of the fluorescent dye.

[0083] In addition, an oligonucleotide probe complementary to the intermediate portion of the target sequence and a combination of two types of fluorescent dyes, a reporter and a quencher, can be used. A reboter is a molecule that emits fluorescence when irradiated with excitation light. However, in the case of an oligonucleotide probe that has a quencher near the reporter, the energy absorbed by the reporter is absorbed by the quencher and the reporter is excited. The fluorescence that would otherwise have been generated does not occur (taenting). When the oligonucleotide probe causing quenching is added to the PCR reaction solution contained in the reaction chamber, it binds to the target sequence. After that, Taq polymerase also synthesizes the extended strand with the 3 'end force of the primer, If it hits the probe in the middle, the probe that has already annealed due to the 5 '→ 3' endonuclease activity will be decomposed, and the reporter and the quencher will be separated adjacent to each other, suppressing the quencher. The reporter who received the light now emits fluorescence. Since this reaction occurs almost in proportion to the PCR cycle, the amount of PCR amplification product can be detected by measuring the fluorescence intensity of the reporter.

[0084] Alternatively, two types of oligonucleotide probes that hybridize adjacent to the target nucleic acid and fluorescent dyes bound thereto can be used. A donor dye is attached to the 3 'end of the 5' probe, and an acceptor dye is attached to the 5 'end of the 3' probe, and two types of probes are adjacent to the target nucleic acid. Then, the donor dye emits fluorescence by the excitation light of the external light source, and the light is absorbed by the acceptor dye, and then the acceptor dye emits light of a different wavelength. As the amount of PCR amplification product increases, the amount of probe that hybridizes to the target nucleic acid also increases. Therefore, the amount of PCR amplification product can be detected by measuring the fluorescence intensity.

[0085] Next, in the reaction device 10, after the lid 12 is detached from the reaction vessel main body 11 by the lid opening / closing part 3, the suction / discharge part 9 is moved in the X-axis direction and the Y-axis by the suction / discharge part moving part 8. The suction / discharge unit 9 is moved above the reaction vessel 1 (open state) held by the reaction vessel holding unit 2.

Next, the reaction apparatus 10 moves the suction / discharge part 9 in the Z-axis direction by the suction / discharge part moving part 8 to react the tip of the pipette tip P attached to the nozzle part 91 of the suction / discharge part 9. After entering the reaction containers la, lb and lc held in the container holding part 2, the inside of the pipette tip P is decompressed by the suction and discharge part 9, and the reaction after PCR in the reaction containers la, lb and lc is performed. Aspirate the reaction solution.

Next, the reaction apparatus 10 moves the suction / discharge part 9 in the Z-axis direction by the suction / discharge part moving part 8 to react the tip of the pipette tip P attached to the nozzle part 91 of the suction / discharge part 9. Containers la, lb and lc forces After retreating, the suction / discharge part 9 is moved in the X-axis direction and the Y-axis direction by the suction / discharge part moving part 8, and the suction / discharge part 9 is moved to the PCR amplification fragment of the container group 200. Is moved above a predetermined container for containing

Next, the reaction apparatus 10 moves the suction / discharge part 9 in the Z-axis direction by the suction / discharge part moving part 8. After moving the tip of the pipette tip P attached to the nozzle portion 91 of the suction / discharge section 9 into the predetermined container, the inside of the pipette tip P is pressurized by the suction / discharge section 9 and the pipette Discharge the reaction solution after PCR in Tochip P.

Next, the reaction apparatus 10 moves the suction / discharge part 9 in the Z-axis direction by the suction / discharge part moving part 8 and moves the tip of the pipette tip P attached to the nozzle part 91 of the suction / discharge part 9 to a predetermined position. Container Evacuate.

[0090] The reaction solution after PCR stored in a predetermined container of the container group 200 may be subjected to further analysis.

For example, as shown in FIG. 7, it can be used for the analysis with a single bead 900 provided on the stage 100.

[0091] The first bead 900 has a cylindrical part 902 having an opening 901 at the upper end, and a part 903 that is in communication with the cylindrical part 902. The beads to which the beads are bound are accommodated in a predetermined order.

[0092] In the same manner as described above, the reaction apparatus 10 sucks the reaction solution after PCR into the pipette chip P attached to the nozzle portion 91 of the suction / discharge portion 9, and then opens the opening 90 of the one-pillar bead 900. Discharge from 1 to the inside. The discharged reaction solution flows into a part of the suspension 903 and reacts with beads to which various probes are bound. The type of PCR amplified fragment can be identified by detecting the presence or absence of binding between the probe and the PCR amplified fragment using, for example, fluorescence.

Various changes can be made in the present embodiment.

For example, the reaction vessel Id shown in FIGS. 8 (a) and (b) can be used as the reaction vessels la, lb and lc.

The reaction vessel body 11 of the reaction vessel Id is the same as the reaction vessel body 11 of the reaction vessel la, lb and lc, but the lid 12d of the reaction vessel Id is different from the lid 12 of the reaction vessel la, lb and lc. The

[0094] As shown in Figs. 8 (a) and (b), the lid body 12d has an opening 122d at one end, which is formed by a continuous wall portion, and has a gas containing portion 121d that can contain a gas. The gas storage portion 121d has a continuous wall portion so that the volume of the gas stored in the gas storage portion 121d is reduced. It can be deformed while maintaining its properties. When the lid 12d is attached to the reaction vessel main body 11, the gas containing portion 121d is attached to the reaction vessel main body 11 so that the opening 122d of the gas containing portion 12 Id and the opening 112a of the cylindrical portion 112 communicate with each other. Being

[0095] When the reaction vessel Id is used, the reaction apparatus 10 preferably includes a pressurizing unit for pressing the lid 12d of the reaction vessel Id (closed state) held by the reaction vessel holding unit 2. . As shown in FIG. 8 (c), the pressurizing unit moves the pressurizing block 800 in the Z-axis direction to press the gas accommodating unit 121d of the reaction vessel Id held by the reaction vessel holding unit 2. Thereby, the gas accommodating part 121d is deformed while maintaining the continuity of the wall so that the volume of the gas accommodated in the gas accommodating part 121d is reduced. That is, the air pressure in the internal space of the reaction vessel Id increases while the hermeticity of the internal space of the reaction vessel Id is maintained. When the pressure in the internal space of the reaction vessel Id increases, it is possible to effectively prevent the generation of bubbles due to the heating of the PCR reagent contained in the reaction vessel Id.

[0096] The form of the lid 12d can be changed, for example, can be changed to the lid 12e shown in Fig. 8 (d).

In order to further secure the sealed state of the reaction vessels la, lb, and lc in the closed state, hook portions that engage with the lid body 12 and the flange portion 113 are provided on the flange portion 113 and the lid body 12, respectively. It may be provided.

[0097] In the present embodiment, the reaction vessel la, lb and lc are moved relative to the temperature control units 4a, 4b and 4c by moving the reaction vessel holding unit 2, but the reaction vessel holding unit The reaction vessels 1a, lb and lc may be moved relative to the temperature control units 4a, 4b and 4c by moving the temperature control units 4a, 4b and 4c without moving 2. Even if the reaction vessels la, lb, and lc are moved relative to the temperature control units 4a, 4b, and 4c by moving the reaction vessel holding unit 2 and the temperature control units 4a, 4b, and 4c together. Good.

In this embodiment, optical detection by the light detection unit 6 is performed after the end of a predetermined PCR cycle, but the optical detection by the light detection unit 6 is performed at a desired time before the end of the PCR cycle. Also good. For example, by performing optical detection every one to several cycles, the progress of PCR can be monitored in real time.

In the present embodiment, the pipette tip P is located between the auxiliary wall portions 1 lc and 11 Id of the liquid storage portion 111. The suction / discharge unit 9 has been moved in the X-axis direction so that the PCR reagent can be discharged into the liquid storage unit 111 while moving, but the reaction container holding unit 2 may be moved in the X-axis direction.

In the present embodiment, the liquid container 111 is stopped at a predetermined position in order to heat or cool the liquid container 111, but the liquid container 111 does not necessarily need to be stopped. By controlling (for example, slowly moving the liquid container 111), the liquid container 111 may be subjected to heating or cooling for a sufficient time.

[0101] In this embodiment, fluorescence is used as an indicator of a reaction result (for example, presence or absence, amount, etc. of a PCR-amplified fragment), but chemiluminescence or the like may be used as an indicator of a reaction result. In the case of chemiluminescence, since it is not necessary to irradiate excitation light, light generated from the reaction liquid stored in the liquid storage unit 111 may be detected by the light detection unit 6.

[0102] In the present embodiment, in order to heat or cool the liquid container 111, the main walls 11 la and 11 lb are brought into contact with the heating surfaces of the calo heat blocks 411 (431) and 451 (471), or The force that allowed the cooling air to blow through the air outlets of the cooling blocks 421 and 461 against the main wall 11 la and 11 lb. As long as it can be heated or cooled to the desired target temperature, the main wall 1 1 la or 11 lb !, one of the sides may be in contact with the heating surface of the heating block 411 (431) or 451 (471) and the main wall 11 la or 11 lb Cooling air may be blown through the blower opening of the rack 421 or 461.

In the present embodiment, the force that has been applied to the main wall portions 11 la and 11 lb of the liquid storage portion 111 through the light supply portion 7 with the excitation light generated from the light source of the light detection portion 6 is the main wall portion 11 la. Or you can irradiate 11 lbs of V.

Claims

The scope of the claims

[1] The first and second main wall portions facing each other and the sub-wall portion continuous with the first and second main wall portions have an opening at one end, and the liquid is formed into a thin layer. A reaction container comprising a reaction container body comprising a liquid container capable of being accommodated, and a cylindrical part having a first opening at one end and a second opening at the other end,

An end portion on the opening side of the liquid container portion and an end portion on the second opening portion side of the cylindrical portion so that the internal space of the liquid storage portion and the internal space of the cylindrical portion communicate with each other. Are continuous,

A reaction vessel in which the first and Z or second main wall portions have thermal conductivity and light transmittance.

[2] The reaction vessel according to claim 1, further comprising a lid capable of sealing the first opening of the cylindrical portion.

[3] The tip of the pipette tip attached to the nozzle of the dispensing device enters from the first opening of the cylindrical part, and the second opening of the cylindrical part and the liquid storage part 2. The reaction container according to claim 1, wherein the reaction container can reach the bottom of the liquid container through the opening.

[4] The anti-J core container according to [2], wherein the lid is attached to the reaction container main body so as to be freely opened and closed.

[5] The lid is brought into contact with an end of the tubular portion on the first opening side without being fitted into the first opening of the tubular portion, whereby the first portion of the tubular portion is arranged. The reaction container according to claim 4, wherein the opening of 1 can be sealed.

6. The lid according to claim 4, wherein the lid is provided with a magnet or a magnetic body, and the lid in the open state can be closed by the magnetic force, and the lid in the closed state can be opened. Reaction vessel.

[7] The lid body has a gas accommodating portion that is formed by a continuous wall portion, has an opening at one end, and can store gas.

The gas accommodating portion can be deformed while maintaining the continuity of the wall portion so that the volume of gas accommodated in the gas accommodating portion is reduced.

5. The reaction container according to claim 4, wherein when the lid is attached to the reaction container main body, the opening of the gas storage part communicates with the first opening of the cylindrical part.

[8] A holding part for holding the reaction vessel according to claim 1, A passage through which the liquid storage part of the reaction vessel held by the holding part can move in the surface direction of the first and second main wall parts;

When the liquid storage part of the reaction container held by the holding part is at a predetermined position of the passage, the first and Z or second main wall parts of the reaction container can be heated or cooled to a predetermined temperature. 1st to nth temperature control units provided at predetermined positions of the passage,

The liquid container of the reaction vessel held by the holding unit can be moved relative to the first to nth temperature control units along the passage, and the first to nth temperatures. The reactor described above at a position where the first to nth temperature controllers are provided such that heating or cooling by the controller is performed on the first and Z or second main walls of the reactor. And a moving part capable of controlling the speed of the liquid storage part.

A holding part for holding the reaction vessel according to claim 1,

A passage through which the liquid storage part of the reaction vessel held by the holding part can move in the surface direction of the first and second main wall parts;

When the liquid storage part of the reaction container held by the holding part is at a predetermined position of the passage, light emitted from the first and Z or second main wall parts of the reaction container can be received. A light receiving portion provided at a predetermined position of the passage;

The liquid storage part of the reaction vessel held by the holding part can be moved relative to the first to nth temperature control parts and the light receiving part along the passage, and the first to nth temperature control parts. heating or cooling by the temperature control unit of the n, and so light by the light receiving unit is performed on the first and Z or the second main wall of the front Symbol reaction vessel, temperature control of the first to _n A moving part capable of controlling the speed of the liquid storage part of the reaction vessel at a position where the light receiving part and the light receiving part are provided;

A light detection unit for detecting light generated by the first and Z or second main wall force of the reaction vessel;

A reaction apparatus comprising: a light supply unit that supplies light from the light receiving unit to the light detection unit. [10] A holding part for holding the reaction vessel according to claim 1,

When the liquid storage part of the reaction container held by the holding part is at a predetermined position of the passage, the first and Z or second main wall parts of the reaction container can be irradiated with light and the reaction container A light irradiation 'light receiving portion provided at a predetermined position of the passage so as to receive light emitted from the first and Z or second main wall portions of

The liquid storage part of the reaction vessel held by the holding part can be moved relative to the first to nth temperature control parts and the light irradiation 'light receiving part along the passage, and The heating or cooling by the first to nth temperature control units, and the light irradiation and light reception by the light receiving unit are performed on the first and Z or second main walls of the reaction vessel. A moving part capable of controlling the speed of the liquid storage part of the reaction container at a position where the first to nth temperature control parts and the light irradiation 'light receiving part are provided;

A light detection unit that generates excitation light and detects light emitted from the first and Z or second main walls of the reaction vessel;

A reaction apparatus comprising: a light supply unit that supplies light from the light detection unit to the light irradiation / light reception unit and a light irradiation / light reception unit to the light detection unit.

[11] A holding unit for holding a plurality of reaction vessels according to claim 1,

A passage through which each reaction vessel held by the holding portion can move in the surface direction of the first and second main wall portions;

When each reaction vessel held by the holding portion is at a predetermined position in the passage, the passage is arranged so that the first and Z or second main wall portions of each reaction vessel can be heated or cooled to a predetermined temperature. First to nth temperature control units provided at predetermined positions,

When each reaction vessel held by the holding portion is at a predetermined position in the passage, the light emitted from the first and Z or second main wall portions of each reaction vessel can be received. A light receiving portion provided at a predetermined position;

The liquid storage part of each reaction vessel held by the holding part can be moved relative to the first to nth temperature control parts and the light receiving part along the passage, and the first to The first to nth temperature controls so that heating or cooling by the n temperature control unit and light reception by the light receiving unit are performed on the first and Z or second main walls of each reaction vessel. And a moving part capable of controlling the speed of the liquid storage part of each reaction container at the position where the light receiving part is provided,

A light detector for detecting the light generated by the first and Z or second main wall force of each reaction vessel; and

A reaction device including a light supply unit configured to supply light from the light receiving unit to the light detection unit;

The light supply unit determines which reaction container receives the light emitted from the first and Z or second main walls, selects the light receiving unit corresponding to the determined reaction container, and selects the selected light receiving unit. The said reaction apparatus which supplies the light received by the part to the said light detection part.

A holding unit for holding a plurality of reaction vessels according to claim 1,

When each reaction vessel held by the holding portion is at a predetermined position in the passage, the passage is arranged so that the first and Z or second main wall portions of each reaction vessel can be heated or cooled to a predetermined temperature. 1st to nth temperature control units provided at predetermined positions,

When each reaction vessel held by the holding unit is at a predetermined position in the passage, the first and Z or the second main wall of each reaction vessel can be irradiated with light, and the first and second of each reaction vessel Light irradiation 'light receiving portion provided at a predetermined position of the passage so that light emitted from Z or the second main wall portion can be received; and

The liquid storage part of each reaction container held by the holding part can be moved relative to the first to nth temperature control parts and the light irradiation 'light receiving part along the passage, and Heating or cooling by the first to nth temperature control units, light irradiation, light irradiation and light reception by the light receiving unit are performed on the first and Z or second main wall portions of each reaction vessel. A moving unit capable of controlling the speed of the liquid storage unit of each reaction container at a position where the first to nth temperature control units and the light irradiation 'light receiving unit are provided,

A light detection unit that generates excitation light and detects light generated by the first and Z or second main wall force of each reaction vessel;

A light supply unit for supplying light from the light detection unit to the light irradiation / light receiving unit and from the light irradiation / light receiving unit to the light detection unit,

The light supply unit determines which of the reaction containers the first and Z or second main wall portions emit light and receives light, selects the light irradiation and light receiving units corresponding to the determined reaction container, The reaction apparatus that supplies excitation light to the selected light irradiation / light receiving unit and supplies light received by the selected light irradiation / light receiving unit to the light detection unit.

[13] When the first to nth temperature control units have a heating surface or a cooling surface, and the reaction vessel held by the holding unit is at a predetermined position of the passage, the heating surface or the cooling surface is 13. The reactor according to any one of claims 8 to 12, which is located in the vicinity of the first and Z or second main walls of the reaction vessel or is in contact with the first and Z or second main walls of the reaction vessel. Reactor.

[14] The reaction device according to any one of [8] to [12], further comprising a pressurization unit that pressurizes the lid of the reaction vessel held by the holding unit against the reaction vessel main body.

[15] The reaction vessel held by the holding portion is the reaction vessel according to claim 6, and the pressurizing portion is provided with a magnet that acts on a magnet or a magnetic body of the lid of the reaction vessel, 15. The reaction device according to claim 14, further comprising a pressurizing unit moving unit that moves the caloric pressure unit so that the lid body is in an open state force closed state or a closed state force open state.

[16] The reaction vessel held by the holding unit is the reaction vessel according to claim 3, wherein a nozzle capable of sucking and discharging a liquid and a tip end portion of a pipette tip attached to the nozzle are provided. The reaction apparatus according to claim 8, further comprising a nozzle moving unit that moves into and out of the liquid storage unit of the reaction vessel held by the holding unit.

[17] The nozzle moving section moves the tip of the pipette tip into the liquid storage section of the reaction container, and then moves it in the surface direction of the first and second main wall sections of the reaction container. Item 16. The reactor according to Item 16.

[18] The reaction vessel according to claim 7, wherein the reaction vessel held by the holding unit is the lid The press part which can press the gas accommodating part of the said cover body and can deform | transform the said gas accommodating part so that the capacity | capacitance of the gas accommodated in the gas accommodating part of a body may be reduced. The reactor according to any one of the above.