WO2008004695A1 - Contenant de réaction et dispositif de réaction - Google Patents

Contenant de réaction et dispositif de réaction Download PDF

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Publication number
WO2008004695A1
WO2008004695A1 PCT/JP2007/063695 JP2007063695W WO2008004695A1 WO 2008004695 A1 WO2008004695 A1 WO 2008004695A1 JP 2007063695 W JP2007063695 W JP 2007063695W WO 2008004695 A1 WO2008004695 A1 WO 2008004695A1
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WO
WIPO (PCT)
Prior art keywords
light
reaction
reaction vessel
unit
passage
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2007/063695
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English (en)
Japanese (ja)
Inventor
Hideji Tajima
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universal Bio Research Co Ltd
Original Assignee
Universal Bio Research Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universal Bio Research Co Ltd filed Critical Universal Bio Research Co Ltd
Priority to JP2008523771A priority Critical patent/JPWO2008004695A1/ja
Publication of WO2008004695A1 publication Critical patent/WO2008004695A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/0332—Cuvette constructions with temperature control
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Rigid containers without fluid transport within
    • B01L3/5082—Test tubes per se
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Rigid containers without fluid transport within
    • B01L3/5082—Test tubes per se
    • B01L3/50825—Closing or opening means, corks, bungs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781—Aspects relating to microreactors
    • B01J2219/00788—Three-dimensional assemblies, i.e. the reactor comprising a form other than a stack of plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/043—Hinged closures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0858—Side walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/14—Means for pressure control
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Rigid containers without fluid transport within
    • B01L3/5085—Rigid containers without fluid transport within for multiple samples, e.g. microtitration plates
    • B01L3/50851—Rigid containers without fluid transport within for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0325—Cells for testing reactions, e.g. containing reagents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters

Definitions

  • 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.
  • PCR Polymerase chain reaction
  • 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.
  • the thermal profile temperature increase / decrease
  • the third stage which is maintained at the temperature at which to ring
  • 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.
  • 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.
  • double-stranded DNA dissociates into single-stranded DNA.
  • the primer and the single-stranded DNA ring.
  • the temperature is raised to the polymerase reaction temperature (72 ° C in the above example)
  • the DNA synthesis reaction by polymerase proceeds.
  • PCR 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.
  • 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.
  • metal block cooling methods one using a compressor and one using a Peltier cooling method.
  • 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.
  • Patent Documents 1 to 5 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 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
  • 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
  • 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.
  • light for example, fluorescence, chemiluminescence, etc.
  • an indicator of the reaction result for example, presence / absence of PCR amplified fragment, amount, etc.
  • the reaction result generated in the reaction solution can be detected.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • reaction solution e.g., PCR reagent
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • a desired time for example, , Chemiluminescence, etc.
  • the reaction result for example, the presence / absence of reaction, degree, etc.
  • Reaction fluid Power is useful in cases where excitation light irradiation is required to generate light (for example, chemiluminescence). It is for.
  • 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.
  • 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.
  • 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.
  • a reactor equipped.
  • 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
  • the reaction can be performed through the first and second main wall parts at a desired time.
  • light for example, fluorescence, etc.
  • 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).
  • 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.
  • the first and Z or second main wall portions of each reaction vessel can be heated or cooled to a predetermined temperature.
  • 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.
  • 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.
  • the light emitted from the reaction liquid stored in the desired reaction container is detected at a desired time.
  • the reaction result for example, the presence or absence, degree of reaction, etc.
  • Multiple specimens can be processed at one time.
  • 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.
  • the first and Z or second main wall portions of each reaction vessel can be heated or cooled to a predetermined temperature.
  • 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 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
  • 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.
  • 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
  • the reaction solution can be rapidly heated or cooled through the first and Z or second main walls.
  • excitation light is emitted to the reaction liquid stored in the desired reaction container at a desired time.
  • reaction fluid light eg fluorescence
  • 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.
  • 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.
  • 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.
  • the reaction vessel held by the holding unit is the reaction vessel of (6)
  • the pressurizing unit has a magnet or magnetic body of a lid of the reaction vessel.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a light supply unit 7 for supplying light (excitation light, fluorescence) to 4c.
  • Container group 200 contains predetermined PCR reagents (eg, buffer, MgCl, dNTP mix, primer, saddle type)
  • predetermined PCR reagents eg, buffer, MgCl, dNTP mix, primer, saddle type
  • 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.
  • 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
  • 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.
  • 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)
  • 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).
  • 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.
  • 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.
  • 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 is usually 0.3 to 3. Omm, preferably 0.5 to 1. Omm.
  • 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.
  • the cylindrical portion 112 has a cylindrical force, but is not limited to this, and may be a rectangular tube shape or the like.
  • 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.
  • 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! /
  • 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.)
  • 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.
  • 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”).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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! /
  • 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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • 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.
  • a temperature at which the double-stranded DNA is dissociated for example, 95 ° C
  • 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.
  • the heating blocks 411 and 451 are heated to a temperature at which an extension reaction by the polymerase occurs (for example, 72 ° C).
  • a temperature at which an extension reaction by the polymerase occurs for example, 72 ° C.
  • 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! /
  • 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).
  • 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!
  • 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.
  • 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.
  • 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.
  • 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.
  • 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!
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the reaction containers la, lb and lc which are in an open state are installed in the reaction container holding part 2.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the tip of is inserted into the reaction vessel la, lb and lc.
  • 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).
  • 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.
  • 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.
  • the PCR reagent 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.
  • 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.
  • the lid 12 is attached to the reaction vessel main body 11 by the lid opening / closing part 3.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.).
  • 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. .
  • one cycle of PCR (eg, 30 seconds at 95 ° C, 30 seconds at 65 ° C, 1 minute at 72 ° C) is completed.
  • 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.
  • 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.
  • the liquid storage unit 111 moves to light irradiation * position between the light receiving units 44 and 48, and stop the liquid storage unit 111 at that position.
  • 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.
  • 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.
  • fluorescent dyes that change properties such as fluorescence intensity and fluorescence wavelength by inter-forced with double-stranded DNA can be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the oligonucleotide probe causing quenching is added to the PCR reaction solution contained in the reaction chamber, it binds to the target sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • reaction solution after PCR stored in a predetermined container of the container group 200 may be subjected to further analysis.
  • FIG. 7 it can be used for the analysis with a single bead 900 provided on the stage 100.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • the form of the lid 12d can be changed, for example, can be changed to the lid 12e shown in Fig. 8 (d).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the liquid container 111 may be subjected to heating or cooling for a sufficient time.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

L'invention concerne un contenant de réaction et un dispositif de réaction apte à contrôler de façon rapide la température de liquide de réaction et à détecter un résultat de réaction obtenu dans le liquide de réaction à un moment désiré dans le temps. Le contenant de réaction comprend une unité de stockage de liquide formée par une première et une seconde paroi principale tournée l'une vers l'autre et une sous-paroi dans la continuité de la première et de la seconde paroi principale et dont une extrémité est munie d'une ouverture pour être apte à stocker du liquide dans une forme de couche mince, et un corps de contenant de réaction doté d'une unité tubulaire présentant une première ouverture à une extrémité et une seconde ouverture à l'autre extrémité, l'extrémité sur le côté d'ouverture de l'unité de stockage de liquide et l'extrémité sur le second côté d'ouverture de l'unité tubulaire étant continues de façon à permettre à l'espace interne de l'unité de stockage de liquide de communiquer avec l'espace interne de l'unité tubulaire, la première paroi principale et/ou la seconde paroi principale présentant une conductivité thermique et une transparence optique.
PCT/JP2007/063695 2006-07-07 2007-07-09 Contenant de réaction et dispositif de réaction Ceased WO2008004695A1 (fr)

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EP2463641A1 (fr) * 2010-12-08 2012-06-13 Qiagen GmbH Tube de traitement de fluide pour analyse optique et procédé d'analyse d'un fluide
JP2012145510A (ja) * 2011-01-14 2012-08-02 Hitachi High-Technologies Corp 核酸分析装置
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JP2013524808A (ja) * 2010-04-23 2013-06-20 ナノバイオシス・インコーポレイテッド ２個の熱ブロックを含むｐｃｒ装置
WO2015053290A1 (fr) * 2013-10-07 2015-04-16 ユニバーサル・バイオ・リサーチ株式会社 Dispositif de mesure de chimiluminescence et son procédé
CN104668006A (zh) * 2011-01-12 2015-06-03 环球生物研究株式会社 反应容器及其制造方法、以及反应容器系统
EP2949394A1 (fr) * 2014-05-28 2015-12-02 Seiko Epson Corporation Appareil de réaction d'amplification d'acide nucléique et récipient
JP2016086751A (ja) * 2014-11-06 2016-05-23 東洋紡株式会社 反応促進装置及び核酸検査装置
KR20170143077A (ko) * 2016-06-17 2017-12-29 주식회사 바이오인프라 튜브 개폐 장치 및 이를 포함하는 분주 시스템
JP2021112134A (ja) * 2020-01-16 2021-08-05 株式会社島津製作所 反応容器
US20230159917A1 (en) * 2019-05-07 2023-05-25 Bio-Rad Laboratories, Inc. System and method for automated single cell processing

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JP2013524808A (ja) * 2010-04-23 2013-06-20 ナノバイオシス・インコーポレイテッド ２個の熱ブロックを含むｐｃｒ装置
US9061285B2 (en) 2010-04-23 2015-06-23 Nanobiosys Inc. PCR device including two heating blocks
JP2012024042A (ja) * 2010-07-27 2012-02-09 Hitachi High-Technologies Corp 核酸検査装置
CN103250042A (zh) * 2010-12-08 2013-08-14 凯杰有限公司 用于光学分析的流体处理管以及分析流体的方法
WO2012076337A1 (fr) * 2010-12-08 2012-06-14 Qiagen Gmbh Tube de traitement de fluide pour analyse optique et méthode d'analyse d'un fluide
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JP2014502362A (ja) * 2010-12-08 2014-01-30 キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング 光学分析用の流体処理チューブ、および流体を分析するための方法
EP2463641A1 (fr) * 2010-12-08 2012-06-13 Qiagen GmbH Tube de traitement de fluide pour analyse optique et procédé d'analyse d'un fluide
CN104668006A (zh) * 2011-01-12 2015-06-03 环球生物研究株式会社 反应容器及其制造方法、以及反应容器系统
JP2012145510A (ja) * 2011-01-14 2012-08-02 Hitachi High-Technologies Corp 核酸分析装置
CN103547666B (zh) * 2011-02-22 2016-04-27 环球生物研究株式会社 反应容器及其制造方法
JPWO2012114562A1 (ja) * 2011-02-22 2014-07-07 ユニバーサル・バイオ・リサーチ株式会社 反応容器およびその製造方法
WO2012114562A1 (fr) * 2011-02-22 2012-08-30 ユニバーサル・バイオ・リサーチ株式会社 Récipient de réaction et son procédé de production
CN103547666A (zh) * 2011-02-22 2014-01-29 环球生物研究株式会社 反应容器及其制造方法
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US9857308B2 (en) 2013-10-07 2018-01-02 Universal Bio Research Co., Ltd. Chemiluminescence measurement device and method for same
WO2015053290A1 (fr) * 2013-10-07 2015-04-16 ユニバーサル・バイオ・リサーチ株式会社 Dispositif de mesure de chimiluminescence et son procédé
JPWO2015053290A1 (ja) * 2013-10-07 2017-03-09 ユニバーサル・バイオ・リサーチ株式会社 化学発光測定装置およびその方法
EP2949394A1 (fr) * 2014-05-28 2015-12-02 Seiko Epson Corporation Appareil de réaction d'amplification d'acide nucléique et récipient
JP2016086751A (ja) * 2014-11-06 2016-05-23 東洋紡株式会社 反応促進装置及び核酸検査装置
KR101878725B1 (ko) 2016-06-17 2018-08-20 주식회사 바이오인프라 튜브 개폐 장치 및 이를 포함하는 분주 시스템
KR20170143077A (ko) * 2016-06-17 2017-12-29 주식회사 바이오인프라 튜브 개폐 장치 및 이를 포함하는 분주 시스템
US11572263B2 (en) 2016-06-17 2023-02-07 Bioinfra Co., Ltd. Tube opening and closing device and dispensing system including same
US20230159917A1 (en) * 2019-05-07 2023-05-25 Bio-Rad Laboratories, Inc. System and method for automated single cell processing
US12410427B2 (en) * 2019-05-07 2025-09-09 Bio-Rad Laboratories, Inc. System and method for automated single cell processing
JP2021112134A (ja) * 2020-01-16 2021-08-05 株式会社島津製作所 反応容器
JP7338485B2 (ja) 2020-01-16 2023-09-05 株式会社島津製作所 反応容器

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