Classifications

• • 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
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• • 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
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/02—Adapting objects or devices to another
  • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
  • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/16—Reagents, handling or storing thereof
• • 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
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/087—Multiple sequential chambers
• • 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
  • B01L2300/16—Surface properties and coatings
  • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
  • B01L2300/165—Specific details about hydrophobic, oleophobic surfaces
• • 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
  • B01L2300/18—Means for temperature control
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0605—Valves, specific forms thereof check valves
  • B01L2400/0616—Ball valves
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0622—Valves, specific forms thereof distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0633—Valves, specific forms thereof with moving parts
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0633—Valves, specific forms thereof with moving parts
  • B01L2400/0644—Valves, specific forms thereof with moving parts rotary valves
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• • 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
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids

Definitions

• the present invention relates to a test container and a nucleic acid testing method.
• test containers such as test cartridges or analysis chips that are used to perform various analyzes on specimens extracted from biological samples are known.
• the test container includes a plurality of chambers (liquid storage portions) that contain liquid, and a flow path that connects the chambers.
• a sample liquid is transferred from chamber to chamber using an external force such as electromagnetic force, centrifugal force, or pressure.
• Japanese Patent Publication No. 2019-515308 discloses a microfluidic chip that uses centrifugal force to send a sample (sample liquid) input from a sample inlet (input port) provided at one end of a sample chamber from the sample chamber to a mixing chamber. is disclosed.
• the microfluidic chip disclosed in Japanese Patent Publication No. 2019-515308 is equipped with a circuit configured to provide an air connection between the sample chamber and the mixing chamber, that is, a vent channel, so that liquid can be smoothly transferred. I have to.
• the present disclosure has been made in view of the above circumstances, and is capable of suppressing backflow of a sample liquid from a downstream chamber to an upstream chamber in a test container for transferring liquid by centrifugal force.
• the purpose is to provide a test container and a nucleic acid testing method.
• the test container of the present disclosure includes an input port into which a sample liquid is input, a first chamber in which the sample liquid sent from the input port is accommodated, and a second chamber in which the sample liquid transferred from the first chamber is accommodated.
• a test container having two chambers, which is attached to a centrifuge, and in which a sample liquid is sequentially sent from the first chamber to the second chamber by the centrifugal force caused by the rotation of the centrifuge, A removable lid that covers the input slot; a liquid reservoir for storing the sample liquid input from the input port in front of the first chamber;
• a valve having a liquid accommodating part disposed between a first chamber and a second chamber and temporarily accommodating a sample liquid to be sent from the first chamber to the second chamber, the valve having a liquid accommodating part disposed between the first chamber and the liquid accommodating part.
• the sealing part is preferably made of a hydrophobic substance that has a lower specific gravity than the sample liquid.
• the hydrophobic substance is preferably a low polar solvent or a thermoplastic substance.
• the hydrophobic substance is preferably provided in the liquid reservoir or the first chamber.
• It may further include a hydrophobic substance accommodating part for accommodating a hydrophobic substance, which is provided on a side closer to the rotation axis of the centrifuge than the liquid reservoir part.
• the volume of the liquid reservoir is V1
• the volume of the first chamber is V2
• the volume of the liquid storage part is V3
• the volume of the second chamber is V4, it is preferable that the relationship is V1 ⁇ V2 ⁇ V3 ⁇ V4. .
• a reagent to be reacted with the sample liquid may be stored in the first chamber, the liquid storage section, or the second chamber.
• the reagent may include an amplification reagent for amplifying a specific nucleic acid sequence and a probe for determining the nucleic acid sequence.
• the probe may be a fluorescent probe.
• the nucleic acid testing method of the present disclosure is a nucleic acid testing method using the test container of the present disclosure, in which a reagent is housed in a liquid container, wherein the valve is in a second state or a second state in an initial state.
• test container and nucleic acid testing method of the present disclosure it is possible to suppress the backflow of the sample liquid from the downstream chamber to the upstream chamber.
• FIG. 1 is a perspective view showing a schematic configuration of a test container 10 according to an embodiment.
• FIG. 2A is a schematic cross-sectional view of the test container 10, and FIG. 2B is a schematic plan view of the flow path structure.
• FIG. 3A is a schematic cross-sectional view of the test container 10, showing a state switched to the first state by the valve 20, and FIG. 3B is a schematic plan view of the flow path structure.
• FIG. 4A is a schematic cross-sectional view of the test container 10, showing a state switched to the second state by the valve 20, and
• FIG. 4B is a schematic plan view of the flow path structure.
• 1 is a diagram showing an aspect of a test container 10.
• FIG. 4 is a diagram showing before and after sending the sample liquid from the liquid reservoir to the first chamber in the flow path structure 11.
• FIG. 7 is a diagram showing before and after the sample liquid is sent from the liquid reservoir to the first chamber in a flow path structure 11A of a modified example. It is a schematic diagram showing a test container provided with a reagent.
• FIG. 7 is a schematic plan view of a modified example of a test container 50.
• FIG. FIG. 7 is a schematic plan view of a modified example of a test container 52.
• FIG. 5 is a diagram illustrating before and after the sample liquid is sent from the liquid reservoir to the first chamber in the flow path structure 11B of the test container 52.
• FIG. FIG. 7 is a schematic plan view of a modified example of a test container 53.
• FIG. FIG. 7 is a diagram illustrating before and after sending the sample liquid from the liquid reservoir to the first chamber in the test container 54 of a modified example.
• FIG. 1 is a diagram showing a schematic configuration of an inspection device 100 according to an embodiment. It is a figure showing the schematic structure of inspection device 101 of a modification. It is a figure showing the schematic structure of inspection device 102 of a modification.
• FIG. 1 is a diagram showing the steps of nucleic acid testing (Part 1). It is a figure showing the process of a nucleic acid test (part 2).
• FIG. 2 is a diagram showing the steps of ⁇ -glucan testing (Part 1).
• FIG. 2 is a diagram showing the process of ⁇ -glucan testing (Part 2).
• FIG. 1 is a perspective view schematically showing a test container 10 of one embodiment.
• FIG. 2A is a schematic cross-sectional view of the test container 10
• FIG. 2B is a schematic plan view showing a simplified flow path structure 11 of the test container 10.
• the test container 10 is a microchannel device that includes a channel structure 11 that includes a plurality of chambers and channels that connect the chambers.
• the test container 10 is attached to a centrifuge, and the liquid inside is transferred from chamber to chamber by centrifugal force caused by rotation of the centrifuge.
• the test container 10 of this example is a test container that is loaded into a test device 100 (see FIG. 14) equipped with a centrifuge and is used for biochemical analysis.
• the test container 10 is composed of a main body member 10A in which recesses, holes, etc. are formed, which constitute a part of a flow path structure 11 including a flow path and a chamber, and a bottom member 10B, which constitutes the bottom surface of the flow path. (See Figure 2).
• the main body member 10A can be made of any known resin-molded plastic material without particular limitation, but from the viewpoint of heat resistance and transparency, polycarbonate, polypropylene, cycloolefin, or silicone resin is preferable.
• the bottom member 10B is formed of, for example, a thin plate or a film.
• any known resin-molded plastic material can be used without particular limitation, but from the viewpoint of adhesion to the main body member 10A, the same material as the main body member 10A is preferable.
• the bottom member 10B is made of a film from the viewpoint of improving close contact with the heating means and enabling efficient heating. It is preferable that the Note that the portions constituting the first chamber 15 and the second chamber 16 on the upper surface side of the main body member 10A may also be made of a film.
• the test container 10 includes an input port 12, a lid 12A, a liquid reservoir 13, a first chamber 15, a second chamber 16, and a valve disposed between the first chamber 15 and the second chamber 16. 20, channels 18a to 18c connecting them, and a sealing part 19.
• the plurality of chambers included in the test container 10 are the liquid reservoir section 13 , the first chamber 15 , the second chamber 16 , and the liquid storage section 24 . In the following, when there is no need to distinguish between the liquid reservoir 13, the first chamber 15, the second chamber 16, and the liquid storage section 24, they may be collectively referred to as chambers.
• the input port 12 is an opening for inputting the sample liquid 40.
• the lid portion 12A is a lid portion that covers the input port 12 and is detachable from the opening of the input port 12.
• the lid portion 12A is formed to be able to be screwed onto the end of the cylindrical portion 14 that forms the input port 12.
• the lid portion 12A may be attached to and detached from the cylindrical portion 14 using a snap-type cap structure or an adhesive.
• the lid part 12A opens the input port 12 when a sample liquid is input, but closes the input port 12 except when a sample liquid is input to eliminate contamination from the outside and prevent evaporation of the sample liquid 40 from inside. do.
• the sample fluid 40 is, for example, a liquid obtained by extracting nucleic acids from a sample collected from a subject's nasal cavity, pharynx, oral cavity, affected area, or the like.
• the surface where the input port 12 is provided will be referred to as the top surface of the test container 10, and the bottom member 10B side will be referred to as the bottom surface of the test container 10.
• the top surface of the main body member 10A is the same as the top surface of the test container 10
• the bottom surface of the main body member 10A is a surface in contact with the top surface of the bottom member 10B
• the bottom surface of the bottom member 10B is the same as the bottom surface of the test container 10. be.
• the liquid reservoir 13 is provided at a position facing the input port 12 and accommodates the sample liquid 40 dropped from the input port 12.
• the shape of the liquid reservoir 13 is not particularly limited, and can be arbitrarily selected from a columnar shape, a conical shape, a truncated cone shape, and the like.
• the input port 12 is constituted by the opening of the cylindrical part 14 that extends through the main body member 10A in the thickness direction and protrudes from the upper surface of the main body member 10A.
• a liquid reservoir 13 is formed by the inner side portion of the member 10A and the bottom member 10B. The liquid reservoir 13 is a space that stores the sample liquid 40 introduced from the input port 12 in front of the first chamber 15 .
• the first chamber 15 is a chamber that can accommodate a liquid, and is a chamber that accommodates the sample liquid 40 that is sent from the input port 12 through the liquid reservoir 13 .
• the first chamber 15 is formed by a recess provided on the lower surface of the main body member 10A and a bottom member 10B.
• the first chamber 15 is, for example, a chamber in which pre-processing in testing is performed, and heat processing is performed by a first heating unit 120 in the testing apparatus 100 (see FIG. 14), which will be described later.
• the second chamber 16 is a chamber that can accommodate a liquid, and is a chamber that accommodates the sample liquid 40 sent from the first chamber 15.
• the sample liquid 40 is sent from the first chamber 15 to the second chamber 16 via a liquid storage section 24 of a valve 20, which will be described later.
• the second chamber 16 is formed by a recess provided on the lower surface of the main body member 10A and a bottom member 10B.
• the second chamber 16 is, for example, a chamber in which reaction and detection processing for testing is performed, and heat processing is performed by the second heating unit 122 in the testing apparatus 100 (see FIG. 14), which will be described later.
• the valve 20 has a liquid storage section 24 that temporarily stores the sample liquid 40 sent from the first chamber 15 to the second chamber 16.
• the valve 20 is a valve that switches the connection state between the first chamber 15 and the second chamber 16 and the liquid storage section 24 to one of the following states: a first state, a second state, and a third state. be.
• the first state is a state in which the first chamber 15 and the liquid storage section 24 are in communication with each other, and the second chamber 16 and the liquid storage section 24 are not in communication with each other.
• the second state is a state in which the second chamber 16 and the liquid storage section 24 are in communication with each other, and the first chamber 15 and the liquid storage section 24 are not in communication with each other.
• the third state is a state in which both the first chamber 15 and the second chamber 16 are not in communication with the liquid storage section 24 .
• FIG. 2A shows a state in which both the first chamber 15 and the second chamber 16 are not in communication with the liquid storage section 24.
• the x shown on the channel 18b schematically indicates that the first chamber 15 and the liquid storage section 24 are in a closed state where they are not communicating with each other.
• an x shown on the flow path 18c schematically indicates that the liquid storage section 24 and the second chamber 16 are in a closed state where they are not communicating with each other.
• the x shown on the flow path means that the chambers connected via the flow path are not communicating with each other.
• the valve 20 in the present test container 10 includes a rotating member 25 disposed in a cylindrical portion 22 consisting of a cylindrical recess provided on the upper surface of the main body member 10A and a portion surrounding the recess and protruding from the upper surface of the main body member 10A.
• the rotating member 25 is a cylindrical member whose outer diameter is slightly smaller than the inner diameter of the cylindrical portion 22, and is rotatably installed within the cylindrical portion 22.
• a liquid storage section 24 is provided inside the rotating member 25, and one hole 24a communicating with the liquid storage section 24 is provided on the bottom surface.
• the bottom surface of the cylindrical portion 22 has a hole 22a communicating with the flow path 18b and a hole 22b communicating with the flow path 18c.
• An O-ring 26 is provided in each of the holes 22a and 22b.
• the upper surface of the rotating member 25 is provided with a knob 25a that is used when rotating the rotating member 25.
• the O-ring 26 aligns the hole 24a on the bottom surface of the rotating member 25 with either the hole 22a or the hole 22b on the bottom surface of the cylindrical portion 22, and communicates the liquid storage portion 24 with the flow path 18b or the flow path 18c. In this case, it functions to prevent leakage from the connection between the hole 24a and the hole 22a or 22b.
• any known sealing member such as a packing or a gasket can be used as a sealing means for preventing leakage without particular limitation.
• the material of the sealing means for example, rubber materials such as nitrile rubber, urethane rubber, silicone rubber, and fluororubber, or known elastomers such as olefin and urethane can be used.
• the seal portion may be integrally formed with the rotating member 25 or the cylindrical portion 22 of the main body member 10A.
• the hole 24a on the bottom surface of the rotating member 25 and the hole 22a on the bottom surface of the cylindrical portion 22 are aligned, the hole 24a is aligned with the hole 22b, and the hole 24a is aligned with the hole 22a.
• 22a and 22b may be set.
• the channels 18a to 18c are all formed by a recess provided on the lower surface of the main body member 10A and the bottom member 10B.
• the flow path 18a is a flow path that connects the liquid reservoir 13 and the first chamber 15.
• the flow path 18b is a flow path that connects the first chamber 15 and the liquid storage portion 24 of the valve 20.
• the flow path 18b communicates with a hole 22a in the bottom surface of the cylindrical portion 22 of the valve 20.
• the flow path 18c is a flow path that connects the second chamber 16 and the liquid storage portion 24 of the valve 20.
• the flow path 18c communicates with a hole 22b in the bottom surface of the cylindrical portion 22.
• the cross-sectional size of the channels 18a to 18c is, for example, approximately 0.5 mm to 1 mm wide and 0.5 mm deep.
• FIG. 3 and 4 are diagrams for explaining the first state and second state switched by the valve 20.
• 3 is an explanatory diagram of the test container 10 in the first state
• FIG. 3A is a schematic cross-sectional view of the test container 10
• FIG. 3B is a schematic plan view of the flow path structure 11.
• FIG. 4 is an explanatory diagram of the test container 10 in the second state, in which FIG. 4A is a schematic cross-sectional view of the test container 10, and FIG. 4B is a schematic plan view of the channel structure 11.
• the hole 24a on the bottom surface of the rotating member 25 coincides with the hole 22a on the bottom surface of the cylindrical portion 22.
• the first chamber 15 and the liquid storage section 24 are in communication with each other, and the second chamber 16 and the liquid storage section 24 are in a first state in which they are not communicated with each other.
• the hole 24a on the bottom surface of the rotating member 25 matches the hole 22b on the bottom surface of the cylindrical portion 22.
• the second chamber 16 and the liquid storage section 24 are in communication with each other, and the first chamber 15 and the liquid storage section 24 are in a second state in which they are not communicated with each other.
• the state in which the hole 24a on the bottom surface of the rotating member 25 is not aligned with either the hole 22a or the hole 22b on the bottom surface of the cylindrical portion 22 is the state in which the first chamber 15 and the second chamber 16 This is a third state in which both and the liquid storage section 24 do not communicate with each other.
• the valve 20 is a rotary valve that can be switched between the first state, the second state, and the third state by rotating the rotary member 25 within the cylindrical portion 22 of the main body member 10A.
• the valve 20 is not limited to the rotary valve having the above structure as long as it can realize the first to third states.
• the sealing part 19 is provided in a part of the channel structure 11 to prevent the sample liquid 40 from flowing backward after the sample liquid 40 is sent from the liquid reservoir 13 to the first chamber 15. ing.
• the sealing part 19 is a hydrophobic substance that has a lighter specific gravity than the sample liquid 40 (hereinafter referred to as hydrophobic substance 19).
• the hydrophobic substance 19 is a low polar solvent or a thermoplastic substance.
• the hydrophobic substance 19 may be a substance that is liquid at room temperature, or a substance that is solid at room temperature but becomes liquid when heated.
• low polar solvents include silicone oil, hydrocarbon solvents, fluorine solvents, and carbon tetrachloride.
• low polarity means that the polarity is such that it does not mix with the specimen liquid 40.
• thermoplastic substances include various waxes and adhesives.
• the hydrophobic substance 19 is provided in the liquid reservoir 13 or the first chamber 15. In the example shown in FIG. 2, the hydrophobic substance 19 is provided in the first chamber 15.
• test container 10 is set in a loading section 111A of a disk-shaped support 111 as shown in FIG. be done.
• the test container 10 is set in the centrifuge 110 in the order of the liquid reservoir 13, the first chamber 15, the liquid storage part 24, and the second chamber 16 from the inner diameter side closest to the axis A, which is the center of rotation.
• the sample liquid 40 inputted from the input port 12 is transferred from the liquid reservoir 13 to the first chamber 15, from the first chamber 15 to the liquid storage section 24, and from the liquid storage section 24 to the second chamber 16.
• the liquid feeding to each of these is realized by the centrifugal force caused by the rotation of the centrifuge 110.
• FIG. 6 shows the state of the flow path structure 11 before and after the sample liquid 40 is sent from the liquid reservoir 13 to the first chamber 15.
• the injection of the sample liquid 40 is performed in a state where the first chamber 15 and the liquid storage portion 24 of the valve 20 are not in communication with each other.
• both the first chamber 15 and the second chamber 16 are in the third state in which they do not communicate with the liquid storage section 24, but they may be in the second state.
• the sample liquid 40 is stored in the liquid reservoir 13.
• the hydrophobic substance 19 is contained in the first chamber 15.
• the sexual substance 19 moves toward the inner diameter side, as a result, the sample liquid 40 is sent from the liquid reservoir 13 to the first chamber 15.
• the test container 10 of this embodiment has a sealing part 19 (in this example, a hydrophobic It is equipped with substance 19).
• a sealing part 19 in this example, a hydrophobic It is equipped with substance 19.
• the hydrophobic substance 19 is provided as the sealing part as in this embodiment, after the sample liquid 40 is sent to the first chamber 15, the sample liquid 40 and the hydrophobic substance 19 are exchanged, and the liquid reservoir 13 is replaced with the hydrophobic substance 19.
• the hydrophobic substance 19 is accommodated in the area. Since the hydrophobic substance 19 is accommodated in the liquid reservoir 13, the vaporized sample liquid 40 flows into the liquid reservoir 13, compared to the case where only air exists in the liquid reservoir 13 without the hydrophobic substance 19. Backflow can be suppressed. The backflow of the sample liquid 40 from the first chamber 15 to the liquid reservoir 13 can be suppressed without providing any mechanical structure, and the sealing part can be realized very simply and at low cost.
• the hydrophobic substance 19 be provided in a volume sufficient to fill the liquid reservoir 13 . If the hydrophobic substance 19 fills the liquid reservoir 13 after sending the sample liquid 40 to the first chamber 15, the backflow of the sample liquid 40 from the first chamber 15 to the liquid reservoir 13 can be more effectively prevented. Can be suppressed.
• the hydrophobic substance 19 When the hydrophobic substance 19 is a low polar solvent, it does not mix with the sample liquid 40, so it is possible to suppress the sample liquid 40 from flowing back into the liquid reservoir 13.
• the hydrophobic substance 19 is a thermoplastic substance
• the hydrophobic substance 19 is fed to the liquid reservoir 13 and the hydrophobic substance 19 is transferred to the liquid reservoir 13. It solidifies when cooled. Thereby, backflow of the sample liquid 40 into the liquid reservoir 13 can be effectively suppressed.
• the hydrophobic substance 19 is provided in the liquid reservoir 13 or the first chamber 15 before the sample liquid 40 is introduced.
• the portion provided with the hydrophobic substance 19 is not limited to these.
• the flow path structure 11A of the modified example includes a hydrophobic substance storage part 17 provided closer to the rotation axis of the centrifuge than the liquid reservoir part 13, and the hydrophobic substance 19 is hydrophobic. It may be accommodated in the substance storage section 17.
• the hydrophobic substance 19 is accommodated in the hydrophobic substance storage unit 17 while the sample liquid 40 is input from the input port 12 and accommodated in the liquid reservoir 13. ing.
• the sample liquid 40 is sent from the liquid reservoir 13 to the first chamber 15, as shown in the right diagram of FIG. The liquid is sent to the 13 side.
• the volume of the liquid reservoir 13 is V1
• the volume of the first chamber 15 is V2
• the volume of the liquid storage section 24 is V3
• the volume of the second chamber 16 is V4.
• the relationship is V1 ⁇ V2 ⁇ V3 ⁇ V4. That is, it is preferable that adjacent chambers have the same volume, or that among a plurality of chambers arranged from the inner diameter side of the centrifugal center to the outer diameter side, the chambers arranged closer to the outer diameter side have smaller volumes.
• the volume V1 of the liquid reservoir 13 is more than 100 ⁇ L to 200 ⁇ L
• the volume V2 of the first chamber 15 is 100 ⁇ L
• the volume V3 of the liquid storage portion 24 is 50 ⁇ L
• the volume V4 of the second chamber 16 is 30 ⁇ L.
• volume V1 of the liquid reservoir 13 is larger than the first chamber 15, more sample liquid 40 can be stored in the liquid reservoir 13 than the volume V2 of the first chamber 15.
• volume relationship of the plurality of chambers arranged from the inner diameter side of the centrifugal center to the outer diameter side is V1 ⁇ V2 ⁇ V3 ⁇ V4, then the outer diameter When the sample liquid 40 is sent to the chamber arranged on the side, the chamber on the outer diameter side (that is, downstream side) can be filled with the sample liquid 40. Therefore, it is possible to prevent the specimen liquid 40 from running out in the second chamber 16 that is ultimately used for detection.
• the test container 10 may include a reagent 32 in the channel structure 11 to react with the sample liquid 40.
• the reagent 32 is a reagent containing an amplification reagent for amplifying a specific nucleic acid sequence and a fluorescent probe for determining the nucleic acid sequence.
• the reagent 32 may be provided in any of the liquid reservoir 13, the first chamber 15, the liquid storage part 24, and the second chamber 16.
• the reagent 32 is preferably provided in the first chamber 15 or the liquid storage section 24, and is particularly preferably provided in the liquid storage section 24.
• a stirring rod for promoting mixing of the sample liquid 40 and the reagent 32 may be included in the chamber containing the reagent 32 or in a chamber downstream thereof.
• test container 10 of the above embodiment has one flow path structure 11 from the input port 12 to the second chamber 16.
• one test container 50 may be provided with a plurality of channel structures 11, as shown in FIG.
• the test container 50 shown in FIG. 9 includes a disc-shaped main body member and a bottom member, and includes four channel structures 11 arranged radially around the rotation axis A.
• the number of channel structures 11 included in one test container 50 is not limited to four.
• the flow path structure 11B may be provided with a vent flow path 35.
• the vent channel 35 is a channel that connects the first chamber 15 and the liquid reservoir 13.
• the vent channel 35 is a channel through which gas passes.
• FIG. 11 shows the state of the flow path structure 11B of the test container 52 before and after liquid feeding.
• the hydrophobic substance 19 fills the liquid reservoir 13, remains partially in the first chamber 15, and vents the first chamber 15.
• vent flow path 35 may not be completely blocked, it is more preferable not to include a vent flow path like the test container 10.
• a plurality of second chambers 16a to 16c may be provided, as in a modified example of a test container 53 shown in FIG.
• test accuracy can be improved by performing the same test on one specimen in the plurality of second chambers 16a to 16c.
• a plurality of test target substances can be tested in parallel on one specimen.
• the first second chamber 16a is tested for influenza
• the second second chamber 16b is tested for the new coronavirus
• the third second chamber 16c is tested for RS (Respiratory syncytial) virus.
• RS Respiratory syncytial
• the hydrophobic substance 19 is provided as the sealing part, but the sealing part is used to prevent the sample liquid 40 from flowing back after the sample liquid 40 is sent from the liquid reservoir 13 to the first chamber 15. Any structure may be used as long as it has the function of preventing this, and a structure such as a check valve may be provided between the liquid reservoir 13 and the first chamber 15.
• FIG. 13 shows a schematic cross-sectional view of a modified test container 54. Further, FIG. 13 shows the state of the flow path structure 11 before and after the sample liquid 40 is sent from the liquid reservoir 13 to the first chamber 15.
• the flow path 29a connecting the liquid reservoir 13 and the first chamber 15 is formed in a tapered shape whose diameter gradually increases from the liquid reservoir 13 side to the first chamber 15 side.
• a sphere 29b is provided within the flow path 29a. The diameter of the sphere 29b is larger than the diameter of the channel 29a closest to the liquid reservoir 13 side.
• a stopper 29c for restricting movement of the sphere 29b toward the first chamber 15 is provided in a portion of the flow path 29a on the first chamber 15 side.
• the flow path 29a, the sphere 29b, and the stopper 29c constitute a sealing part 29 that prevents the sample liquid 40 from flowing back after the sample liquid 40 is sent from the liquid reservoir 13 to the first chamber 15. There is.
• the sphere 29b is movably arranged in the flow path 29a before liquid feeding.
• a sample liquid 40 is input from the input port 12 and stored in the liquid reservoir 13.
• the test container 54 is rotated in a centrifuge while the first chamber 15 and the liquid storage section 24 are closed. Thereby, as shown in the lower diagram of FIG. 13, the specimen liquid 40 is sent to the first chamber 15 by centrifugal force.
• the sphere 29b is gradually pushed toward the liquid reservoir 13 by the sample liquid 40, and is moved to a portion having the same diameter as the sphere 29b. Thereby, the sphere 29b becomes a check valve that closes the flow path 29a.
• the first chamber 15 when the first chamber 15 is heated for pretreatment of the sample liquid 40 while the sample liquid 40 is stored in the first chamber 15, for example, 90° C.
• the sample liquid 40 When heated to a high temperature above, if the sample liquid 40 is vaporized and the pressure inside the first chamber 15 increases, the sphere 29b will be pushed further toward the liquid reservoir 13 and seal the flow path 29a. Become. Therefore, it is possible to prevent the sample liquid 40 from flowing back into the liquid reservoir 13.
• the bottom member 10B that constitutes the bottom surface of the flow path 18c connecting the liquid reservoir 13 and the first chamber 15 is made of a film, and the bottom member 10B is pressed from the outside to form the flow path. It may be provided with a configuration that makes it possible to close 18c.
• the channel structures 11, 11A, and 11B in each of the test containers 10, 50, 52, and 54 described above may further include a deformable chamber that communicates with the second chamber 16.
• a deformable chamber that communicates with the second chamber 16.
• the inside of the deformable chamber and the second chamber 16 communicating with the deformable chamber can be pressurized.
• pressurizing the inside of the second chamber 16 the temperature raising and cooling characteristics of the liquid contained in the second chamber 16 can be improved.
• FIG. 14 is a diagram showing a schematic configuration of the inspection device 100.
• the testing device 100 is, for example, a nucleic acid testing device.
• the inspection device 100 includes a centrifuge 110, a first heating section 120, a second heating section 122, a valve control section 124, and a detection section 126.
• the centrifuge 110 includes a pedestal 112 equipped with a rotation mechanism such as a motor, a disk-shaped support 111 constituting a rotation table equipped with a loading section 111A (see FIG. 5) for loading the test container 10, and a rotation mechanism.
• a rotary coupler 114 is connected to the shaft and rotatably supports the disc-shaped support 111. By driving the rotation mechanism, the disk-shaped support 111 is rotated about the axis A. Due to the centrifugal force accompanying this rotation, liquid delivery is realized in the channel structure within the test container 10.
• the disk-shaped support 111 has a presser foot 111B that presses down the test container 10 set in the loading section 111A.
• the first heating unit 120 is provided so as to be movable between a position where it contacts the bottom surface of the first chamber 15 of the test container 10 set on the disc-shaped support 111 and a position away from it.
• the first heating unit 120 is disposed at a position apart from the bottom surface of the first chamber 15 while the disc-shaped support 111 is rotating, and is located at a position apart from the bottom surface of the first chamber 15 when the disc-shaped support 111 is stopped. It is placed in a position where it makes contact with the bottom surface.
• the first heating unit 120 heats the liquid contained in the first chamber 15 .
• the liquid contained in the first chamber 15 is the sample liquid 40.
• the first heating unit 120 heats the sample liquid 40 to a high temperature of, for example, 90° C. or higher for pretreatment.
• the second heating unit 122 is provided so as to be movable between a position in which it contacts the bottom surface of the second chamber 16 of the test container 10 set on the disk-shaped support 111 and a position away from it.
• the second heating unit 122 is disposed at a position apart from the bottom surface of the second chamber 16 while the disc-shaped support 111 is rotating, and is located at a position apart from the bottom surface of the second chamber 16 when the disc-shaped support 111 is stopped. It is placed in a position where it makes contact with the bottom surface.
• the second heating unit 122 heats the liquid contained in the second chamber 16.
• the liquid contained in the second chamber 16 is, for example, a mixed liquid of the specimen liquid 40 and the reagent 32.
• the second heating unit 122 heats the mixture of the sample liquid 40 and the reagent 32 to promote nucleic acid amplification.
• the second heating unit 122 is equipped with a Peltier device and the like, and is capable of temperature control, and performs temperature cycles in the nucleic acid amplification process.
• the first heating section 120 does not require a temperature cycle like the second heating section 122, and is composed of, for example, a heater.
• the heating mechanisms used for each of the first heating section 120 and the second heating section 122 are not particularly limited and can be any known heating mechanism.
• the valve control unit 124 includes a mechanism for controlling the valve 20 to switch the communication state of the first chamber 15, second chamber 16, and liquid storage unit 24 to a first state, a second state, and a third state.
• the valve control unit 124 includes, for example, a gripping portion that grips the knob 25a of the rotating member 25, a rotation mechanism that rotates the gripping portion while the gripping portion grips the knob 25a, and a rotation mechanism that rotates the gripping portion as necessary in the inspection process. and a processor that drives the rotation mechanism accordingly.
• the detection unit 126 detects whether a detection target object is contained in the sample liquid 40 in the second chamber 16.
• the detection unit 126 includes a light source 126a, a wavelength selection filter 126b, and a photodetector 126c.
• the detection unit 126 is arranged above the second chamber 16 of the test container 10. Detection by the detection unit 126 is also carried out in a state where rotation by the centrifuge 110 is stopped.
• the light source 126a irradiates the second chamber 16 with excitation light L1 of a specific wavelength via the wavelength selection filter 126b.
• the photodetector 126c is excited by the excitation light L1 and detects fluorescence L2 generated from the fluorescent probe.
• Excitation light L1 is selected according to the excitation wavelength of the fluorescent probe. Further, if necessary, it may include a filter for adjusting the intensity and light amount, a lens for converging the excitation light L1 and condensing the fluorescence L2 derived from the detection probe onto the photodetector 126c, or an optical system.
• the light source 126a an LED, a laser, or the like is used.
• the wavelength selection filter 126b is a filter that transmits only the wavelength corresponding to the excitation wavelength of the probe out of the light emitted from the light source 126a.
• the photodetector 126c for example, a photodiode or a photomultiplier tube is applied.
• testing container 10 can also be used in the testing device 101 shown in FIG. 15, or the testing device 102 shown in FIG. 16.
• FIGS. 15 and 16 components equivalent to those of the inspection apparatus 100 shown in FIG. 14 are denoted by the same reference numerals, and detailed description thereof will be omitted.
• the inspection device 101 and the inspection device 102 each include centrifuges 110A and 110B that have a different structure from the centrifuge 110 of the inspection device 100.
• the inspection apparatus 101 shown in FIG. 15 is configured such that a centrifuge 110A rotates the disk-shaped support 111 with its surface tilted from the horizontal plane.
• the rotation axis A of the centrifuge 110A is configured to be inclined from a direction perpendicular to a horizontal plane.
• the centrifuge 110B is equipped with an umbrella-shaped support 131 instead of the disk-shaped support 111.
• the umbrella-shaped support 131 includes a horizontal portion supported by the rotary coupler 114 and an inclined portion surrounding the horizontal portion with the outer peripheral side located vertically downward with respect to the horizontal portion.
• test container 10 can be applied to any of the configurations of the various centrifuges 110A, 110B, and 110C shown in FIGS. 14 to 16.
• the testing devices 100, 101, and 102 are described as nucleic acid testing devices used for nucleic acid testing that involves nucleic acid amplification processing.
• the first heating section 120, the second heating section 122, and the detection section 126 are configured to be suitable for various tests, so that the testing devices 100, 101, and 102 can be used for various tests other than nucleic acid tests. I can do it.
• nucleic acid testing method An embodiment of a nucleic acid testing method using the test container 10 of the above embodiment will be described with reference to FIGS. 17 and 18.
• a test container 10 (see FIG. 8) is used, in which the first chamber 15 is provided with a hydrophobic substance 19 as a sealing part, and the liquid storage part 24 is provided with a reagent 32 for nucleic acid testing.
• a specimen 40a collected from a living body using a collection tool 60 such as a swab is placed in an extract solution 62 containing a surfactant and ProK (proteinase K). to extract nucleic acids and treat impurities.
• a liquid containing the sample 40a obtained by mixing the sample 40a with the extract liquid 62 and performing nucleic acid extraction in the extract liquid 62 will be referred to as a sample liquid 40.
• the specimen 40a is collected, for example, from the nasal cavity, pharynx, oral cavity, or affected area of the subject using a collection tool. or body fluids such as nasal, pharynx, or oral cavity cleaning fluids, saliva, urine, or blood.
• a nucleic acid extraction method any known nucleic acid extraction method can be used without particular limitations. Examples include a method using a surfactant or a chaotropic substance, and a method applying physical shear such as ultrasonic waves or a bead mill.
• the valve 20 In the initial state of the test container 10 before the sample liquid 40 is introduced, the valve 20 is placed in the second or third state in which at least the first chamber 15 and the liquid storage section 24 do not communicate with each other. In this embodiment, a third state is set in which neither the first chamber 15 nor the second chamber 16 communicates with the liquid storage section 24 .
• the valve 20 is left in the initial state as described above, and the filter 64 for removing coarse impurities is placed in the container 63 containing the sample liquid 40 containing the sample 40a from which the nucleic acid has been extracted.
• a dripping cap 66 equipped with a dripping cap 66 is attached, and the sample liquid 40 filtered through the filter 64 is poured into the test container 10 from the input port 12 (step ST1).
• the sample liquid 40 is stored in the liquid reservoir 13.
• the input port 12 is closed by the lid part 12A, and the internal space of the test container 10 is sealed (step ST2).
• the test container 10 is loaded into the test apparatus 100 with the sample liquid 40 added thereto and the inlet 12 closed by the lid 12A. The following processing is performed within the inspection device 100.
• the test container 10 is rotated by the centrifuge 110 of the test device 100, and the sample liquid 40 is sent from the liquid reservoir 13 to the first chamber 15 (step ST3).
• the sample liquid 40 is sent to the first chamber 15, and the hydrophobic substance 19 contained in the first chamber 15 is moved to the liquid reservoir 13.
• Step ST4 This heat treatment deactivates ProK.
• the heating temperature is preferably, for example, 80°C or higher, more preferably 85°C or higher, and even more preferably 90°C or higher.
• the upper limit temperature is preferably 100°C or lower, more preferably 95°C or lower, from the viewpoint of evaporation of the sample liquid.
• valve control unit 124 switches the valve 20 to the first state (step ST5). That is, the first chamber 15 and the liquid storage section 24 communicate with each other, and the liquid storage section 24 and the second chamber 16 do not communicate with each other.
• test container 10 is rotated by the centrifuge 110 of the test device 100, and the sample liquid 40 is sent from the first chamber 15 to the liquid storage section 24 of the valve 20 (step ST6).
• a reagent 32 is stored in the liquid storage section 24 , and the supplied sample liquid 40 and the reagent 32 are mixed in the liquid storage section 24 .
• the valve control unit 124 switches the valve 20 to the second state (step ST7). That is, the second chamber 16 and the liquid storage section 24 communicate with each other, and the liquid storage section 24 and the first chamber 15 do not communicate with each other.
• test container 10 is rotated by the centrifuge 110 of the test device 100, and the mixed liquid 42 of the sample liquid 40 and the reagent 32 is sent from the liquid storage part 24 of the valve 20 to the second chamber 16 (step ST8). .
• the valve control unit 124 switches the valve 20 to the third state (step ST9).
• the second chamber 16 and the liquid storage section 24 are not in communication with each other.
• neither the first chamber 15 nor the second chamber 16 communicates with the liquid storage section 24, but since it is sufficient that the second chamber 16 and the liquid storage section 24 do not communicate with each other, It does not matter if it is in the 1 state.
• the nucleic acid amplification process and fluorescence detection are performed on the mixed liquid 42 in a state where the second chamber 16 and the liquid storage section 24 are not in communication with each other (step ST10).
• the nucleic acid amplification process is a process in which the mixed liquid 42 in the second chamber 16 is heated by the second heating unit 122 to amplify a specific nucleic acid sequence.
• the nucleic acid amplification treatment method is not limited, but for example, RT-PCR method or PCR method is used.
• PCR method there are a step of dissociating double-stranded DNA into single-stranded DNA at high temperature (thermal denaturation step), a step of lowering the temperature and binding the primer to the single-stranded DNA (annealing step), and Using the stranded DNA as a template, a polymerase repeats the step (elongation step) of newly synthesizing double-stranded DNA.
• one cycle is 94° C. for 1 minute, 50 to 60° C. for 1 minute, and 72° C. for 1 to 5 minutes, which is repeated 20 to 50 times. It will be done. Further, the heat denaturation step and the annealing step may be performed at one temperature.
• An example of such a temperature cycle is, for example, one cycle consisting of 94° C. for 1 minute and 60° C. for 1 minute, which is repeated 20 to 50 times.
• the temperature and time of the temperature cycle in the amplification step are not particularly limited and can be arbitrarily selected depending on the performance of the polymerase and primers.
• Fluorescence detection is performed by the detection unit 126 located above the second chamber 16. Excitation light L1 of a specific wavelength is irradiated into the second chamber 16 from the light source 126a via the wavelength selection filter 126b. The photodetector 126c detects fluorescence L2 generated from the fluorescent probe excited by the excitation light L1. For example, when the nucleic acid amplification process is a PCR method, fluorescence detection is performed every cycle of the above-mentioned temperature cycle to monitor the amplification status in real time.
• the nucleic acid sequence is amplified in the amplification step, and the fluorescent probe labeled with this specific nucleic acid sequence is irradiated with excitation light L1, whereby fluorescence L2 is detected. be done.
• the fluorescent probe labeled with this specific nucleic acid sequence is irradiated with excitation light L1, whereby fluorescence L2 is detected.
• the excitation light L1 is irradiated.
• the sample liquid 40 sent from the liquid reservoir 13 to the first chamber 15 is pretreated by high-temperature heating in the first chamber 15. Since the sample liquid 40 is provided with a sealing portion (hydrophobic substance 19 in this example) that prevents the sample liquid 40 from flowing back into the liquid reservoir 13, the back flow of the sample liquid 40 can be suppressed. Thereby, it is possible to suppress a decrease in the amount of the sample liquid 40 that is finally processed in the second chamber 16, so that the test accuracy can be improved.
• a sealing portion hydrophobic substance 19 in this example
• the presence or absence of nucleic acids is determined by a fluorescence method using a fluorescent probe.
• Other detection methods such as , light scattering, sequence methods and electrochemical methods may also be used. These can be realized by appropriately changing the detection section, and the test container may be equipped with a reagent containing a probe corresponding to each detection method instead of the fluorescent probe.
• the nucleic acid test was mentioned as a test using the test container 10, but the test container 10 is applicable not only to nucleic acid tests but also to other genetic tests, microbial tests, etc.
• test container 10 As an example of a microbial test, an example in which the test container 10 is used for a ⁇ -glucan test will be described with reference to FIGS. 19 and 20.
• a test container 10 (see FIG. 8) is used, in which the first chamber 15 is equipped with a hydrophobic substance 19 as a sealing part, and the liquid storage part 24 is equipped with an LAL (Limulus Amebocyte Lysate) reagent as a reagent 32.
• LAL Local Amebocyte Lysate
• an inspection device having the same configuration as inspection device 100 can be used.
• a sample liquid 40 is prepared by mixing the sample and a pretreatment liquid.
• a dripping cap 66 equipped with a filter 64 is attached to the container 63 containing the sample liquid 40, and the sample liquid 40 filtered through the filter 64 is poured into the test container 10 from the inlet (step ST11).
• the sample liquid 40 is stored in the liquid reservoir 13.
• the input port 12 is closed by the lid portion 12A, and the internal space of the test container 10 is sealed (step ST12).
• the test container 10 is loaded into the test apparatus 100 with the sample liquid 40 added thereto and the inlet 12 closed by the lid 12A. The following processing is performed within the inspection device 100.
• the test container 10 is rotated by the centrifuge 110 of the test device 100, and the sample liquid 40 is sent from the liquid reservoir 13 to the first chamber 15 (step ST13).
• the sample liquid 40 is sent to the first chamber 15, and the hydrophobic substance 19 contained in the first chamber 15 is moved to the liquid reservoir 13.
• the first heating unit 120 heats the first chamber 15 to raise the sample liquid 40 to 70°C for 10 minutes. Temperature control is carried out by heating and cooling to 3° C. (step ST14).
• the pretreatment carried out here is a treatment in which the specimen liquid 40 is heated to stop the reaction cascade of endotoxin.
• valve control unit 124 switches the valve 20 to the first state (step ST15). That is, the first chamber 15 and the liquid storage section 24 communicate with each other, and the liquid storage section 24 and the second chamber 16 do not communicate with each other.
• the test container 10 is rotated by the centrifuge 110 of the test device 100, and the sample liquid 40 is sent from the first chamber 15 to the liquid storage part 24 of the valve 20 (step ST16).
• a reagent 32 is stored in the liquid storage section 24 , and the supplied sample liquid 40 and the reagent 32 are mixed in the liquid storage section 24 .
• the reagent 32 is a freeze-dried reagent, and is dissolved and mixed with the sample liquid 40.
• valve 20 is switched to the second state by the valve control unit 124 (see FIG. 14) (step ST17). That is, the second chamber 16 and the liquid storage section 24 communicate with each other, and the liquid storage section 24 and the first chamber 15 do not communicate with each other.
• test container 10 is rotated by the centrifuge 110 of the test device 100, and the mixed liquid 42 of the sample liquid 40 and the reagent 32 is sent from the liquid storage part 24 of the valve 20 to the second chamber 16 (step ST18). .
• the valve control unit 124 switches the valve 20 to the third state (step ST19).
• the second chamber 16 and the liquid storage section 24 are not in communication with each other.
• neither the first chamber 15 nor the second chamber 16 communicates with the liquid storage section 24, but since it is sufficient that the second chamber 16 and the liquid storage section 24 do not communicate with each other, It does not matter if it is in the 1 state.
• the LAL reaction is detected for the mixed liquid 42 by a colorimetric method (step ST20). Detection using the colorimetric method is performed by the detection section 126 disposed on the second chamber 16. Irradiation light L having a specific wavelength is irradiated into the second chamber 16 from the light source 126a via the wavelength selection filter 126b. The LAL reaction is detected by detecting the absorbance (or transmittance) of the irradiated light L using the photodetector 126c.

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