WO2017154349A1 - Nucleic acid extraction device, nucleic acid extraction unit, and nucleic acid extraction method - Google Patents

Nucleic acid extraction device, nucleic acid extraction unit, and nucleic acid extraction method Download PDF

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Publication number
WO2017154349A1
WO2017154349A1 PCT/JP2017/001164 JP2017001164W WO2017154349A1 WO 2017154349 A1 WO2017154349 A1 WO 2017154349A1 JP 2017001164 W JP2017001164 W JP 2017001164W WO 2017154349 A1 WO2017154349 A1 WO 2017154349A1
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WIPO (PCT)
Prior art keywords
nucleic acid
container
acid extraction
unit
specimen
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PCT/JP2017/001164
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French (fr)
Japanese (ja)
Inventor
宏明 橘
成正 岩本
徹 馬場
章吾 澁谷
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パナソニックＩｐマネジメント株式会社
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Priority to JP2018504026A priority Critical patent/JP6771161B2/en
Publication of WO2017154349A1 publication Critical patent/WO2017154349A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/10—Devices for withdrawing samples in the liquid or fluent state
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q

Definitions

  • the present invention relates to a nucleic acid extraction apparatus for extracting nucleic acid (deoxyribonucleic acid, ribonucleic acid) from a specimen, a nucleic acid extraction unit used in the nucleic acid extraction apparatus, and a nucleic acid extraction method for extracting nucleic acid from a specimen.
  • Nucleic acid amplification methods such as polymerase chain reaction (PCR) are used for testing microorganisms such as bacteria contained in beverages or foods.
  • the nucleic acid amplification method has an advantage that the inspection process can be greatly speeded up and simplified as compared with the culture method.
  • a pretreatment for extracting nucleic acid from a specimen is required before performing nucleic acid amplification.
  • a method of capturing a test object such as bacteria contained in the sample by filtering the sample (specimen stock solution) with a filter and extracting the nucleic acid from the test object using a nucleic acid extraction reagent.
  • Known for example, Patent Document 1.
  • the test object is filtered and concentrated through a filter in a syringe containing a sample, and then the filter is removed and placed in another syringe containing a nucleic acid extraction reagent.
  • the nucleic acid is extracted by reattaching and eluting the test object.
  • the work is complicated and there is a risk that impurities such as bacteria different from the test object are mixed in the nucleic acid extract containing the extracted nucleic acid (contamination) during the work. is there.
  • the present invention has been made to solve such problems, and provides a nucleic acid extraction apparatus, a nucleic acid extraction unit, and the like that are simple in workability and can greatly reduce the risk of impurities being mixed into a nucleic acid extract. For the purpose.
  • one aspect of a nucleic acid extraction apparatus includes a nucleic acid extraction unit for extracting nucleic acid from a specimen, and the nucleic acid extraction unit injects a specimen into the container and the container.
  • Split 1 container part and 2nd container part, the 1st inlet and the 2nd inlet are provided in the 1st container part, the 1st outlet and the 2nd outlet are It is provided in the second container part.
  • nucleic acid extraction unit used in a nucleic acid extraction apparatus for extracting nucleic acid from a specimen, and includes a container and a first for injecting the specimen into the container.
  • Nucleic acid is extracted from the nucleic acid extraction target captured by the inlet, the capture unit that is disposed in the container and captures the nucleic acid extraction target contained in the sample injected from the first injection port, and A second injection port for injecting a nucleic acid extraction reagent for extraction into the container; and a nucleic acid extract containing the nucleic acid extracted from the nucleic acid extraction object by the nucleic acid extraction reagent from the container.
  • a first discharge port and a second discharge port for discharging the specimen from the container includes a first container part for dividing the internal space of the container with the capture part as a boundary.
  • the first inlet and the second inlet are provided in the first container part, and the first outlet and the second outlet are provided in the second container part. .
  • one aspect of the nucleic acid extraction method includes a capture step of capturing the nucleic acid extraction target contained in the sample by the capture unit by passing the sample through the capture unit, and a nucleic acid extraction reagent in the capture unit.
  • the workability is simple and the risk of impurities being mixed into the nucleic acid extract can be greatly reduced.
  • FIG. 1 is a perspective view schematically showing a nucleic acid extraction apparatus according to an embodiment.
  • FIG. 2 is an enlarged perspective view of a main part showing a peripheral structure of the nucleic acid extraction unit in the nucleic acid extraction apparatus according to the embodiment.
  • FIG. 3 is a perspective view of the nucleic acid extraction unit according to the embodiment.
  • FIG. 4 is a cross-sectional view of the nucleic acid extraction unit according to the embodiment.
  • FIG. 5 is a flowchart of the nucleic acid extraction method according to the embodiment.
  • FIG. 6A is a diagram illustrating a state when a specimen is injected into a container in the nucleic acid extraction method according to the embodiment.
  • FIG. 6A is a diagram illustrating a state when a specimen is injected into a container in the nucleic acid extraction method according to the embodiment.
  • FIG. 6B is a diagram illustrating the nucleic acid extraction target captured by the capturing unit in the nucleic acid extraction method according to the embodiment.
  • FIG. 6C is a diagram showing a state when the nucleic acid extraction reagent is injected into the container in the nucleic acid extraction method according to the embodiment.
  • FIG. 6D is a diagram illustrating a state where the nucleic acid extraction reagent is held in the capturing unit in the nucleic acid extraction method according to the embodiment.
  • FIG. 6E is a diagram showing a state when the nucleic acid extract is moved from the first container part to the second container part in the nucleic acid extraction method according to the embodiment.
  • FIG. 6F is a diagram showing a state of the nucleic acid extract moved to the second container part in the nucleic acid extraction method according to the embodiment.
  • FIG. 6G is a diagram showing a state when the nucleic acid extraction liquid is discharged from the container in the nucleic acid extraction method according to the embodiment.
  • FIG. 7 is a cross-sectional view of a nucleic acid extraction unit according to Modification 1.
  • FIG. 8 is a cross-sectional view of a nucleic acid extraction unit according to Modification 2.
  • FIG. 9 is a cross-sectional view of a nucleic acid extraction unit according to Modification 3.
  • FIG. 10 is a cross-sectional view of a nucleic acid extraction unit according to Modification 4.
  • FIG. 11 is a cross-sectional view of a nucleic acid extraction unit according to Modification 5.
  • FIG. 1 is a perspective view schematically showing a nucleic acid extraction apparatus 100 according to an embodiment.
  • FIG. 2 is an enlarged perspective view of a main part showing a peripheral structure of the nucleic acid extraction unit 1 in the nucleic acid extraction apparatus 100, and shows the nucleic acid extraction unit 1 installed in the nucleic acid extraction apparatus 100.
  • FIG. 3 is a perspective view of the nucleic acid extraction unit 1 according to the embodiment, and FIG. 4 is a cross-sectional view of the nucleic acid extraction unit 1.
  • the nucleic acid extraction apparatus 100 includes a nucleic acid extraction unit 1 for extracting nucleic acid from a measurement object contained in a specimen, a vacuum pump 2, and a vibration device 3.
  • An object to be measured contained in a specimen is an object (nucleic acid extraction object) from which nucleic acid is extracted, and is, for example, a microorganism such as a bacterium, virus, or tissue cell.
  • a sample stock solution containing microorganisms can be collected from a beverage, for example.
  • Microorganisms such as bacteria, viruses or tissue cells are inspection objects to be inspected by nucleic acid amplification by PCR or the like. That is, the nucleic acid extraction apparatus 100 is an apparatus used for pretreatment before performing nucleic acid amplification, and the nucleic acid extracted by the nucleic acid extraction apparatus 100 is used for a desired test. In addition, in the nucleic acid extraction apparatus 100 in this Embodiment, it collect
  • the nucleic acid extraction unit 1 is a processing unit for performing a nucleic acid extraction process from a specimen, and is installed in the reaction box 4 of the nucleic acid extraction apparatus 100 as shown in FIG.
  • the nucleic acid extraction unit 1 is a replaceable cartridge and can be removed from the reaction box 4.
  • the nucleic acid extraction unit 1 is replaced for each nucleic acid extraction process.
  • FIG. 1 shows a state where the entire lid of the reaction box 4 is opened.
  • the nucleic acid extraction unit 1 includes a container 10, a first inlet 21, a second inlet 22, a first outlet 31, a second outlet 32, and a capturing unit. 40.
  • the container 10 is a processing container for performing a nucleic acid extraction process from a specimen by using a specimen to be injected and a nucleic acid extraction reagent.
  • the nucleic acid extracted from the specimen is discharged from the container 10 as a nucleic acid extract.
  • the material of the container 10 is not particularly limited, but the container 10 has a high heat resistance such as polypropylene (PP) or polycarbonate (PC) in order to enable heat treatment by nucleic acid extraction treatment. It is good to be comprised with resin materials, metal materials, such as aluminum or stainless steel, or inorganic materials, such as glass or a ceramic. In addition, when a resin material is used as the container 10, there is an advantage that the weight can be reduced at low cost. Further, when a metal material or a high thermal conductive resin is used as the container 10, the thermal conductivity of the container 10 can be improved. For this reason, when performing heat processing, it is good for at least one part of the container 10 to be comprised with the metal material or highly heat conductive resin.
  • the container 10 may be configured by combining the above materials.
  • the inner surface of the container 10 is preferably non-hydrophilic.
  • the inner surface of the container 10 may be hydrophobic.
  • the container 10 may be formed using a hydrophobic material, or the inner surface of the container 10 may be made hydrophobic by surface treatment, or a hydrophobic film is coated on the inner surface of the container 10. Also good.
  • the container 10 includes a first container part 11 and a second container part 12.
  • the first container part 11 and the second container part 12 divide the internal space of the container 10 with the capturing part 40 as a boundary.
  • the container 10 is divided into two parts in the vertical direction with the main body 41 of the capturing part 40 as a boundary.
  • the first container part 11 is an upper part and the second container part 12 is a lower part. It has become.
  • the first container part 11 is further divided into two parts, but is not limited thereto.
  • the first inlet 21, the second inlet 22, the first outlet 31, the second outlet 32, and the capturing unit 40 are provided in the container 10. Specifically, the first inlet 21, the second inlet 22, the first outlet 31, and the second outlet 32 are provided in the partition wall of the container 10. 10 is fixed to the container 10 so as to be located inside.
  • the first injection port 21 is a sample injection port for injecting a sample into the container 10.
  • the first inlet 21 is provided on the upper wall of the first container part 11.
  • a specimen (specimen stock solution) is injected into the first container section 11 through the first inlet 21 from a specimen insertion cup 200 installed in the nucleic acid extraction apparatus 100.
  • the first inlet 21 is a pipe that communicates the outside of the container 10 and the internal space of the first container part 11. Specifically, one end portion of the first injection port 21 is connected to the first container portion 11, and the other end portion of the first injection port 21 is connected to the sample insertion cup 200. In the present embodiment, the first inlet 21 is formed integrally with the first container part 11. The first inlet 21 may be a through hole provided in the upper wall of the container 10 (first container portion 11).
  • the second injection port 22 is a nucleic acid extraction reagent injection port for injecting the nucleic acid extraction reagent into the container 10.
  • the second inlet 22 is provided on the upper wall of the first container portion 11 similarly to the first inlet 21, but is provided at a position different from the first inlet 21.
  • the nucleic acid extraction reagent injected from the second injection port 22 is a liquid reagent for extracting nucleic acid from the nucleic acid extraction target captured by the capturing unit 40.
  • the nucleic acid extraction reagent functions to extract nucleic acid from the cell membrane of the nucleic acid extraction target.
  • the nucleic acid extraction reagent is injected from the nucleic acid extraction reagent container 300 installed in the nucleic acid extraction apparatus 100 into the first container unit 11 through the second injection port 22.
  • the second inlet 22 is a through hole provided in the first container part 11.
  • the second inlet 22 which is a through hole is provided with a rubber stopper 22a which closes the second inlet 22 (through hole).
  • the rubber stopper 22a is made of a rubber material that can be penetrated by a needle for injecting the nucleic acid extraction reagent into the first container portion 11.
  • the needle provided in the nucleic acid extraction reagent container 300 is made to penetrate the rubber stopper 22a.
  • the liquid reagent can be injected from the nucleic acid extraction reagent container 300 into the first container part 11 through the second injection port 22.
  • the rubber material of the rubber plug 22a is not particularly limited, but, for example, silicone rubber or fluorine rubber is used.
  • the angle of the corner portion of the wall surface of the internal space of the first container part 11 is preferably 90 degrees or more. That is, the angle formed by any two wall surfaces in the first container portion 11 is preferably 90 degrees or more.
  • the first discharge port 31 is a nucleic acid extract discharge port for discharging a nucleic acid extract containing a nucleic acid extracted from a nucleic acid extraction target by a nucleic acid extraction reagent from the container 10.
  • the first discharge port 31 is provided on the lower wall of the second container portion 12.
  • the first discharge port 31 is a pipe that communicates the outside of the container 10 and the internal space of the second container part 12. Specifically, one end of the first outlet 31 is connected to the second container part 12, and the other end of the first outlet 31 is installed in the nucleic acid extraction apparatus 100 via a pipe. It is connected to a recovery container 400 (nucleic acid extract recovery container). In the present embodiment, the first discharge port 31 is formed integrally with the second container portion 12.
  • the nucleic acid extract discharged from the first outlet 31 is recovered in the recovery container 400 through a pipe connected to the first outlet 31.
  • the nucleic acid extract is collected by being fed from the container 10 to the collection container 400 by the liquid feed pump 5.
  • the second discharge port 32 is a sample discharge port for discharging the sample from the container 10. From the second discharge port 32, the waste liquid of the sample injected into the container 10 is discharged. That is, the sample (filtrate) injected from the first container part 11 and passing through the capturing part 40 is discharged from the second discharge port 32.
  • the second discharge port 32 is provided on the side wall of the second container portion 12. That is, the second discharge port 32 is provided at a position closer to the capturing unit 40 than the first discharge port 31.
  • the opening diameter (inner diameter) of the second outlet 32 is preferably at least twice as large as the opening diameter (inner diameter) of the first outlet 31.
  • the sample waste liquid has a large volume (for example, about 100 ml), and the nucleic acid extract collected for nucleic acid amplification has a small volume (for example, about 200 ⁇ l).
  • the discharge path of the sample waste liquid and the nucleic acid extract is divided into the first discharge port 31 and the second discharge port 32, and the opening diameter of the second discharge port 32 is set to the first diameter.
  • the nucleic acid extract can be discharged with little loss.
  • the opening diameter of the second discharge port 32 is 2 mm, and the opening diameter of the first discharge port 31 is 0.5 mm.
  • the opening diameter of the second discharge port 32 may not be twice or more than the opening diameter of the first discharge port 31.
  • the opening diameter of the second discharge port 32 is the same as the opening diameter of the first discharge port 31. It may be smaller than the opening diameter of the first discharge port 31.
  • the second discharge port 32 is a pipe that communicates the outside of the container 10 and the internal space of the second container part 12. Specifically, one end of the second discharge port 32 is connected to the second container part 12, and the other end of the second discharge port 32 is installed in the nucleic acid extraction apparatus 100 via a pipe. It is connected to a collection container 500 (specimen waste liquid collection container). In the present embodiment, the second discharge port 32 is formed integrally with the second container portion 12.
  • the specimen discharged from the second discharge port 32 is collected in the collection container 500 through a pipe connected to the second discharge port 32.
  • the waste liquid of the specimen is recovered by being sent from the container 10 to the recovery container 500 by the vacuum pump 2.
  • the capture unit 40 is a capture unit for capturing a nucleic acid extraction target contained in a sample injected from the first injection port 21, and at least a part of the capture unit 40 is disposed in the container 10. In the present embodiment, the capture unit 40 captures the nucleic acid extraction target contained in the specimen by filtration.
  • the capturing unit 40 includes a main body 41 for capturing and holding the nucleic acid extraction target and a support 42 for supporting the main body 41.
  • the capturing part 40 is fixed to the container 10 such that the main body part 41 is located inside the container 10.
  • the main body 41 is placed on a plate-like support 42.
  • a plurality of through holes (eyes) are formed in a central portion corresponding to the main body portion 41 of the support portion 42. That is, the support part 42 is an eye plate. Further, the peripheral part of the support part 42 is sandwiched between the first container part 11 and the second container part 12. Thereby, the support portion 42 is fixed to the container 10.
  • the support part 42, the first container part 11, and the second container part 12 are fixed by, for example, four screws.
  • the main body 41 is a filter unit for filtering and capturing a nucleic acid extraction target (microorganism) from a specimen.
  • the main body 41 is a filter having a plurality of fine holes (eyes) smaller than the size of the nucleic acid extraction target.
  • the main body 41 (filter) can reliably capture the nucleic acid extraction target.
  • the hole diameter of the main body 41 is, for example, 0.45 ⁇ m.
  • the nucleic acid extraction target captured by the main body 41 is held on the main body 41.
  • the size of the hole of the main body 41 is preferably larger than the size of the nucleic acid.
  • a membrane filter made of a material such as cellulose acetate, polyvinylidene fluoride (PVDF), polyethersulfone (PES), cellulose acetate, or the like can be used.
  • a capture filter having a function of adsorbing a nucleic acid extraction target contained in a specimen may be used. By using such a capture filter, the speed at which the nucleic acid extraction target is captured from the specimen can be improved.
  • the main body 41 (filter) is a planar filter, but is not limited to this, and may be a filter having another shape such as a cylindrical shape.
  • At least a part of the support portion 42 may be made of a metal material such as aluminum or a high thermal conductive resin.
  • the support part 42 may be entirely made of a metal material or a high thermal conductive resin.
  • the metal material may be embedded in the resin.
  • the surface of the support part 42 is good to be hydrophilic.
  • the vacuum pump 2 is an example of a pressure adjusting unit for adjusting the pressure in the container 10.
  • the piping of the vacuum pump 2 is connected to the second container portion 12. Therefore, when the liquid is accumulated on the capturing part 40 and the internal space of the second container part 12 is sealed, the vacuum pump 2 adjusts the pressure in the second container part 12.
  • the pressure in the container 10 or the second container part 12 can be adjusted by the leak valve 6 provided in the piping of the vacuum pump 2. That is, the degree of pressure reduction by the vacuum pump 2 can be adjusted by the leak valve 6.
  • the piping of the vacuum pump 2 is connected to the second discharge port 32. That is, the decompression port (decompression suction port) of the vacuum pump 2 also serves as the second discharge port 32 provided in the second container portion 12.
  • the vibration device 3 has a function for vibrating the container 10. By vibrating the container 10 by the vibration device 3, the liquid in the container 10 can be stirred.
  • the nucleic acid extraction reaction can be effectively performed by stirring the nucleic acid extraction reagent containing the nucleic acid extraction target.
  • the vibration device 3 is provided so as to contact the reaction box 4.
  • FIG. 5 is a flowchart of the nucleic acid extraction method according to the embodiment.
  • 6A to 6G are schematic cross-sectional views for explaining the nucleic acid extraction method according to the embodiment. 6A to 6G, the first injection port 21 and the second injection port 22 (rubber plug 22a) are illustrated in the correct positions for easy understanding of the flow of the liquid of the specimen 210 and the nucleic acid extraction reagent. Not.
  • the nucleic acid extraction method is a method of extracting nucleic acid from a nucleic acid extraction target contained in a specimen using the nucleic acid extraction unit 1, and as shown in FIG. 5, at least a capture step S1 and nucleic acid extraction.
  • recovery process S3 are included.
  • the capturing step S1 is a step of capturing the nucleic acid extraction target contained in the sample by the capturing unit 40 by passing the sample through the capturing unit 40.
  • the nucleic acid extraction step S2 is a step of extracting the nucleic acid from the nucleic acid extraction target captured by the capturing unit 40 by introducing a nucleic acid extraction reagent into the capturing unit 40 after the capturing step S1.
  • the recovery step S3 is a step of recovering the nucleic acid extract containing the nucleic acid extracted in the nucleic acid extraction step S2 through the capture unit 40 after the nucleic acid extraction step S2.
  • a specimen 210 is injected into the nucleic acid extraction unit 1 as shown in FIG. 6A.
  • the sample input cup 200 containing the sample 210 (sample stock solution) including the nucleic acid extraction object 220 is placed at a predetermined position of the nucleic acid extraction apparatus 100 (see FIG. 1), and the container is removed from the sample input cup 200. 10 into the specimen 210.
  • the valve 5 a provided in the pipe connected to the first discharge port 31 is closed.
  • the valve 5a is provided, for example, in the liquid feed pump 5 in FIG. 1, but is not limited thereto.
  • the specimen 210 can be produced, for example, by suspending a sample containing microorganisms in sterilized diluted water or the like.
  • the amount of solution of the specimen 210 is, for example, 10 ml to 500 ml, but is not limited thereto.
  • the specimen 210 may be subjected to a pretreatment such as removing a solid component as necessary.
  • the specimen 210 introduced from the specimen insertion cup 200 is injected into the first container section 11 through the first injection port 21, passes through the capturing section 40 and flows into the second container section 12.
  • the specimen 210 is concentrated by the capturing unit 40. That is, the specimen 210 can be concentrated by passing the specimen 210 through the capturing unit 40.
  • the nucleic acid extraction target 220 contained in the sample 210 is captured by the capturing unit 40.
  • the nucleic acid extraction target 220 contained in the specimen 210 is captured by the main body 41 and passes over the main body 41 when passing through the main body 41 (filter) of the capturing unit 40.
  • the specimen 210 (filtrate) that has passed through the capture unit 40 is discharged from the second container unit 12 through the second discharge port 32 as a waste solution, and the nucleic acid extraction apparatus 100 (see FIG. 1). It is collected in a collection container 500 installed at a predetermined position.
  • the nucleic acid extraction target 220 contained in the specimen 210 is captured by the capturing unit 40 by suction filtration.
  • the air in the container 10 is exhausted (suctioned) from the first discharge port 31 by the vacuum pump 2 to depressurize the second container part 12, and the pressure in the first container part 11 is set to the second pressure.
  • the pressure in the second container portion 12 is adjusted in the specimen discharge mode in which the pressure is higher than the pressure in the container portion 12.
  • the specimen 210 injected into the first container section 11 can be quickly passed through the capturing section 40 and moved to the second container section 12, and the waste liquid of the specimen 210 can be promptly removed from the second container section 12. It can be discharged. Therefore, the specimen 210 can be concentrated efficiently, and the waste liquid of the specimen 210 can be quickly discharged from the container 10.
  • the specimen 210 when concentrating the specimen 210, the specimen 210 may be further pressurized from above. Thereby, the specimen 210 can be more efficiently concentrated. For example, the space region above the specimen 210 in the specimen insertion cup 200 may be pressurized.
  • the nucleic acid extraction target 220 contained in the sample 210 is captured by the capturing unit 40 by allowing the sample 210 to pass through the capturing unit 40.
  • the capture unit 40 After passing the specimen 210 through the capture unit 40, if necessary, further introducing sterile diluted water from the first injection port 21 or the like into the first container unit 11 and allowing the nucleic acid to pass through the capture unit 40.
  • the extraction object 220 can be washed.
  • nucleic acid extraction step S2 the nucleic acid 230 is extracted from the nucleic acid extraction target 220 captured by the capturing unit 40.
  • the nucleic acid extraction reagent 310 is put into the container 10 from the nucleic acid extraction reagent container 300 installed at a predetermined position of the nucleic acid extraction apparatus 100 (see FIG. 1).
  • the nucleic acid extraction reagent 310 for example, a simple DNA extraction kit (Kaneka Corporation) or Cellize (Biocosm Corporation) can be used.
  • the amount of the nucleic acid extraction reagent 310 to be injected is, for example, 50 ⁇ l to 200 ⁇ l. Note that when the nucleic acid extraction reagent 310 is put into the container 10, the valve 5a of the pipe connected to the first outlet 31 remains closed.
  • the nucleic acid extraction reagent 310 introduced from the nucleic acid extraction reagent container 300 is injected into the first container part 11 through the second injection port 22. Specifically, the needle of the nucleic acid extraction reagent container 300 is passed through a rubber stopper 22a provided in the second inlet 22 (through hole), and the nucleic acid extraction reagent is inserted from the nucleic acid extraction reagent container 300 into the first container portion 11. Inject 310. In addition, before injecting the nucleic acid extraction reagent 310 into the container 10, a dead bacteria inactive treatment reagent or the like may be injected into the container 10.
  • the nucleic acid extraction target 220 captured by the capturing unit 40 reacts with the nucleic acid extraction reagent 310.
  • the nucleic acid 230 can be extracted from the nucleic acid extraction object 220. That is, a nucleic acid extraction reaction in which the nucleic acid 230 is extracted from the nucleic acid extraction target 220 is performed. That is, the cell membrane of the nucleic acid extraction target 220 and the nucleic acid 230 are separated.
  • the nucleic acid 230 is eluted from the nucleic acid extraction target 220 to the nucleic acid extraction reagent 310, and a nucleic acid extract 320 containing the nucleic acid 230 is generated.
  • the nucleic acid extraction reagent 310 may be injected into the container 10 to mix the nucleic acid extraction target 220 and the nucleic acid extraction reagent 310, and then appropriately heated. Thereby, since the reaction between the nucleic acid extraction reagent 310 and the nucleic acid extraction target 220 can be promoted and the nucleic acid 230 can be efficiently eluted, the nucleic acid 230 can be extracted efficiently.
  • the nucleic acid extraction reagent 310 can be heated by heating the container 10 by the heating / cooling unit 7 shown in FIG.
  • the heating / cooling unit 7 is provided, for example, in the reaction box 4 of the nucleic acid extraction apparatus 100, and is connected to, for example, the container 10 and the support part 42 of the capturing part 40.
  • a Peltier element or the like can be used as the heating / cooling unit 7, for example, a Peltier element or the like.
  • the container 10 may be vibrated by the vibration device 3 to stir the nucleic acid extraction reagent 310.
  • the nucleic acid 230 can be extracted more efficiently.
  • the nucleic acid extraction target 220 is subjected to the nucleic acid extraction reaction by introducing the nucleic acid extraction reagent 310 into the capturing unit 40, and the nucleic acid 230 is extracted from the nucleic acid extraction target 220.
  • a nucleic acid extract 320 containing the extracted nucleic acid 230 is generated.
  • the nucleic acid extraction reaction of the nucleic acid extraction target 220 may be performed by holding the nucleic acid extraction reagent 310 injected into the container 10 on the capturing unit 40.
  • the nucleic acid extraction reagent 310 stays on the capture unit 40.
  • the nucleic acid extraction object 220 is immersed in the nucleic acid extraction reagent 310 for a certain period of time. Thereby, since the nucleic acid 230 can be easily eluted, the extraction (elution) of the nucleic acid 230 can be performed efficiently.
  • the vacuum pump 2 adjusts the pressure in the second container unit 12 in the nucleic acid extraction reagent holding mode in which the nucleic acid extraction reagent 310 is held on the capture unit 40.
  • the inside of the second container part 12 may be pressurized by the vacuum pump 2 so that the pressure in the second container part 12 is higher than the pressure in the first container part 11. Thereby, the nucleic acid extraction reagent 310 injected into the container 10 can be held on the capture unit 40.
  • a filter that allows the nucleic acid extraction reagent 310 to pass through the main body 41 during normal times (when the second container 12 is not pressurized) can be used.
  • the pressurization in the second container unit 12 by the vacuum pump 2 is stopped, whereby FIG.
  • the nucleic acid extract 320 remaining on the capture unit 40 can be moved to the second container unit 12 through the capture unit 40.
  • the pressure in the second container portion 12 is adjusted so that the inside of the second container portion 12 is at a normal pressure, or the pressure reduction degree is lower than the pressure reduction degree in the specimen discharge mode. Just do it.
  • the method for holding the nucleic acid extraction reagent 310 on the capturing unit 40 is not limited to the method for adjusting the pressure in the second container unit 12 as described above.
  • a filter that holds the nucleic acid extraction reagent 310 on the main body 41 when the inside of the second container 12 is not decompressed as the main body 41 of the capturing unit 40 can also be realized by using.
  • the second container unit 12 is used by the vacuum pump 2. What is necessary is just to decompress the inside and make the pressure in the 1st container part 11 higher than the pressure in the 2nd container part 12.
  • FIG. Thereby, the nucleic acid extraction solution 320 that has completed the nucleic acid extraction reaction and stays on the capturing unit 40 can be moved to the second container unit 12. That is, the nucleic acid extract 320 can be moved to the second container part 12 using the pressure difference.
  • the nucleic acid extract 320 in the capture unit 40 is reduced by reducing the pressure in the second container part 12 by the vacuum pump 2 so that the pressure in the first container part 11 is higher than the pressure in the second container part 12. It is good to adjust the pressure in the 2nd container part 12 in the nucleic acid extraction liquid movement mode which moves to the 2nd container part 12 via the capture
  • the degree of decompression by the vacuum pump 2 in the nucleic acid extract transfer mode is preferably lower than the degree of decompression by the vacuum pump 2 in the specimen discharge mode for discharging the specimen 210.
  • a centrifugal force may be applied to the 2nd container part 12 in a sample discharge
  • the nucleic acid extract 320 containing the nucleic acid 230 is discharged from the second container part 12 through the first discharge port 31, and the recovery set at a predetermined position of the nucleic acid extraction device 100 (see FIG. 1).
  • the nucleic acid extract 320 is collected in the container 400.
  • the nucleic acid extract 320 in the second container portion 12 is sent to the collection container 400 by the liquid feed pump 5 and collected.
  • a nucleic acid test (nucleic acid analysis) is performed using the collected nucleic acid extract 320.
  • a nucleic acid test reagent is mixed with the nucleic acid extract 320 in accordance with the method for testing the nucleic acid 230 to obtain a test solution.
  • a PCR reagent is used as the nucleic acid test reagent.
  • the PCR reagent includes, for example, a nucleic acid staining fluorescent reagent for examining the amount of the nucleic acid 230 with fluorescence emission intensity, a reaction reagent for amplifying the nucleic acid, and the like.
  • the reaction reagent is, for example, a PCR primer, a polymerase enzyme, a buffer or the like.
  • the nucleic acid test by the PCR method can be performed using, for example, a test chip (PCR chip or the like) in which a micro flow channel that is a test flow channel is formed.
  • a mixed solution of the nucleic acid extract 320 and the nucleic acid test reagent is introduced into the test chip, the nucleic acid 230 is amplified by flow PCR, and the nucleic acid 230 amplified by the optical detection device is measured.
  • the object 220 microorganism
  • the nucleic acid extraction method according to the present embodiment can be used for a nucleic acid test method.
  • the nucleic acid 230 can be identified with high sensitivity and high accuracy by inspecting the nucleic acid 230 by the PCR method.
  • the nucleic acid extraction apparatus 100 includes the nucleic acid extraction unit 1 for extracting the nucleic acid 230 from the specimen 210.
  • the nucleic acid extraction unit 1 includes a container 10, a first inlet 21 for injecting a specimen into the container 10, and a nucleic acid extraction contained in the specimen 210 that is disposed in the container 10 and injected from the first inlet 21.
  • a capture unit 40 that captures the object 220, and a second injection port 22 that injects a nucleic acid extraction reagent 310 for extracting the nucleic acid 230 from the nucleic acid extraction object 220 captured by the capture unit 40 into the container 10.
  • the container 10 has the 1st container part 11 and the 2nd container part 12 which divide
  • the first discharge port 31 and the second discharge port 32 are provided in the second container portion 12.
  • the specimen 210 and the nucleic acid extraction reagent 310 can be injected into the upper first container section 11, and the waste liquid specimen 210 and the nucleic acid extraction liquid 320 can be injected from the lower second container section 12 via the capturing section 40. And can be discharged.
  • the nucleic acid extract 320 containing the nucleic acid 230 can be easily recovered after the sample 210 is concentrated and the nucleic acid 230 is extracted using the same container 10.
  • the operation can be performed without transferring the container during the nucleic acid extraction step, it is possible to prevent impurities from being mixed into the nucleic acid extract 320.
  • the nucleic acid extraction target 220 can be easily captured by the capturing section 40 from the specimen 210.
  • the workability is simple and the risk of impurities being mixed into the nucleic acid extract 320 can be greatly reduced.
  • the nucleic acid 230 can be extracted with high sensitivity and high accuracy. Therefore, the accuracy of the inspection using the nucleic acid extract 320 can be improved.
  • the nucleic acid extraction device 100 further includes the vacuum pump 2 as a pressure adjusting unit for adjusting the pressure in the second container unit 12.
  • the capturing unit 40 includes a filter having a plurality of holes smaller than the nucleic acid extraction target 220 as the main body 41.
  • the second discharge port 32 is provided at a position closer to the capturing unit 40 than the first discharge port 31 and also serves as a decompression port of the vacuum pump 2.
  • the nucleic acid extract 320 can be efficiently recovered even though the nucleic acid extract 320 necessary for the test and the sample 210 as the waste liquid are discharged from the same second container portion 12.
  • the vacuum pump 2 was inject
  • a specimen discharge mode in which the specimen 210 is moved from the first container section 11 through the capturing section 40 to the second container section 12, and the pressure in the first container section 11 is reduced by reducing the pressure in the second container section 12.
  • the nucleic acid extract solution 320 in the capture unit 40 is moved to the second container unit 12 via the capture unit 40 in the nucleic acid extract solution movement mode. Adjust pressure.
  • the degree of decompression by the vacuum pump 2 in the nucleic acid extract moving mode is lower than the degree of decompression by the vacuum pump 2 in the specimen discharge mode.
  • nucleic acid extract 320 can be efficiently recovered with a simple structure.
  • the vacuum pump 2 pressurizes the inside of the 2nd container part 12, and makes the container 10 the pressure in the 2nd container part 12 higher than the pressure in the 1st container part 11.
  • the pressure in the second container unit 12 is adjusted in the nucleic acid extraction reagent holding mode in which the injected nucleic acid extraction reagent 310 is held on the capturing unit 40.
  • the nucleic acid extraction reaction can be performed in a state where the nucleic acid extraction reagent 310 is held on the capture unit 40 by adjusting the pressure in the second container unit 12, so that the nucleic acid extract 320 can be efficiently recovered. Can do.
  • the second inlet 22 is a through hole provided in the first container portion 11, and the through hole is provided with a rubber plug 22a that closes the through hole.
  • 22a is made of a rubber material through which a needle for injecting the nucleic acid extraction reagent 310 into the first container part 11 can penetrate.
  • the capturing unit 40 includes a main body 41 for capturing and holding the nucleic acid extraction target 220 contained in the specimen 210, and a support 42 for supporting the main body 41. Yes.
  • the nucleic acid extraction target 220 contained in the specimen 210 can be captured in the main body 41.
  • At least a part of the support portion 42 may be made of metal. Or at least one part of the support part 42 may be comprised with highly heat conductive resin.
  • the heating / cooling unit 7 connected to the support portion 42 can efficiently heat or cool the specimen 210, the nucleic acid extraction target 220 or the nucleic acid extraction reagent 310 on the main body portion 41.
  • the support portion 42 is made of a high thermal conductive resin and is not made of a metal, it is possible to reduce that the inspection is hindered by the metal itself or metal corrosion.
  • the surface of the support part 42 of the capturing part 40 may be hydrophilic.
  • the speed at which the nucleic acid extraction target 220 is captured from the specimen 210 can be improved. That is, the time required to capture the nucleic acid extraction target 220 from the specimen 210 can be shortened.
  • At least a part of the container 10 may be made of a high thermal conductive resin.
  • the sample 210, the nucleic acid extraction target 220 or the nucleic acid extraction reagent 310 on the main body 41 can be efficiently heated or cooled by the heating / cooling unit 7 connected to the container 10. Further, by not using a metal as the container 10, it is possible to reduce that the inspection is hindered by the metal itself or metal corrosion.
  • angular part in the wall surface of the internal space of the 1st container part 11 is good in it being 90 degree
  • the specimen 210 and the nucleic acid extraction reagent 310 are injected into the first container section 11, it is possible to suppress the specimen 210 and the nucleic acid extraction reagent 310 from staying on the wall surface of the internal space of the first container section 11. That is, the liquid loss of the specimen 210 and the nucleic acid extraction reagent 310 can be reduced.
  • the inner surface of the container 10 may be non-hydrophilic.
  • the specimen 210 and the nucleic acid extraction reagent 310 are injected into the first container part 11, it is possible to suppress the specimen 210 and the nucleic acid extraction reagent 310 from adhering to the wall surface of the internal space of the first container part 11. . That is, the liquid loss of the specimen 210 and the nucleic acid extraction reagent 310 can be reduced.
  • the nucleic acid extraction device 100 further includes a vibration device 3 for vibrating the container 10.
  • the nucleic acid extraction reagent 310 and the nucleic acid extraction target object 220 can be stirred and mixed, the nucleic acid 230 can be extracted efficiently, and the sample 210 and the nucleic acid are placed on the wall surface of the internal space of the first container portion 11. It can suppress that the extraction reagent 310 adheres.
  • the nucleic acid extraction method includes a capture step S1 in which the nucleic acid extraction target 220 contained in the specimen 210 is captured by the capture section 40 by passing the specimen 210 through the capture section 40, and a nucleic acid in the capture section 40.
  • a nucleic acid extraction step S2 for extracting the nucleic acid 230 from the nucleic acid extraction target 220 captured by the capture unit 40 by introducing the extraction reagent 310, and a nucleic acid extract 320 containing the extracted nucleic acid 230 are passed through the capture unit 40 and recovered.
  • the workability is simple, and the risk of impurities being mixed into the nucleic acid extract 320 can be greatly reduced. Therefore, the nucleic acid 230 can be extracted with high sensitivity and high accuracy.
  • the end of the tube constituting the second discharge port 32 on the second container part 12 side is flush with the inner wall of the second container part 12. At least a part of the constituting tube on the second container part 12 side may protrude from the inner wall of the second container part 12 toward the inside of the second container part 12.
  • a projecting portion 12 a is provided so that the entire circumference of the end portion on the second container portion 12 side of the tube constituting the second discharge port 32 projects from the inner wall of the second container portion 12.
  • the protruding portion is formed so that only the upper portion of the end portion on the second container portion 12 side of the tube constituting the second discharge port 32 protrudes from the inner wall of the second container portion 12. 12b may be provided.
  • the nucleic acid extract 320 can be prevented from entering the second outlet 32 when the nucleic acid extract 320 is moved from the first container 11 to the second container 12. That is, the protrusions 12 a and 12 b function as eaves, and the nucleic acid extract 320 can be prevented from entering the tube of the second outlet 32. Therefore, the nucleic acid extract 320 can be recovered with little loss.
  • a dead bacteria removing step of inactivating and removing dead bacteria contained in the specimen may be performed.
  • the dead bacteria removing step is a step of inactivating the dead bacteria nucleic acid by introducing a dead bacteria removing reagent (for example, a reagent that inhibits amplification of dead bacteria nucleic acid) into the capturing unit 40 to remove the dead bacteria. And is performed after the capturing step S1. Specifically, the dead bacteria removal step is performed between the capture step S1 and the nucleic acid extraction step S2.
  • the killing microorganism removing reagent is introduced into the container 10 from the first injection port 21 or the second injection port 22 to capture the nucleic acid extraction target microorganism.
  • the dead bacteria removing reagent is introduced onto the part 40 and allowed to stand for 5 to 20 minutes while cooling. Thereby, dead bacteria can be removed and only living bacteria can be collected in the capturing part 40.
  • the dead bacteria removing reagent for example, “Viable Bacteria Selection Kit for PCR” manufactured by Takara Bio Inc. can be used.
  • a step of lightly sucking or stirring with the vacuum pump 2 may be added as appropriate. Furthermore, the dead bacteria removing step may be repeated several times as necessary. Note that the dead bacteria removing reagent may be discharged from the second discharge port 32 or the like through the main body 41 after the dead bacteria removing step is completed. Thus, only the nucleic acid of a living microbe can be extracted by performing a dead bacteria removal process after capture process S1.
  • the container 10 is formed of a light shielding member, but is not limited thereto.
  • the container 10 may be made of a transparent material.
  • a light irradiation unit 8 for irradiating light inside the container 10 can be provided in the reaction box 4 or around the reaction box 4.
  • the light irradiation unit 8 is used, for example, in the above killed bacteria removal step. Specifically, the reaction between the dead bacteria and the dead bacteria removing reagent can be promoted by irradiating the dead light with the light irradiation unit 8 after introducing the dead bacteria removing reagent into the container 10 and allowing it to stand. Thereby, dead bacteria can be removed efficiently.
  • the first injection port 21 for injecting the specimen into the container 10 and the second injection port 22 for injecting the nucleic acid extraction reagent into the container 10 are separately provided. It is not a thing. Moreover, although the 1st discharge port 31 for discharging
  • the first inlet 21 and the second inlet 22 may be configured as one inlet 23 as shown in FIG. That is, one inlet 23 that serves as both the first inlet 21 and the second inlet 22 may be provided in the container 10 (first container portion 11). From the injection port 23, a specimen is injected or a nucleic acid extraction reagent is injected. In addition to the specimen 210 and the nucleic acid extraction reagent 310, other liquids such as a dead bacteria removal reagent may be injected from the injection port 23.
  • first outlet 31 and the second outlet 32 may be configured as one outlet 33 as shown in FIG. That is, one discharge port 33 that doubles as the first discharge port 31 and the second discharge port 32 may be provided in the container 10 (second container portion 12). From the outlet 33, the nucleic acid extract is discharged or the sample is discharged. In addition to the nucleic acid extract and the sample, other liquids introduced into the container 10 may be discharged from the discharge port 33.
  • the sample is input to the injection port 23 provided on the upstream side of the capturing unit 40 via the valve 600.
  • the cup 200 and the nucleic acid extraction reagent container 300 may be connected.
  • the discharge port 33 provided on the downstream side of the capturing unit 40 is connected to a pipe 5b for collecting a nucleic acid extract in a collection container 400 (not shown) via a valve 700 and a sample waste liquid in the collection container 500.
  • a pipe 2b for collecting (filtrate) may be connected.
  • the specimen 210 is directly passed through the capturing unit 40, but the present invention is not limited to this.
  • the specimen 210 may be passed through a prefilter as a pretreatment before the specimen 210 is passed through the capturing unit 40.
  • a pipe having a prefilter may be inserted between the specimen insertion cup 200 and the first inlet 21. In this manner, by passing the specimen 210 through the prefilter before passing through the capturing unit 40, unnecessary components having a large size can be removed. Thereby, capture of the nucleic acid extraction target 220 from the specimen 210 can be performed efficiently.
  • Nucleic acid extraction unit 2 Vacuum pump (pressure adjustment unit) DESCRIPTION OF SYMBOLS 10 Container 11 1st container part 12 2nd container part 21 1st inlet 22 2nd inlet 23 Injector (1st inlet, 2nd inlet) 22a Rubber plug 31 First discharge port 32 Second discharge port 33 Discharge port (first discharge port, second discharge port) 40 Nucleic Acid Extraction Device 230 Nucleic Acid Extraction Object 230 Nucleic Acid 310 Nucleic Acid Extraction Reagent 320 Nucleic Acid Extraction Solution

Abstract

A nucleic acid extraction unit (1) in a nucleic acid extraction device (100) is provided with: a vessel (10); a first inlet (21) through which a sample is injected into the vessel (10); a capturing unit (40) for capturing a nucleic acid extraction material (i.e., a material from which a nucleic acid is to be extracted) contained in the sample that is injected through the first inlet (21); a second inlet (22) through which a nucleic acid extraction reagent for extracting a nucleic acid from the nucleic acid extraction material captured by the capturing unit (40) is injected into the vessel 10; a first outlet (31) through which a nucleic acid extract, which contains the nucleic acid extracted from the nucleic acid extraction material with the nucleic acid extraction reagent, is discharged from the vessel (10); and a second outlet (32) through which the sample is discharged from the vessel (10). The vessel (10) is provided with a first vessel unit (11) and a second vessel unit (12), the first inlet (21) and the second inlet (22) are provided in the first vessel unit (11), and the first outlet (31) and the second outlet (32) are provided in the second vessel unit (12).

Description

核酸抽出装置、核酸抽出ユニット及び核酸抽出方法Nucleic acid extraction apparatus, nucleic acid extraction unit and nucleic acid extraction method
　本発明は、検体から核酸（デオキシリボ核酸、リボ核酸）を抽出するための核酸抽出装置、核酸抽出装置に用いられる核酸抽出ユニット、及び、検体から核酸を抽出するための核酸抽出方法に関する。 The present invention relates to a nucleic acid extraction apparatus for extracting nucleic acid (deoxyribonucleic acid, ribonucleic acid) from a specimen, a nucleic acid extraction unit used in the nucleic acid extraction apparatus, and a nucleic acid extraction method for extracting nucleic acid from a specimen.
　飲料又は食品等に含まれる細菌等の微生物の検査においては、ポリメラーゼ連鎖反応（ＰＣＲ：ｐｏｌｙｍｅｒａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ）等の核酸増幅法が利用されている。核酸増幅法は、培養法と比べて大幅に検査工程を高速化及び簡略化することができる利点がある。 Nucleic acid amplification methods such as polymerase chain reaction (PCR) are used for testing microorganisms such as bacteria contained in beverages or foods. The nucleic acid amplification method has an advantage that the inspection process can be greatly speeded up and simplified as compared with the culture method.
　核酸増幅法を利用した検査を実施するためには、核酸増幅を行う前に検体から核酸を抽出する前処理が必要となる。従来、このような前処理としては、検体（検体原液）をフィルタでろ過することで検体に含まれる細菌等の検査対象物を捕捉し、核酸抽出試薬によって検査対象物から核酸を抽出する方法が知られている（例えば特許文献１）。 In order to perform a test using a nucleic acid amplification method, a pretreatment for extracting nucleic acid from a specimen is required before performing nucleic acid amplification. Conventionally, as such pretreatment, there is a method of capturing a test object such as bacteria contained in the sample by filtering the sample (specimen stock solution) with a filter and extracting the nucleic acid from the test object using a nucleic acid extraction reagent. Known (for example, Patent Document 1).
特開平４－３６１９７号公報JP-A-4-36197
　特許文献１に開示された従来の核酸抽出方法（前処理）は、検体を入れたシリンジにフィルタを通して検査対象物をろ過濃縮した後、このフィルタを取り外して核酸抽出試薬を入れた別のシリンジに再度取り付けて検査対象物を溶出して核酸を抽出するものである。 In the conventional nucleic acid extraction method (pretreatment) disclosed in Patent Document 1, the test object is filtered and concentrated through a filter in a syringe containing a sample, and then the filter is removed and placed in another syringe containing a nucleic acid extraction reagent. The nucleic acid is extracted by reattaching and eluting the test object.
　しかしながら、従来の核酸抽出方法では、作業が煩雑な上に、作業中に検査対象物とは異なる菌等の不純物が、抽出した核酸を含む核酸抽出液に混入すること（コンタミ）が生じるリスクがある。 However, in the conventional nucleic acid extraction method, the work is complicated and there is a risk that impurities such as bacteria different from the test object are mixed in the nucleic acid extract containing the extracted nucleic acid (contamination) during the work. is there.
　本発明は、このような課題を解決するためになされたものであり、作業性が簡便で、核酸抽出液に不純物が混入するリスクを大幅に軽減できる核酸抽出装置及び核酸抽出ユニット等を提供することを目的とする。 The present invention has been made to solve such problems, and provides a nucleic acid extraction apparatus, a nucleic acid extraction unit, and the like that are simple in workability and can greatly reduce the risk of impurities being mixed into a nucleic acid extract. For the purpose.
　上記目的を達成するために、本発明に係る核酸抽出装置の一態様は、検体から核酸を抽出するための核酸抽出ユニットを備え、前記核酸抽出ユニットは、容器と、前記容器内に検体を注入するための第１注入口と、少なくとも一部が前記容器内に配置され、前記第１注入口から注入された検体に含まれる核酸抽出対象物を捕捉する捕捉部と、前記捕捉部で捕捉された前記核酸抽出対象物から核酸を抽出するための核酸抽出試薬を前記容器内に注入するための第２注入口と、前記核酸抽出試薬によって前記核酸抽出対象物から抽出された核酸が含まれる核酸抽出液を前記容器から排出するための第１排出口と、前記検体を前記容器から排出するための第２排出口とを有し、前記容器は、前記捕捉部を境界として当該容器の内部空間を分割する第１容器部と第２容器部とを有し、前記第１注入口及び前記第２注入口は、前記第１容器部に設けられており、前記第１排出口及び前記第２排出口は、前記第２容器部に設けられている。 In order to achieve the above object, one aspect of a nucleic acid extraction apparatus according to the present invention includes a nucleic acid extraction unit for extracting nucleic acid from a specimen, and the nucleic acid extraction unit injects a specimen into the container and the container. A first injection port for capturing, a capture unit that is at least partially disposed in the container, captures a nucleic acid extraction target contained in a sample injected from the first injection port, and is captured by the capture unit A second injection port for injecting a nucleic acid extraction reagent for extracting nucleic acid from the nucleic acid extraction object into the container, and a nucleic acid containing the nucleic acid extracted from the nucleic acid extraction object by the nucleic acid extraction reagent It has a first outlet for discharging the extract from the container and a second outlet for discharging the specimen from the container, and the container has an internal space of the container with the capture section as a boundary. Split 1 container part and 2nd container part, the 1st inlet and the 2nd inlet are provided in the 1st container part, the 1st outlet and the 2nd outlet are It is provided in the second container part.
　また、本発明に係る核酸抽出ユニットの一態様は、検体から核酸を抽出するための核酸抽出装置に用いられる核酸抽出ユニットであって、容器と、前記容器内に検体を注入するための第１注入口と、前記容器内に配置され、前記第１注入口から注入された検体に含まれる核酸抽出対象物を捕捉する捕捉部と、前記捕捉部で捕捉された前記核酸抽出対象物から核酸を抽出するための核酸抽出試薬を前記容器内に注入するための第２注入口と、前記核酸抽出試薬によって前記核酸抽出対象物から抽出された核酸が含まれる核酸抽出液を前記容器から排出するための第１排出口と、前記検体を前記容器から排出するための第２排出口とを備え、前記容器は、前記捕捉部を境界として当該容器の内部空間を分割するための第１容器部と第２容器部とを有し、前記第１注入口及び前記第２注入口は、前記第１容器部に設けられており、前記第１排出口及び前記第２排出口は、前記第２容器部に設けられている。 Moreover, one aspect of the nucleic acid extraction unit according to the present invention is a nucleic acid extraction unit used in a nucleic acid extraction apparatus for extracting nucleic acid from a specimen, and includes a container and a first for injecting the specimen into the container. Nucleic acid is extracted from the nucleic acid extraction target captured by the inlet, the capture unit that is disposed in the container and captures the nucleic acid extraction target contained in the sample injected from the first injection port, and A second injection port for injecting a nucleic acid extraction reagent for extraction into the container; and a nucleic acid extract containing the nucleic acid extracted from the nucleic acid extraction object by the nucleic acid extraction reagent from the container. A first discharge port and a second discharge port for discharging the specimen from the container, and the container includes a first container part for dividing the internal space of the container with the capture part as a boundary. With the second container part The first inlet and the second inlet are provided in the first container part, and the first outlet and the second outlet are provided in the second container part. .
　また、本発明に係る核酸抽出方法の一態様は、捕捉部に検体を通過させることによって前記検体に含まれる核酸抽出対象物を前記捕捉部で捕捉する捕捉工程と、前記捕捉部に核酸抽出試薬を導入することによって前記捕捉部で捕捉した前記核酸抽出対象物から核酸を抽出する核酸抽出工程と、抽出した前記核酸を含む核酸抽出液を前記捕捉部を通過させて回収する回収工程とを含む。 Moreover, one aspect of the nucleic acid extraction method according to the present invention includes a capture step of capturing the nucleic acid extraction target contained in the sample by the capture unit by passing the sample through the capture unit, and a nucleic acid extraction reagent in the capture unit. A nucleic acid extraction step for extracting nucleic acid from the nucleic acid extraction target captured by the capture unit by introducing a nucleic acid, and a recovery step for recovering the nucleic acid extract containing the extracted nucleic acid through the capture unit .
　本発明によれば、作業性が簡便で、核酸抽出液に不純物が混入するリスクを大幅に軽減できる。 According to the present invention, the workability is simple and the risk of impurities being mixed into the nucleic acid extract can be greatly reduced.
図１は、実施の形態に係る核酸抽出装置を模式的に示す斜視図である。FIG. 1 is a perspective view schematically showing a nucleic acid extraction apparatus according to an embodiment. 図２は、実施の形態に係る核酸抽出装置における核酸抽出ユニットの周辺構造を示す要部拡大斜視図である。FIG. 2 is an enlarged perspective view of a main part showing a peripheral structure of the nucleic acid extraction unit in the nucleic acid extraction apparatus according to the embodiment. 図３は、実施の形態に係る核酸抽出ユニットの斜視図である。FIG. 3 is a perspective view of the nucleic acid extraction unit according to the embodiment. 図４は、実施の形態に係る核酸抽出ユニットの断面図である。FIG. 4 is a cross-sectional view of the nucleic acid extraction unit according to the embodiment. 図５は、実施の形態に係る核酸抽出方法のフローチャートである。FIG. 5 is a flowchart of the nucleic acid extraction method according to the embodiment. 図６Ａは、実施の形態に係る核酸抽出方法において、検体を容器に注入するときの様子を示す図である。FIG. 6A is a diagram illustrating a state when a specimen is injected into a container in the nucleic acid extraction method according to the embodiment. 図６Ｂは、実施の形態に係る核酸抽出方法において、捕捉部で捕捉された核酸抽出対象物を示す図である。FIG. 6B is a diagram illustrating the nucleic acid extraction target captured by the capturing unit in the nucleic acid extraction method according to the embodiment. 図６Ｃは、実施の形態に係る核酸抽出方法において、核酸抽出試薬を容器に注入するときの様子を示す図である。FIG. 6C is a diagram showing a state when the nucleic acid extraction reagent is injected into the container in the nucleic acid extraction method according to the embodiment. 図６Ｄは、実施の形態に係る核酸抽出方法において、核酸抽出試薬を捕捉部に保持させるときの様子を示す図である。FIG. 6D is a diagram illustrating a state where the nucleic acid extraction reagent is held in the capturing unit in the nucleic acid extraction method according to the embodiment. 図６Ｅは、実施の形態に係る核酸抽出方法において、核酸抽出液を第１容器部から第２容器部に移動させるときの様子を示す図である。FIG. 6E is a diagram showing a state when the nucleic acid extract is moved from the first container part to the second container part in the nucleic acid extraction method according to the embodiment. 図６Ｆは、実施の形態に係る核酸抽出方法において、第２容器部に移動させた核酸抽出液の様子を示す図である。FIG. 6F is a diagram showing a state of the nucleic acid extract moved to the second container part in the nucleic acid extraction method according to the embodiment. 図６Ｇは、実施の形態に係る核酸抽出方法において、核酸抽出液の容器から排出するときの様子を示す図である。FIG. 6G is a diagram showing a state when the nucleic acid extraction liquid is discharged from the container in the nucleic acid extraction method according to the embodiment. 図７は、変形例１に係る核酸抽出ユニットの断面図である。FIG. 7 is a cross-sectional view of a nucleic acid extraction unit according to Modification 1. 図８は、変形例２に係る核酸抽出ユニットの断面図である。FIG. 8 is a cross-sectional view of a nucleic acid extraction unit according to Modification 2. 図９は、変形例３に係る核酸抽出ユニットの断面図である。FIG. 9 is a cross-sectional view of a nucleic acid extraction unit according to Modification 3. 図１０は、変形例４に係る核酸抽出ユニットの断面図である。FIG. 10 is a cross-sectional view of a nucleic acid extraction unit according to Modification 4. 図１１は、変形例５に係る核酸抽出ユニットの断面図である。FIG. 11 is a cross-sectional view of a nucleic acid extraction unit according to Modification 5.
　以下、本発明の実施の形態について、図面を参照しながら説明する。なお、以下に説明する実施の形態は、いずれも本発明の好ましい一具体例を示すものである。したがって、以下の実施の形態で示される、数値、形状、材料、構成要素、構成要素の配置位置及び接続形態、並びに、ステップ（工程）及びステップの順序などは、一例であって本発明を限定する主旨ではない。よって、以下の実施の形態における構成要素のうち、本発明の最上位概念を示す独立請求項に記載されていない構成要素については、任意の構成要素として説明される。 Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that each of the embodiments described below shows a preferred specific example of the present invention. Therefore, numerical values, shapes, materials, components, arrangement positions and connection forms of components, steps (steps) and order of steps, and the like shown in the following embodiments are examples and limit the present invention. It is not the purpose to do. Therefore, among the constituent elements in the following embodiments, constituent elements that are not described in the independent claims showing the highest concept of the present invention are described as optional constituent elements.
　なお、各図は、模式図であり、必ずしも厳密に図示されたものではない。また、各図において、実質的に同一の構成に対しては同一の符号を付しており、重複する説明は省略又は簡略化する。 Each figure is a schematic diagram and is not necessarily shown strictly. Moreover, in each figure, the same code | symbol is attached | subjected to the substantially same structure, The overlapping description is abbreviate | omitted or simplified.
　（実施の形態）
　［核酸抽出装置］
　実施の形態に係る核酸抽出装置１００及び核酸抽出ユニット１の構成について、図１～図４を用いて説明する。図１は、実施の形態に係る核酸抽出装置１００を模式的に示す斜視図である。図２は、同核酸抽出装置１００における核酸抽出ユニット１の周辺構造を示す要部拡大斜視図であり、核酸抽出装置１００に設置された状態の核酸抽出ユニット１を示している。図３は、実施の形態に係る核酸抽出ユニット１の斜視図であり、図４は、同核酸抽出ユニット１の断面図である。 (Embodiment)
[Nucleic acid extraction equipment]
The configurations of the nucleic acid extraction apparatus 100 and the nucleic acid extraction unit 1 according to the embodiment will be described with reference to FIGS. FIG. 1 is a perspective view schematically showing a nucleic acid extraction apparatus 100 according to an embodiment. FIG. 2 is an enlarged perspective view of a main part showing a peripheral structure of the nucleic acid extraction unit 1 in the nucleic acid extraction apparatus 100, and shows the nucleic acid extraction unit 1 installed in the nucleic acid extraction apparatus 100. FIG. 3 is a perspective view of the nucleic acid extraction unit 1 according to the embodiment, and FIG. 4 is a cross-sectional view of the nucleic acid extraction unit 1.
　図１に示すように、核酸抽出装置１００は、検体に含まれる被測定物から核酸を抽出するための核酸抽出ユニット１と、真空ポンプ２と、振動装置３とを備える。 As shown in FIG. 1, the nucleic acid extraction apparatus 100 includes a nucleic acid extraction unit 1 for extracting nucleic acid from a measurement object contained in a specimen, a vacuum pump 2, and a vibration device 3.
　検体（検体原液）に含まれる被測定物は、核酸を抽出する対象物（核酸抽出対象物）であって、例えば、細菌、ウイルス又は組織細胞等の微生物である。微生物を含む検体原液は、例えば、飲料から採取することができる。 An object to be measured contained in a specimen (specimen stock solution) is an object (nucleic acid extraction object) from which nucleic acid is extracted, and is, for example, a microorganism such as a bacterium, virus, or tissue cell. A sample stock solution containing microorganisms can be collected from a beverage, for example.
　細菌、ウイルス又は組織細胞等の微生物は、ＰＣＲ等による核酸増幅によって検査が行われる検査対象物である。つまり、核酸抽出装置１００は、核酸増幅を行う前の前処理のために用いられる装置であり、核酸抽出装置１００で抽出された核酸は、所望の検査に利用される。なお、本実施の形態における核酸抽出装置１００では、抽出された核酸を含む液体（核酸抽出液）として回収される。 Microorganisms such as bacteria, viruses or tissue cells are inspection objects to be inspected by nucleic acid amplification by PCR or the like. That is, the nucleic acid extraction apparatus 100 is an apparatus used for pretreatment before performing nucleic acid amplification, and the nucleic acid extracted by the nucleic acid extraction apparatus 100 is used for a desired test. In addition, in the nucleic acid extraction apparatus 100 in this Embodiment, it collect | recovers as a liquid (nucleic acid extract) containing the extracted nucleic acid.
　核酸抽出ユニット１は、検体から核酸の抽出処理を行うための処理ユニットであり、図２に示すように、核酸抽出装置１００の反応ボックス４に設置される。本実施の形態において、核酸抽出ユニット１は、交換可能なカートリッジであり、反応ボックス４から取り外すことができる。核酸抽出ユニット１は、例えば、１回の核酸抽出処理ごとに交換される。なお、図１では、反応ボックス４の全面蓋を開いた状態を示している。 The nucleic acid extraction unit 1 is a processing unit for performing a nucleic acid extraction process from a specimen, and is installed in the reaction box 4 of the nucleic acid extraction apparatus 100 as shown in FIG. In the present embodiment, the nucleic acid extraction unit 1 is a replaceable cartridge and can be removed from the reaction box 4. For example, the nucleic acid extraction unit 1 is replaced for each nucleic acid extraction process. FIG. 1 shows a state where the entire lid of the reaction box 4 is opened.
　核酸抽出ユニット１は、図３及び図４に示すように、容器１０と、第１注入口２１と、第２注入口２２と、第１排出口３１と、第２排出口３２と、捕捉部４０とを備える。 As shown in FIGS. 3 and 4, the nucleic acid extraction unit 1 includes a container 10, a first inlet 21, a second inlet 22, a first outlet 31, a second outlet 32, and a capturing unit. 40.
　容器１０は、注入される検体及び核酸抽出試薬によって検体から核酸の抽出処理を行うための処理容器である。検体から抽出された核酸は、核酸抽出液となって容器１０から排出される。 The container 10 is a processing container for performing a nucleic acid extraction process from a specimen by using a specimen to be injected and a nucleic acid extraction reagent. The nucleic acid extracted from the specimen is discharged from the container 10 as a nucleic acid extract.
　容器１０の材質は、特に限定されるものではないが、核酸抽出処理で加熱処理を行うことを可能とするために、容器１０は、ポリプロピレン（ＰＰ）又はポリカーボネート（ＰＣ）等の耐熱性の高い樹脂材料、アルミニウム又はステンレス等の金属材料、あるいは、ガラス又はセラミック等の無機材料によって構成されているとよい。また、容器１０として樹脂材料を用いた場合には、安価で軽量化を図ることができるという利点もある。また、容器１０として金属材料又は高熱伝導性樹脂を用いた場合には、容器１０の熱伝導性を向上させることができる。このため、加熱処理を行う場合には、容器１０の少なくとも一部は、金属材料又は高熱伝導性樹脂によって構成されているとよい。なお、容器１０は、上記の材料を組み合わせて構成されていてもよい。  The material of the container 10 is not particularly limited, but the container 10 has a high heat resistance such as polypropylene (PP) or polycarbonate (PC) in order to enable heat treatment by nucleic acid extraction treatment. It is good to be comprised with resin materials, metal materials, such as aluminum or stainless steel, or inorganic materials, such as glass or a ceramic. In addition, when a resin material is used as the container 10, there is an advantage that the weight can be reduced at low cost. Further, when a metal material or a high thermal conductive resin is used as the container 10, the thermal conductivity of the container 10 can be improved. For this reason, when performing heat processing, it is good for at least one part of the container 10 to be comprised with the metal material or highly heat conductive resin. The container 10 may be configured by combining the above materials.
　また、容器１０の内面は、非親水性であるとよい。例えば、容器１０の内面は、疎水性であるとよい。この場合、疎水性材料を用いて容器１０を形成してもよいし、表面処理によって容器１０の内面を疎水性にしてもよいし、疎水性を有する膜が容器１０の内面にコーティングされていてもよい。 Moreover, the inner surface of the container 10 is preferably non-hydrophilic. For example, the inner surface of the container 10 may be hydrophobic. In this case, the container 10 may be formed using a hydrophobic material, or the inner surface of the container 10 may be made hydrophobic by surface treatment, or a hydrophobic film is coated on the inner surface of the container 10. Also good.
　容器１０は、第１容器部１１と第２容器部１２とによって構成されている。第１容器部１１と第２容器部１２とは、捕捉部４０を境界として容器１０の内部空間を分割している。本実施の形態において、容器１０は、捕捉部４０の本体部４１を境界として上下方向に二分割されており、第１容器部１１が上側の部品で、第２容器部１２が下側の部品となっている。なお、第１容器部１１については、さらに２つに分割されているが、これに限るものではない。 The container 10 includes a first container part 11 and a second container part 12. The first container part 11 and the second container part 12 divide the internal space of the container 10 with the capturing part 40 as a boundary. In the present embodiment, the container 10 is divided into two parts in the vertical direction with the main body 41 of the capturing part 40 as a boundary. The first container part 11 is an upper part and the second container part 12 is a lower part. It has become. The first container part 11 is further divided into two parts, but is not limited thereto.
　第１注入口２１、第２注入口２２、第１排出口３１、第２排出口３２及び捕捉部４０は、容器１０に設けられている。具体的には、第１注入口２１、第２注入口２２、第１排出口３１及び第２排出口３２は、容器１０の隔壁に設けられており、捕捉部４０は、本体部４１が容器１０の内部に位置するように容器１０に固定されている。 The first inlet 21, the second inlet 22, the first outlet 31, the second outlet 32, and the capturing unit 40 are provided in the container 10. Specifically, the first inlet 21, the second inlet 22, the first outlet 31, and the second outlet 32 are provided in the partition wall of the container 10. 10 is fixed to the container 10 so as to be located inside.
　第１注入口２１は、容器１０内に検体を注入するための検体注入口である。本実施の形態において、第１注入口２１は、第１容器部１１の上壁に設けられている。検体（検体原液）は、核酸抽出装置１００に設置される検体投入カップ２００から第１注入口２１を介して第１容器部１１に注入される。 The first injection port 21 is a sample injection port for injecting a sample into the container 10. In the present embodiment, the first inlet 21 is provided on the upper wall of the first container part 11. A specimen (specimen stock solution) is injected into the first container section 11 through the first inlet 21 from a specimen insertion cup 200 installed in the nucleic acid extraction apparatus 100.
　第１注入口２１は、容器１０の外部と第１容器部１１の内部空間とを連通する配管である。具体的には、第１注入口２１の一方の端部は、第１容器部１１に接続され、第１注入口２１の他方の端部は、検体投入カップ２００に接続される。本実施の形態において、第１注入口２１は、第１容器部１１と一体的に形成されている。なお、第１注入口２１は、容器１０（第１容器部１１）の上壁に設けられた貫通孔であってもよい。 The first inlet 21 is a pipe that communicates the outside of the container 10 and the internal space of the first container part 11. Specifically, one end portion of the first injection port 21 is connected to the first container portion 11, and the other end portion of the first injection port 21 is connected to the sample insertion cup 200. In the present embodiment, the first inlet 21 is formed integrally with the first container part 11. The first inlet 21 may be a through hole provided in the upper wall of the container 10 (first container portion 11).
　第２注入口２２は、容器１０内に核酸抽出試薬を注入するための核酸抽出試薬注入口である。本実施の形態において、第２注入口２２は、第１注入口２１と同様に第１容器部１１の上壁に設けられているが、第１注入口２１と異なる位置に設けられている。 The second injection port 22 is a nucleic acid extraction reagent injection port for injecting the nucleic acid extraction reagent into the container 10. In the present embodiment, the second inlet 22 is provided on the upper wall of the first container portion 11 similarly to the first inlet 21, but is provided at a position different from the first inlet 21.
　第２注入口２２から注入される核酸抽出試薬は、捕捉部４０で捕捉された核酸抽出対象物から核酸を抽出するための液体試薬であり、例えば核酸抽出対象物の細胞膜から核酸を取り出す作用を持つ。核酸抽出試薬は、核酸抽出装置１００に設置される核酸抽出試薬容器３００から第２注入口２２を介して第１容器部１１に注入される。 The nucleic acid extraction reagent injected from the second injection port 22 is a liquid reagent for extracting nucleic acid from the nucleic acid extraction target captured by the capturing unit 40. For example, the nucleic acid extraction reagent functions to extract nucleic acid from the cell membrane of the nucleic acid extraction target. Have. The nucleic acid extraction reagent is injected from the nucleic acid extraction reagent container 300 installed in the nucleic acid extraction apparatus 100 into the first container unit 11 through the second injection port 22.
　第２注入口２２は、第１容器部１１に設けられた貫通孔である。貫通孔である第２注入口２２には、第２注入口２２（貫通孔）を塞ぐゴム栓２２ａが設けられている。ゴム栓２２ａは、核酸抽出試薬を第１容器部１１内に注入するための針が貫通可能なゴム材料によって構成されている。核酸抽出試薬容器３００内の核酸抽出試薬を第１容器部１１内に注入する際、核酸抽出試薬容器３００に設けられた針をゴム栓２２ａに貫通させる。これにより、核酸抽出試薬容器３００から第２注入口２２を介して液体試薬を第１容器部１１内に注入することができる。なお、ゴム栓２２ａのゴム材料は、特に限定されるものではないが、例えば、シリコーンゴム又はフッ素ゴム等が用いられる。 The second inlet 22 is a through hole provided in the first container part 11. The second inlet 22 which is a through hole is provided with a rubber stopper 22a which closes the second inlet 22 (through hole). The rubber stopper 22a is made of a rubber material that can be penetrated by a needle for injecting the nucleic acid extraction reagent into the first container portion 11. When the nucleic acid extraction reagent in the nucleic acid extraction reagent container 300 is injected into the first container part 11, the needle provided in the nucleic acid extraction reagent container 300 is made to penetrate the rubber stopper 22a. Thereby, the liquid reagent can be injected from the nucleic acid extraction reagent container 300 into the first container part 11 through the second injection port 22. The rubber material of the rubber plug 22a is not particularly limited, but, for example, silicone rubber or fluorine rubber is used.
　このように、第１容器部１１には検体及び核酸抽出試薬の液体が注入されるので、第１容器部１１の内部空間の壁面における角部分の角度は、９０度以上であるとよい。つまり、第１容器部１１における任意の２つの壁面のなす角が９０度以上であるとよい。これにより、検体及び核酸抽出試薬を第１容器部１１に注入したときに、第１容器部１１の内部空間の壁面に検体及び核酸抽出試薬が留まることを抑制することができる。 Thus, since the liquid of the sample and the nucleic acid extraction reagent is injected into the first container part 11, the angle of the corner portion of the wall surface of the internal space of the first container part 11 is preferably 90 degrees or more. That is, the angle formed by any two wall surfaces in the first container portion 11 is preferably 90 degrees or more. Thereby, when the sample and the nucleic acid extraction reagent are injected into the first container part 11, it can be suppressed that the sample and the nucleic acid extraction reagent remain on the wall surface of the internal space of the first container part 11.
　第１排出口３１は、核酸抽出試薬によって核酸抽出対象物から抽出された核酸が含まれる核酸抽出液を容器１０から排出するための核酸抽出液排出口である。本実施の形態において、第１排出口３１は、第２容器部１２の下壁に設けられている。 The first discharge port 31 is a nucleic acid extract discharge port for discharging a nucleic acid extract containing a nucleic acid extracted from a nucleic acid extraction target by a nucleic acid extraction reagent from the container 10. In the present embodiment, the first discharge port 31 is provided on the lower wall of the second container portion 12.
　第１排出口３１は、容器１０の外部と第２容器部１２の内部空間とを連通する配管である。具体的には、第１排出口３１の一方の端部は、第２容器部１２に接続され、第１排出口３１の他方の端部は、配管を介して核酸抽出装置１００に設置された回収容器４００（核酸抽出液回収容器）に接続される。本実施の形態において、第１排出口３１は、第２容器部１２と一体的に形成されている。 The first discharge port 31 is a pipe that communicates the outside of the container 10 and the internal space of the second container part 12. Specifically, one end of the first outlet 31 is connected to the second container part 12, and the other end of the first outlet 31 is installed in the nucleic acid extraction apparatus 100 via a pipe. It is connected to a recovery container 400 (nucleic acid extract recovery container). In the present embodiment, the first discharge port 31 is formed integrally with the second container portion 12.
　第１排出口３１から排出される核酸抽出液は、第１排出口３１に接続された配管を通じて回収容器４００に回収される。例えば、核酸抽出液は、送液ポンプ５によって容器１０から回収容器４００に送液されることで回収される。 The nucleic acid extract discharged from the first outlet 31 is recovered in the recovery container 400 through a pipe connected to the first outlet 31. For example, the nucleic acid extract is collected by being fed from the container 10 to the collection container 400 by the liquid feed pump 5.
　第２排出口３２は、検体を容器１０から排出するための検体排出口である。第２排出口３２からは、容器１０に注入された検体の廃液が排出される。つまり、第１容器部１１から注入されて捕捉部４０を通過した後の検体（ろ液）が第２排出口３２から排出される。本実施の形態において、第２排出口３２は、第２容器部１２の側壁に設けられている。つまり、第２排出口３２は、第１排出口３１よりも捕捉部４０に近い位置に設けられている。 The second discharge port 32 is a sample discharge port for discharging the sample from the container 10. From the second discharge port 32, the waste liquid of the sample injected into the container 10 is discharged. That is, the sample (filtrate) injected from the first container part 11 and passing through the capturing part 40 is discharged from the second discharge port 32. In the present embodiment, the second discharge port 32 is provided on the side wall of the second container portion 12. That is, the second discharge port 32 is provided at a position closer to the capturing unit 40 than the first discharge port 31.
　また、第２排出口３２の開口径（内径）は、第１排出口３１の開口径（内径）の２倍以上であるとよい。一般的に、検体の廃液は大容量（例えば約１００ｍｌ）で、核酸増幅のために回収される核酸抽出液は小容量（例えば約２００μｌ）である。このため、本実施の形態のように、検体の廃液と核酸抽出液との排出経路を第１排出口３１と第２排出口３２とに分けるとともに、第２排出口３２の開口径を第１排出口３１の開口径の２倍以上とすることで、核酸抽出液が容器１０の壁面に付着する等のロスを低減することができる。つまり、第１排出口３１の開口径を小さくすることで、ロスが少なく核酸抽出液を排出することができる。例えば、第２排出口３２の開口径は２ｍｍであり、第１排出口３１の開口径は０．５ｍｍである。なお、第２排出口３２の開口径は、第１排出口３１の開口径の２倍以上でなくてもよく、第２排出口３２の開口径は、第１排出口３１の開口径と同じであってもよいし、第１排出口３１の開口径よりも小さくてもよい。 Further, the opening diameter (inner diameter) of the second outlet 32 is preferably at least twice as large as the opening diameter (inner diameter) of the first outlet 31. Generally, the sample waste liquid has a large volume (for example, about 100 ml), and the nucleic acid extract collected for nucleic acid amplification has a small volume (for example, about 200 μl). For this reason, as in the present embodiment, the discharge path of the sample waste liquid and the nucleic acid extract is divided into the first discharge port 31 and the second discharge port 32, and the opening diameter of the second discharge port 32 is set to the first diameter. By making the opening diameter of the discharge port 31 twice or more, a loss such as adhesion of the nucleic acid extract to the wall surface of the container 10 can be reduced. That is, by reducing the opening diameter of the first discharge port 31, the nucleic acid extract can be discharged with little loss. For example, the opening diameter of the second discharge port 32 is 2 mm, and the opening diameter of the first discharge port 31 is 0.5 mm. The opening diameter of the second discharge port 32 may not be twice or more than the opening diameter of the first discharge port 31. The opening diameter of the second discharge port 32 is the same as the opening diameter of the first discharge port 31. It may be smaller than the opening diameter of the first discharge port 31.
　第２排出口３２は、容器１０の外部と第２容器部１２の内部空間とを連通する配管である。具体的には、第２排出口３２の一方の端部は、第２容器部１２に接続され、第２排出口３２の他方の端部は、配管を介して核酸抽出装置１００に設置された回収容器５００（検体廃液回収容器）に接続される。本実施の形態において、第２排出口３２は、第２容器部１２と一体的に形成されている。 The second discharge port 32 is a pipe that communicates the outside of the container 10 and the internal space of the second container part 12. Specifically, one end of the second discharge port 32 is connected to the second container part 12, and the other end of the second discharge port 32 is installed in the nucleic acid extraction apparatus 100 via a pipe. It is connected to a collection container 500 (specimen waste liquid collection container). In the present embodiment, the second discharge port 32 is formed integrally with the second container portion 12.
　第２排出口３２から排出される検体は、第２排出口３２に接続された配管を通じて、回収容器５００に回収される。例えば、検体の廃液は、真空ポンプ２によって容器１０から回収容器５００に送液されることで回収される。 The specimen discharged from the second discharge port 32 is collected in the collection container 500 through a pipe connected to the second discharge port 32. For example, the waste liquid of the specimen is recovered by being sent from the container 10 to the recovery container 500 by the vacuum pump 2.
　捕捉部４０は、第１注入口２１から注入された検体に含まれる核酸抽出対象物を捕捉するための捕捉ユニットであり、少なくとも一部が容器１０内に配置されている。本実施の形態において、捕捉部４０は、検体に含まれる核酸抽出対象物をろ過によって捕捉する。 The capture unit 40 is a capture unit for capturing a nucleic acid extraction target contained in a sample injected from the first injection port 21, and at least a part of the capture unit 40 is disposed in the container 10. In the present embodiment, the capture unit 40 captures the nucleic acid extraction target contained in the specimen by filtration.
　捕捉部４０は、核酸抽出対象物を捕捉して保持するための本体部４１と、本体部４１を支持する支持部４２とを有する。捕捉部４０は、本体部４１が容器１０の内部に位置するように容器１０に固定されている。 The capturing unit 40 includes a main body 41 for capturing and holding the nucleic acid extraction target and a support 42 for supporting the main body 41. The capturing part 40 is fixed to the container 10 such that the main body part 41 is located inside the container 10.
　本体部４１は、板状の支持部４２の上に載置されている。支持部４２の本体部４１に対応する中央部分には複数の貫通孔（目）が形成されている。つまり、支持部４２は目皿である。また、支持部４２の周辺部分は、第１容器部１１と第２容器部１２とで挟持されている。これにより、支持部４２が容器１０に固定されている。支持部４２と第１容器部１１と第２容器部１２とは、例えば、４本のネジによって固定されている。 The main body 41 is placed on a plate-like support 42. A plurality of through holes (eyes) are formed in a central portion corresponding to the main body portion 41 of the support portion 42. That is, the support part 42 is an eye plate. Further, the peripheral part of the support part 42 is sandwiched between the first container part 11 and the second container part 12. Thereby, the support portion 42 is fixed to the container 10. The support part 42, the first container part 11, and the second container part 12 are fixed by, for example, four screws.
　本実施の形態において、本体部４１は、検体から核酸抽出対象物（微生物）をろ過して捕捉するためのフィルタ部である。具体的には、本体部４１は、核酸抽出対象物の大きさより小さい微細な孔（目）を複数有するフィルタである。これにより。本体部４１（フィルタ）によって、核酸抽出対象物を確実に捕捉することができる。本体部４１の孔径は、例えば、０．４５μｍである。本体部４１で捕捉された核酸抽出対象物は、本体部４１上に保持される。また、本実施の形態では、核酸抽出対象物から抽出された核酸を本体部４１に通過させるので、本体部４１の孔の大きさは核酸の大きさよりも大きくしておくとよい。 In the present embodiment, the main body 41 is a filter unit for filtering and capturing a nucleic acid extraction target (microorganism) from a specimen. Specifically, the main body 41 is a filter having a plurality of fine holes (eyes) smaller than the size of the nucleic acid extraction target. By this. The main body 41 (filter) can reliably capture the nucleic acid extraction target. The hole diameter of the main body 41 is, for example, 0.45 μm. The nucleic acid extraction target captured by the main body 41 is held on the main body 41. In this embodiment, since the nucleic acid extracted from the nucleic acid extraction target is passed through the main body 41, the size of the hole of the main body 41 is preferably larger than the size of the nucleic acid.
　本体部４１としては、酢酸セルロース、ポリフッ化ビニリデン（ＰＶＤＦ：ｐｏｌｙｖｉｎｙｌｉｄｅｎｅ　ｄｉｆｌｕｏｒｉｄｅ）、ポリエーテルサルフォン（ＰＥＳ：ｐｏｌｙｅｔｈｅｒｓｕｌｆｏｎｅ）、セルロースアセテート等の材質で作られたメンブレンフィルタ等を用いることができる。また、本体部４１としては、検体に含まれる核酸抽出対象物を吸着させる機能を有する捕捉フィルタを用いてもよい。このような捕捉フィルタを用いることにより、検体から核酸抽出対象物を捕捉する速度を向上させることができる。なお、本実施の形態において、本体部４１（フィルタ）は、平面状のフィルタであるが、これに限るものではなく、円筒状等のその他の形状のフィルタであってもよい。 As the main body portion 41, a membrane filter made of a material such as cellulose acetate, polyvinylidene fluoride (PVDF), polyethersulfone (PES), cellulose acetate, or the like can be used. As the main body 41, a capture filter having a function of adsorbing a nucleic acid extraction target contained in a specimen may be used. By using such a capture filter, the speed at which the nucleic acid extraction target is captured from the specimen can be improved. In the present embodiment, the main body 41 (filter) is a planar filter, but is not limited to this, and may be a filter having another shape such as a cylindrical shape.
　支持部４２の少なくとも一部は、アルミニウム等の金属材料又は高熱伝導性樹脂によって構成されているとよい。この場合、支持部４２は、全部が金属材料又は高熱伝導性樹脂によって構成されていてもよい。支持部４２の一部に金属材料を用いる場合、樹脂内に金属材料が埋め込まれたものでもよい。また、支持部４２の表面は、親水性であるとよい。 At least a part of the support portion 42 may be made of a metal material such as aluminum or a high thermal conductive resin. In this case, the support part 42 may be entirely made of a metal material or a high thermal conductive resin. When a metal material is used for a part of the support portion 42, the metal material may be embedded in the resin. Moreover, the surface of the support part 42 is good to be hydrophilic.
　真空ポンプ２は、容器１０内の圧力を調整するための圧力調整部の一例である。本実施の形態において、真空ポンプ２の配管は、第２容器部１２に接続されている。したがって、捕捉部４０の上に液体が溜まっていて第２容器部１２の内部空間が密閉されている場合、真空ポンプ２は、第２容器部１２内の圧力を調整する。容器１０内又は第２容器部１２内の圧力は、真空ポンプ２の配管に設けられたリークバルブ６によって調整することができる。つまり、真空ポンプ２による減圧度は、リークバルブ６によって調整することができる。 The vacuum pump 2 is an example of a pressure adjusting unit for adjusting the pressure in the container 10. In the present embodiment, the piping of the vacuum pump 2 is connected to the second container portion 12. Therefore, when the liquid is accumulated on the capturing part 40 and the internal space of the second container part 12 is sealed, the vacuum pump 2 adjusts the pressure in the second container part 12. The pressure in the container 10 or the second container part 12 can be adjusted by the leak valve 6 provided in the piping of the vacuum pump 2. That is, the degree of pressure reduction by the vacuum pump 2 can be adjusted by the leak valve 6.
　また、本実施の形態において、真空ポンプ２の配管は、第２排出口３２に接続されている。つまり、真空ポンプ２の減圧口（減圧吸引口）は、第２容器部１２に設けられた第２排出口３２を兼ねている。 In the present embodiment, the piping of the vacuum pump 2 is connected to the second discharge port 32. That is, the decompression port (decompression suction port) of the vacuum pump 2 also serves as the second discharge port 32 provided in the second container portion 12.
　振動装置３は、容器１０を振動させるための機能を有する。振動装置３によって容器１０を振動させることで、容器１０内の液体を撹拌させることができる。例えば、核酸抽出対象物が含まれる核酸抽出試薬を撹拌させることで核酸抽出反応を効果的に行うことができる。なお、振動装置３は、反応ボックス４に接触するように設けられている。 The vibration device 3 has a function for vibrating the container 10. By vibrating the container 10 by the vibration device 3, the liquid in the container 10 can be stirred. For example, the nucleic acid extraction reaction can be effectively performed by stirring the nucleic acid extraction reagent containing the nucleic acid extraction target. The vibration device 3 is provided so as to contact the reaction box 4.
　［核酸抽出方法］
　次に、核酸抽出装置１００を用いた核酸抽出方法について、図１を参照しながら、図５及び図６Ａ～図６Ｇを用いて説明する。図５は、実施の形態に係る核酸抽出方法のフローチャートである。図６Ａ～図６Ｇは、実施の形態に係る核酸抽出方法を説明するための模式断面図である。なお、図６Ａ～図６Ｇにおいては、第１注入口２１及び第２注入口２２（ゴム栓２２ａ）については、検体２１０及び核酸抽出試薬の液の流れを分かりやすくするために正しい位置に図示されていない。 [Nucleic acid extraction method]
Next, a nucleic acid extraction method using the nucleic acid extraction apparatus 100 will be described with reference to FIG. 1 and FIGS. 5 and 6A to 6G. FIG. 5 is a flowchart of the nucleic acid extraction method according to the embodiment. 6A to 6G are schematic cross-sectional views for explaining the nucleic acid extraction method according to the embodiment. 6A to 6G, the first injection port 21 and the second injection port 22 (rubber plug 22a) are illustrated in the correct positions for easy understanding of the flow of the liquid of the specimen 210 and the nucleic acid extraction reagent. Not.
　本実施の形態における核酸抽出方法は、核酸抽出ユニット１を用いて検体に含まれる核酸抽出対象物から核酸を抽出する方法であり、図５に示すように、少なくとも、捕捉工程Ｓ１と、核酸抽出工程Ｓ２と、回収工程Ｓ３とを含む。 The nucleic acid extraction method according to the present embodiment is a method of extracting nucleic acid from a nucleic acid extraction target contained in a specimen using the nucleic acid extraction unit 1, and as shown in FIG. 5, at least a capture step S1 and nucleic acid extraction. Process S2 and collection | recovery process S3 are included.
　捕捉工程Ｓ１は、捕捉部４０に検体を通過させることによって検体に含まれる核酸抽出対象物を捕捉部４０で捕捉する工程である。 The capturing step S1 is a step of capturing the nucleic acid extraction target contained in the sample by the capturing unit 40 by passing the sample through the capturing unit 40.
　核酸抽出工程Ｓ２は、捕捉工程Ｓ１の後、捕捉部４０に核酸抽出試薬を導入することによって捕捉部４０で捕捉した核酸抽出対象物から核酸を抽出する工程である。 The nucleic acid extraction step S2 is a step of extracting the nucleic acid from the nucleic acid extraction target captured by the capturing unit 40 by introducing a nucleic acid extraction reagent into the capturing unit 40 after the capturing step S1.
　回収工程Ｓ３は、核酸抽出工程Ｓ２の後、核酸抽出工程Ｓ２で抽出した核酸を含む核酸抽出液を、捕捉部４０を通過させて回収する工程である。 The recovery step S3 is a step of recovering the nucleic acid extract containing the nucleic acid extracted in the nucleic acid extraction step S2 through the capture unit 40 after the nucleic acid extraction step S2.
　以下、図６Ａ～図６Ｇを用いて、具体的な核酸抽出方法について説明する。 Hereinafter, a specific nucleic acid extraction method will be described with reference to FIGS. 6A to 6G.
　　［捕捉工程］
　捕捉工程Ｓ１では、まず、図６Ａに示すように、核酸抽出ユニット１に検体２１０を注入する。具体的には、核酸抽出対象物２２０を含む検体２１０（検体原液）が入った検体投入カップ２００を核酸抽出装置１００（図１参照）の所定の位置に設置して、検体投入カップ２００から容器１０に検体２１０に投入する。検体２１０を容器１０に投入する際、第１排出口３１に接続された配管に設けられたバルブ５ａは閉じられている。バルブ５ａは、例えば図１における送液ポンプ５に備えられているが、これに限るものではない。 [Capture process]
In the capture step S1, first, a specimen 210 is injected into the nucleic acid extraction unit 1 as shown in FIG. 6A. Specifically, the sample input cup 200 containing the sample 210 (sample stock solution) including the nucleic acid extraction object 220 is placed at a predetermined position of the nucleic acid extraction apparatus 100 (see FIG. 1), and the container is removed from the sample input cup 200. 10 into the specimen 210. When the sample 210 is put into the container 10, the valve 5 a provided in the pipe connected to the first discharge port 31 is closed. The valve 5a is provided, for example, in the liquid feed pump 5 in FIG. 1, but is not limited thereto.
　検体２１０は、例えば、微生物を含む試料を滅菌希釈水等に懸濁することで作製することができる。検体２１０の溶液量は、例えば、１０ｍｌ～５００ｍｌであるが、これに限るものではない。なお、検体２１０に対しては、必要に応じて、固形成分を除去する等の前処理を行ってもよい。 The specimen 210 can be produced, for example, by suspending a sample containing microorganisms in sterilized diluted water or the like. The amount of solution of the specimen 210 is, for example, 10 ml to 500 ml, but is not limited thereto. Note that the specimen 210 may be subjected to a pretreatment such as removing a solid component as necessary.
　検体投入カップ２００から投入される検体２１０は、第１注入口２１を介して第１容器部１１に注入され、捕捉部４０を通過して第２容器部１２に流れる。 The specimen 210 introduced from the specimen insertion cup 200 is injected into the first container section 11 through the first injection port 21, passes through the capturing section 40 and flows into the second container section 12.
　このとき、検体２１０は、捕捉部４０によって濃縮される。つまり、検体２１０を捕捉部４０に通過させることで検体２１０を濃縮することができる。 At this time, the specimen 210 is concentrated by the capturing unit 40. That is, the specimen 210 can be concentrated by passing the specimen 210 through the capturing unit 40.
　具体的には、検体２１０が捕捉部４０を通過する際、検体２１０に含まれる核酸抽出対象物２２０が捕捉部４０に捕捉される。本実施の形態において、検体２１０に含まれる核酸抽出対象物２２０は、捕捉部４０の本体部４１（フィルタ）を通過する際に本体部４１に捕捉されて本体部４１の上に留まる。 Specifically, when the sample 210 passes through the capturing unit 40, the nucleic acid extraction target 220 contained in the sample 210 is captured by the capturing unit 40. In the present embodiment, the nucleic acid extraction target 220 contained in the specimen 210 is captured by the main body 41 and passes over the main body 41 when passing through the main body 41 (filter) of the capturing unit 40.
　図６Ａに示すように、捕捉部４０を通過した検体２１０（ろ液）は、廃液として第２排出口３２を介して第２容器部１２から排出され、核酸抽出装置１００（図１参照）の所定の位置に設置された回収容器５００に回収される。 As shown in FIG. 6A, the specimen 210 (filtrate) that has passed through the capture unit 40 is discharged from the second container unit 12 through the second discharge port 32 as a waste solution, and the nucleic acid extraction apparatus 100 (see FIG. 1). It is collected in a collection container 500 installed at a predetermined position.
　このとき、本実施の形態では、吸引ろ過によって検体２１０に含まれる核酸抽出対象物２２０を捕捉部４０で捕捉している。具体的には、真空ポンプ２によって第１排出口３１から容器１０内の空気を排気（吸引）することで第２容器部１２内を減圧して、第１容器部１１内の圧力を第２容器部１２内の圧力よりも高くする検体排出モードで第２容器部１２内の圧力を調整している。このように容器１０内の圧力を調整することで、容器１０内に注入された検体２１０は、吸引されながら捕捉部４０でろ過される。これにより、第１容器部１１に注入された検体２１０を速やかに捕捉部４０を通過させて第２容器部１２に移動させることができるとともに、検体２１０の廃液を速やかに第２容器部１２から排出させることができる。したがって、効率良く検体２１０を濃縮することができるとともに、検体２１０の廃液を速やかに容器１０から排出させることができる。 At this time, in this embodiment, the nucleic acid extraction target 220 contained in the specimen 210 is captured by the capturing unit 40 by suction filtration. Specifically, the air in the container 10 is exhausted (suctioned) from the first discharge port 31 by the vacuum pump 2 to depressurize the second container part 12, and the pressure in the first container part 11 is set to the second pressure. The pressure in the second container portion 12 is adjusted in the specimen discharge mode in which the pressure is higher than the pressure in the container portion 12. By adjusting the pressure in the container 10 in this way, the specimen 210 injected into the container 10 is filtered by the capturing unit 40 while being sucked. Thus, the specimen 210 injected into the first container section 11 can be quickly passed through the capturing section 40 and moved to the second container section 12, and the waste liquid of the specimen 210 can be promptly removed from the second container section 12. It can be discharged. Therefore, the specimen 210 can be concentrated efficiently, and the waste liquid of the specimen 210 can be quickly discharged from the container 10.
　なお、検体２１０を濃縮する際は、さらに、検体２１０を上部から加圧するとよい。これにより、さらに効率良く検体２１０を濃縮することができる。例えば、検体投入カップ２００内の検体２１０の上部の空間領域を加圧するとよい。 In addition, when concentrating the specimen 210, the specimen 210 may be further pressurized from above. Thereby, the specimen 210 can be more efficiently concentrated. For example, the space region above the specimen 210 in the specimen insertion cup 200 may be pressurized.
　このように、検体２１０を容器１０に注入して排出させると、図６Ｂに示すように、捕捉部４０の本体部４１の上には、本体部４１で捕捉された核酸抽出対象物２２０が溜まることになる。 In this way, when the specimen 210 is injected into the container 10 and discharged, the nucleic acid extraction target 220 captured by the main body 41 is accumulated on the main body 41 of the capturing unit 40 as shown in FIG. 6B. It will be.
　このようにして、捕捉工程Ｓ１では、捕捉部４０に検体２１０を通過させることによって検体２１０に含まれる核酸抽出対象物２２０を捕捉部４０で捕捉させている。 Thus, in the capturing step S1, the nucleic acid extraction target 220 contained in the sample 210 is captured by the capturing unit 40 by allowing the sample 210 to pass through the capturing unit 40.
　なお、検体２１０を捕捉部４０に通過させた後は、必要に応じて、さらに滅菌希釈水を第１注入口２１等から第１容器部１１に導入して捕捉部４０を通過させることで核酸抽出対象物２２０を洗浄することができる。 In addition, after passing the specimen 210 through the capture unit 40, if necessary, further introducing sterile diluted water from the first injection port 21 or the like into the first container unit 11 and allowing the nucleic acid to pass through the capture unit 40. The extraction object 220 can be washed.
　　［核酸抽出工程］
　核酸抽出工程Ｓ２では、捕捉部４０で捕捉した核酸抽出対象物２２０から核酸２３０を抽出する。 [Nucleic acid extraction step]
In the nucleic acid extraction step S2, the nucleic acid 230 is extracted from the nucleic acid extraction target 220 captured by the capturing unit 40.
　具体的には、まず、図６Ｃに示すように、核酸抽出装置１００（図１参照）の所定の位置に設置された核酸抽出試薬容器３００から容器１０に核酸抽出試薬３１０を投入する。核酸抽出試薬３１０としては、例えば、簡易ＤＮＡ抽出キット（株式会社カネカ）又はセルイーズ（Ｂｉｏｃｏｓｍ株式会社）を用いることができる。注入する核酸抽出試薬３１０の量は、例えば５０μｌ～２００μｌである。なお、核酸抽出試薬３１０を容器１０に投入する際、第１排出口３１に接続された配管のバルブ５ａは閉じられたままである。 Specifically, first, as shown in FIG. 6C, the nucleic acid extraction reagent 310 is put into the container 10 from the nucleic acid extraction reagent container 300 installed at a predetermined position of the nucleic acid extraction apparatus 100 (see FIG. 1). As the nucleic acid extraction reagent 310, for example, a simple DNA extraction kit (Kaneka Corporation) or Cellize (Biocosm Corporation) can be used. The amount of the nucleic acid extraction reagent 310 to be injected is, for example, 50 μl to 200 μl. Note that when the nucleic acid extraction reagent 310 is put into the container 10, the valve 5a of the pipe connected to the first outlet 31 remains closed.
　核酸抽出試薬容器３００から投入される核酸抽出試薬３１０は、第２注入口２２を介して第１容器部１１に注入される。具体的には、核酸抽出試薬容器３００の針を第２注入口２２（貫通孔）に設けられたゴム栓２２ａに貫通させて、核酸抽出試薬容器３００から第１容器部１１内に核酸抽出試薬３１０を注入する。なお、核酸抽出試薬３１０を容器１０に注入する前に、死菌の不活性処理試薬等を容器１０内に注入してもよい。 The nucleic acid extraction reagent 310 introduced from the nucleic acid extraction reagent container 300 is injected into the first container part 11 through the second injection port 22. Specifically, the needle of the nucleic acid extraction reagent container 300 is passed through a rubber stopper 22a provided in the second inlet 22 (through hole), and the nucleic acid extraction reagent is inserted from the nucleic acid extraction reagent container 300 into the first container portion 11. Inject 310. In addition, before injecting the nucleic acid extraction reagent 310 into the container 10, a dead bacteria inactive treatment reagent or the like may be injected into the container 10.
　容器１０内に核酸抽出試薬３１０を注入して静置すると、捕捉部４０に捕捉された核酸抽出対象物２２０が核酸抽出試薬３１０に反応する。これにより、核酸抽出対象物２２０から核酸２３０を抽出することができる。つまり、核酸抽出対象物２２０から核酸２３０が抽出される核酸抽出反応が行われる。つまり、核酸抽出対象物２２０の細胞膜と核酸２３０とが分離される。具体的には、図６Ｄに示すように、核酸抽出対象物２２０から核酸抽出試薬３１０に核酸２３０が溶出し、核酸２３０が含まれる核酸抽出液３２０が生成される。 When the nucleic acid extraction reagent 310 is injected into the container 10 and allowed to stand, the nucleic acid extraction target 220 captured by the capturing unit 40 reacts with the nucleic acid extraction reagent 310. Thereby, the nucleic acid 230 can be extracted from the nucleic acid extraction object 220. That is, a nucleic acid extraction reaction in which the nucleic acid 230 is extracted from the nucleic acid extraction target 220 is performed. That is, the cell membrane of the nucleic acid extraction target 220 and the nucleic acid 230 are separated. Specifically, as shown in FIG. 6D, the nucleic acid 230 is eluted from the nucleic acid extraction target 220 to the nucleic acid extraction reagent 310, and a nucleic acid extract 320 containing the nucleic acid 230 is generated.
　なお、核酸抽出試薬３１０を容器１０に注入して核酸抽出対象物２２０と核酸抽出試薬３１０とを混合させた後、適宜加熱してもよい。これにより、核酸抽出試薬３１０と核酸抽出対象物２２０との反応を促進させて核酸２３０を効率良く溶出させることができるので、核酸２３０の抽出を効率良く行うことができる。この場合、例えば、図１に示される加熱冷却ユニット７によって容器１０を加熱することで核酸抽出試薬３１０を加熱することができる。加熱冷却ユニット７は、例えば、核酸抽出装置１００の反応ボックス４に設けられており、例えば容器１０及び捕捉部４０の支持部４２に接続されている。加熱冷却ユニット７としては、例えばペルチェ素子等を用いることができる。 Note that the nucleic acid extraction reagent 310 may be injected into the container 10 to mix the nucleic acid extraction target 220 and the nucleic acid extraction reagent 310, and then appropriately heated. Thereby, since the reaction between the nucleic acid extraction reagent 310 and the nucleic acid extraction target 220 can be promoted and the nucleic acid 230 can be efficiently eluted, the nucleic acid 230 can be extracted efficiently. In this case, for example, the nucleic acid extraction reagent 310 can be heated by heating the container 10 by the heating / cooling unit 7 shown in FIG. The heating / cooling unit 7 is provided, for example, in the reaction box 4 of the nucleic acid extraction apparatus 100, and is connected to, for example, the container 10 and the support part 42 of the capturing part 40. As the heating / cooling unit 7, for example, a Peltier element or the like can be used.
　また、核酸抽出対象物２２０と核酸抽出試薬３１０とを混合させた後に、振動装置３によって容器１０を振動させて核酸抽出試薬３１０を撹拌させるとよい。これにより、核酸２３０の抽出をさらに効率良く行うことができる。 In addition, after mixing the nucleic acid extraction target 220 and the nucleic acid extraction reagent 310, the container 10 may be vibrated by the vibration device 3 to stir the nucleic acid extraction reagent 310. Thereby, the nucleic acid 230 can be extracted more efficiently.
　このように、本実施の形態では、捕捉部４０に核酸抽出試薬３１０を導入することによって核酸抽出対象物２２０の核酸抽出反応が行われ、核酸抽出対象物２２０から核酸２３０が抽出される。これにより、抽出された核酸２３０を含む核酸抽出液３２０が生成される。 Thus, in this embodiment, the nucleic acid extraction target 220 is subjected to the nucleic acid extraction reaction by introducing the nucleic acid extraction reagent 310 into the capturing unit 40, and the nucleic acid 230 is extracted from the nucleic acid extraction target 220. As a result, a nucleic acid extract 320 containing the extracted nucleic acid 230 is generated.
　このとき、図６Ｄに示すように、核酸抽出対象物２２０の核酸抽出反応は、容器１０に注入された核酸抽出試薬３１０を捕捉部４０の上に保持させて行うとよい。核酸抽出試薬３１０を捕捉部４０の上に保持させることで、核酸抽出試薬３１０が捕捉部４０の上に滞留する。この結果、核酸抽出対象物２２０が核酸抽出試薬３１０に一定時間浸されることになる。これにより、核酸２３０を溶出させやすくできるので、核酸２３０の抽出（溶出）を効率良く行うことができる。 At this time, as shown in FIG. 6D, the nucleic acid extraction reaction of the nucleic acid extraction target 220 may be performed by holding the nucleic acid extraction reagent 310 injected into the container 10 on the capturing unit 40. By holding the nucleic acid extraction reagent 310 on the capture unit 40, the nucleic acid extraction reagent 310 stays on the capture unit 40. As a result, the nucleic acid extraction object 220 is immersed in the nucleic acid extraction reagent 310 for a certain period of time. Thereby, since the nucleic acid 230 can be easily eluted, the extraction (elution) of the nucleic acid 230 can be performed efficiently.
　核酸抽出試薬３１０を捕捉部４０の上に保持させる場合、真空ポンプ２は、核酸抽出試薬３１０を捕捉部４０の上に保持させる核酸抽出試薬保持モードで第２容器部１２内の圧力を調整するとよい。具体的には、真空ポンプ２によって第２容器部１２内を加圧して第２容器部１２内の圧力を第１容器部１１内の圧力よりも高くすればよい。これにより、容器１０に注入された核酸抽出試薬３１０を捕捉部４０上に保持させることができる。 When the nucleic acid extraction reagent 310 is held on the capture unit 40, the vacuum pump 2 adjusts the pressure in the second container unit 12 in the nucleic acid extraction reagent holding mode in which the nucleic acid extraction reagent 310 is held on the capture unit 40. Good. Specifically, the inside of the second container part 12 may be pressurized by the vacuum pump 2 so that the pressure in the second container part 12 is higher than the pressure in the first container part 11. Thereby, the nucleic acid extraction reagent 310 injected into the container 10 can be held on the capture unit 40.
　この場合、捕捉部４０の本体部４１としては、通常時（第２容器部１２を加圧していない時）に核酸抽出試薬３１０が本体部４１を通過するようなフィルタを用いることができる。 In this case, as the main body 41 of the capturing unit 40, a filter that allows the nucleic acid extraction reagent 310 to pass through the main body 41 during normal times (when the second container 12 is not pressurized) can be used.
　　［回収工程］
　核酸抽出試薬３１０の注入が完了して捕捉部４０上の核酸抽出対象物２２０の核酸抽出反応が完了した後は、真空ポンプ２による第２容器部１２内の加圧を停止することで、図６Ｅに示すように、捕捉部４０上に滞留している核酸抽出液３２０を、捕捉部４０を通過させて第２容器部１２に移動させることができる。例えば、リークバルブ６を開放することで、第２容器部１２内を常圧にしたり、検体排出モード時の減圧度よりも低い減圧度となるように第２容器部１２内の圧力を調整したりすればよい。 [Recovery process]
After the injection of the nucleic acid extraction reagent 310 is completed and the nucleic acid extraction reaction of the nucleic acid extraction target 220 on the capture unit 40 is completed, the pressurization in the second container unit 12 by the vacuum pump 2 is stopped, whereby FIG. As shown in 6E, the nucleic acid extract 320 remaining on the capture unit 40 can be moved to the second container unit 12 through the capture unit 40. For example, by opening the leak valve 6, the pressure in the second container portion 12 is adjusted so that the inside of the second container portion 12 is at a normal pressure, or the pressure reduction degree is lower than the pressure reduction degree in the specimen discharge mode. Just do it.
　なお、核酸抽出試薬３１０を捕捉部４０の上に保持させる方法としては、上記のような第２容器部１２内の圧力を調整する方法に限るものではない。例えば、核酸抽出試薬保持モードを実行する代わりに、捕捉部４０の本体部４１として、第２容器部１２内を減圧していない時に核酸抽出試薬３１０が本体部４１上に保持されるようなフィルタを用いることでも実現できる。 Note that the method for holding the nucleic acid extraction reagent 310 on the capturing unit 40 is not limited to the method for adjusting the pressure in the second container unit 12 as described above. For example, instead of executing the nucleic acid extraction reagent holding mode, a filter that holds the nucleic acid extraction reagent 310 on the main body 41 when the inside of the second container 12 is not decompressed as the main body 41 of the capturing unit 40. It can also be realized by using.
　この場合、図示しないが、捕捉部４０の上に保持させた核酸抽出試薬３１０によって捕捉部４０上の核酸抽出対象物２２０の核酸抽出反応が完了した後は、真空ポンプ２によって第２容器部１２内を減圧して第１容器部１１内の圧力を第２容器部１２内の圧力よりも高くすればよい。これにより、核酸抽出反応が完了して捕捉部４０上に滞留している核酸抽出液３２０を第２容器部１２に移動させることができる。つまり、圧力差を利用して核酸抽出液３２０を第２容器部１２に移動させることができる。このように、真空ポンプ２によって第２容器部１２内を減圧して第１容器部１１内の圧力を第２容器部１２内の圧力よりも高くすることで、捕捉部４０における核酸抽出液３２０を捕捉部４０を介して第２容器部１２に移動させる核酸抽出液移動モードで第２容器部１２内の圧力を調整するとよい。 In this case, although not shown, after the nucleic acid extraction reaction of the nucleic acid extraction target 220 on the capture unit 40 is completed by the nucleic acid extraction reagent 310 held on the capture unit 40, the second container unit 12 is used by the vacuum pump 2. What is necessary is just to decompress the inside and make the pressure in the 1st container part 11 higher than the pressure in the 2nd container part 12. FIG. Thereby, the nucleic acid extraction solution 320 that has completed the nucleic acid extraction reaction and stays on the capturing unit 40 can be moved to the second container unit 12. That is, the nucleic acid extract 320 can be moved to the second container part 12 using the pressure difference. Thus, the nucleic acid extract 320 in the capture unit 40 is reduced by reducing the pressure in the second container part 12 by the vacuum pump 2 so that the pressure in the first container part 11 is higher than the pressure in the second container part 12. It is good to adjust the pressure in the 2nd container part 12 in the nucleic acid extraction liquid movement mode which moves to the 2nd container part 12 via the capture | acquisition part 40.
　このとき、核酸抽出液移動モードにおける真空ポンプ２による減圧度は、上記の検体２１０を排出させる検体排出モードにおける真空ポンプ２による減圧度よりも低くするとよい。 At this time, the degree of decompression by the vacuum pump 2 in the nucleic acid extract transfer mode is preferably lower than the degree of decompression by the vacuum pump 2 in the specimen discharge mode for discharging the specimen 210.
　なお、検体排出モード及び核酸抽出液移動モードにおいて第２容器部１２に遠心力がかかるように制御してもよい。これにより、検体２１０及び核酸抽出液３２０を効果的に回収することができる。 In addition, you may control so that a centrifugal force may be applied to the 2nd container part 12 in a sample discharge | emission mode and a nucleic acid extract movement mode. Thereby, the specimen 210 and the nucleic acid extract 320 can be effectively recovered.
　次に、図６Ｆに示すように、核酸２３０が含まれる核酸抽出液３２０が全て第２容器部１２に移動した後は、図６Ｇに示すように、第１排出口３１に接続された配管のバルブ５ａを開けることによって核酸抽出液３２０を回収する。 Next, as shown in FIG. 6F, after all of the nucleic acid extract 320 containing the nucleic acid 230 has moved to the second container part 12, as shown in FIG. 6G, the pipe connected to the first outlet 31 is removed. The nucleic acid extract 320 is recovered by opening the valve 5a.
　具体的には、核酸２３０が含まれる核酸抽出液３２０を第１排出口３１を介して第２容器部１２から排出し、核酸抽出装置１００（図１参照）の所定の位置に設置された回収容器４００に核酸抽出液３２０を回収する。例えば、第２容器部１２内の核酸抽出液３２０は、送液ポンプ５によって回収容器４００に送液されて回収される。 Specifically, the nucleic acid extract 320 containing the nucleic acid 230 is discharged from the second container part 12 through the first discharge port 31, and the recovery set at a predetermined position of the nucleic acid extraction device 100 (see FIG. 1). The nucleic acid extract 320 is collected in the container 400. For example, the nucleic acid extract 320 in the second container portion 12 is sent to the collection container 400 by the liquid feed pump 5 and collected.
　その後、図示しないが、回収した核酸抽出液３２０を用いて核酸検査（核酸分析）を行う。この場合、核酸２３０を検査する方法に合わせて核酸抽出液３２０に核酸検査試薬を混合させて検査溶液を得る。例えば、ＰＣＲ法によって核酸検査を行う場合、核酸検査試薬として、ＰＣＲ試薬を用いる。ＰＣＲ試薬には、例えば、核酸２３０の量を蛍光発光強度で検査するための核酸染色蛍光試薬及び核酸を増幅させるための反応試薬等が含まれている。反応試薬は、例えば、ＰＣＲプライマやポリメラーゼ酵素、バッファー等である。 Thereafter, although not shown, a nucleic acid test (nucleic acid analysis) is performed using the collected nucleic acid extract 320. In this case, a nucleic acid test reagent is mixed with the nucleic acid extract 320 in accordance with the method for testing the nucleic acid 230 to obtain a test solution. For example, when performing a nucleic acid test by the PCR method, a PCR reagent is used as the nucleic acid test reagent. The PCR reagent includes, for example, a nucleic acid staining fluorescent reagent for examining the amount of the nucleic acid 230 with fluorescence emission intensity, a reaction reagent for amplifying the nucleic acid, and the like. The reaction reagent is, for example, a PCR primer, a polymerase enzyme, a buffer or the like.
　ＰＣＲ法による核酸検査は、例えば、検査流路であるマイクロ流路が形成された検査チップ（ＰＣＲチップ等）を用いて行うことができる。この場合、例えば、核酸抽出液３２０と核酸検査試薬との混合溶液を検査チップに導入してフローＰＣＲによって核酸２３０を増幅し、光学検出装置によって増幅された核酸２３０を測定することで、核酸抽出対象物２２０（微生物）を同定することができる。 The nucleic acid test by the PCR method can be performed using, for example, a test chip (PCR chip or the like) in which a micro flow channel that is a test flow channel is formed. In this case, for example, a mixed solution of the nucleic acid extract 320 and the nucleic acid test reagent is introduced into the test chip, the nucleic acid 230 is amplified by flow PCR, and the nucleic acid 230 amplified by the optical detection device is measured. The object 220 (microorganism) can be identified.
　このように、本実施の形態における核酸抽出方法は、核酸検査方法に利用することができる。この場合、ＰＣＲ法によって核酸２３０を検査することで、高感度及び高精度で核酸２３０の同定が可能となる。 Thus, the nucleic acid extraction method according to the present embodiment can be used for a nucleic acid test method. In this case, the nucleic acid 230 can be identified with high sensitivity and high accuracy by inspecting the nucleic acid 230 by the PCR method.
　［まとめ］
　以上、本実施の形態に係る核酸抽出装置１００によれば、検体２１０から核酸２３０を抽出するための核酸抽出ユニット１を備える。核酸抽出ユニット１は、容器１０と、容器１０内に検体を注入するための第１注入口２１と、容器１０内に配置され、第１注入口２１から注入された検体２１０に含まれる核酸抽出対象物２２０を捕捉する捕捉部４０と、捕捉部４０で捕捉された核酸抽出対象物２２０から核酸２３０を抽出するための核酸抽出試薬３１０を容器１０内に注入するための第２注入口２２と、核酸抽出試薬３１０によって核酸抽出対象物２２０から抽出された核酸２３０が含まれる核酸抽出液３２０を容器１０から排出するための第１排出口３１と、検体２１０を容器１０から排出するための第２排出口３２とを有する。そして、容器１０は、捕捉部４０を境界として容器１０の内部空間を分割する第１容器部１１と第２容器部１２とを有し、第１注入口２１及び第２注入口２２は、第１容器部１１に設けられており、第１排出口３１及び第２排出口３２は、第２容器部１２に設けられている。 [Summary]
As described above, the nucleic acid extraction apparatus 100 according to the present embodiment includes the nucleic acid extraction unit 1 for extracting the nucleic acid 230 from the specimen 210. The nucleic acid extraction unit 1 includes a container 10, a first inlet 21 for injecting a specimen into the container 10, and a nucleic acid extraction contained in the specimen 210 that is disposed in the container 10 and injected from the first inlet 21. A capture unit 40 that captures the object 220, and a second injection port 22 that injects a nucleic acid extraction reagent 310 for extracting the nucleic acid 230 from the nucleic acid extraction object 220 captured by the capture unit 40 into the container 10. The first outlet 31 for discharging the nucleic acid extract 320 containing the nucleic acid 230 extracted from the nucleic acid extraction target 220 by the nucleic acid extraction reagent 310 from the container 10 and the first outlet 31 for discharging the specimen 210 from the container 10. 2 outlets 32. And the container 10 has the 1st container part 11 and the 2nd container part 12 which divide | segment the interior space of the container 10 by making the capture | acquisition part 40 into a boundary, and the 1st inlet 21 and the 2nd inlet 22 are the 1st. The first discharge port 31 and the second discharge port 32 are provided in the second container portion 12.
　これにより、上部の第１容器部１１に検体２１０と核酸抽出試薬３１０とを注入することができるとともに、捕捉部４０を介して下部の第２容器部１２から廃液の検体２１０と核酸抽出液３２０とを排出することができる。これにより、同じ容器１０を用いて検体２１０の濃縮と核酸２３０の抽出とを行った上で核酸２３０を含む核酸抽出液３２０を簡便に回収することができる。また、核酸抽出工程中に容器を移し替えることなく作業を行うことができるので、核酸抽出液３２０に不純物が混入することを抑制できる。しかも、上部の第１容器部１１から下部の第２容器部１２に検体２１０が流れるので、検体２１０から核酸抽出対象物２２０を捕捉部４０で容易に捕捉することもできる。このように、本実施の形態によれば、作業性が簡便で、核酸抽出液３２０に不純物が混入するリスクを大幅に軽減することができる。これにより、高感度及び高精度で核酸２３０を抽出することができる。したがって、核酸抽出液３２０を用いた検査の精度を向上させることができる。 As a result, the specimen 210 and the nucleic acid extraction reagent 310 can be injected into the upper first container section 11, and the waste liquid specimen 210 and the nucleic acid extraction liquid 320 can be injected from the lower second container section 12 via the capturing section 40. And can be discharged. Thus, the nucleic acid extract 320 containing the nucleic acid 230 can be easily recovered after the sample 210 is concentrated and the nucleic acid 230 is extracted using the same container 10. In addition, since the operation can be performed without transferring the container during the nucleic acid extraction step, it is possible to prevent impurities from being mixed into the nucleic acid extract 320. In addition, since the specimen 210 flows from the upper first container section 11 to the lower second container section 12, the nucleic acid extraction target 220 can be easily captured by the capturing section 40 from the specimen 210. Thus, according to the present embodiment, the workability is simple and the risk of impurities being mixed into the nucleic acid extract 320 can be greatly reduced. Thereby, the nucleic acid 230 can be extracted with high sensitivity and high accuracy. Therefore, the accuracy of the inspection using the nucleic acid extract 320 can be improved.
　また、本実施の形態において、核酸抽出装置１００は、さらに、第２容器部１２内の圧力を調整するための圧力調整部として真空ポンプ２を備えている。また、捕捉部４０は、本体部４１として、核酸抽出対象物２２０より小さい孔を複数有するフィルタを有する。そして、第２排出口３２は、第１排出口３１よりも捕捉部４０に近い位置に設けられており、かつ、真空ポンプ２の減圧口を兼ねている。 Further, in the present embodiment, the nucleic acid extraction device 100 further includes the vacuum pump 2 as a pressure adjusting unit for adjusting the pressure in the second container unit 12. The capturing unit 40 includes a filter having a plurality of holes smaller than the nucleic acid extraction target 220 as the main body 41. The second discharge port 32 is provided at a position closer to the capturing unit 40 than the first discharge port 31 and also serves as a decompression port of the vacuum pump 2.
　これにより、検査に必要な核酸抽出液３２０と廃液となる検体２１０とを同じ第２容器部１２から排出する構成でありながらも、核酸抽出液３２０を効率良く回収することができる。 Thus, the nucleic acid extract 320 can be efficiently recovered even though the nucleic acid extract 320 necessary for the test and the sample 210 as the waste liquid are discharged from the same second container portion 12.
　また、本実施の形態において、真空ポンプ２は、第２容器部１２内を減圧して第１容器部１１内の圧力を第２容器部１２内の圧力よりも高くすることで、注入された検体２１０を第１容器部１１から捕捉部４０を通過させて第２容器部１２に移動させる検体排出モードと、第２容器部１２内を減圧して第１容器部１１内の圧力を第２容器部１２内の圧力よりも高くすることで、捕捉部４０における核酸抽出液３２０を捕捉部４０を介して第２容器部１２に移動させる核酸抽出液移動モードとで第２容器部１２内の圧力を調整する。この場合、核酸抽出液移動モードにおける真空ポンプ２による減圧度は、検体排出モードにおける真空ポンプ２による減圧度よりも低くしている。 Moreover, in this Embodiment, the vacuum pump 2 was inject | poured by decompressing the inside of the 2nd container part 12, and making the pressure in the 1st container part 11 higher than the pressure in the 2nd container part 12. A specimen discharge mode in which the specimen 210 is moved from the first container section 11 through the capturing section 40 to the second container section 12, and the pressure in the first container section 11 is reduced by reducing the pressure in the second container section 12. By making the pressure higher than the pressure in the container unit 12, the nucleic acid extract solution 320 in the capture unit 40 is moved to the second container unit 12 via the capture unit 40 in the nucleic acid extract solution movement mode. Adjust pressure. In this case, the degree of decompression by the vacuum pump 2 in the nucleic acid extract moving mode is lower than the degree of decompression by the vacuum pump 2 in the specimen discharge mode.
　これにより、簡便な構造で、効率良く核酸抽出液３２０を回収することができる。 Thereby, the nucleic acid extract 320 can be efficiently recovered with a simple structure.
　また、本実施の形態において、真空ポンプ２は、第２容器部１２内を加圧して第２容器部１２内の圧力を第１容器部１１内の圧力よりも高くすることで、容器１０に注入された核酸抽出試薬３１０を捕捉部４０上に保持させる核酸抽出試薬保持モードで第２容器部１２内の圧力を調整している。 Moreover, in this Embodiment, the vacuum pump 2 pressurizes the inside of the 2nd container part 12, and makes the container 10 the pressure in the 2nd container part 12 higher than the pressure in the 1st container part 11. The pressure in the second container unit 12 is adjusted in the nucleic acid extraction reagent holding mode in which the injected nucleic acid extraction reagent 310 is held on the capturing unit 40.
　これにより、第２容器部１２内の圧力を調整して捕捉部４０上に核酸抽出試薬３１０を保持させた状態で核酸抽出反応を行うことができるので、核酸抽出液３２０を効率良く回収することができる。 As a result, the nucleic acid extraction reaction can be performed in a state where the nucleic acid extraction reagent 310 is held on the capture unit 40 by adjusting the pressure in the second container unit 12, so that the nucleic acid extract 320 can be efficiently recovered. Can do.
　また、本実施の形態において、第２注入口２２は、第１容器部１１に設けられた貫通孔であり、貫通孔には、当該貫通孔を塞ぐゴム栓２２ａが設けられており、ゴム栓２２ａは、核酸抽出試薬３１０を第１容器部１１内に注入するための針が貫通可能なゴム材料によって構成されている。 Further, in the present embodiment, the second inlet 22 is a through hole provided in the first container portion 11, and the through hole is provided with a rubber plug 22a that closes the through hole. 22a is made of a rubber material through which a needle for injecting the nucleic acid extraction reagent 310 into the first container part 11 can penetrate.
　これにより、容器１０に注入された検体２１０及び核酸抽出試薬３１０が容器１０から蒸発したり攪拌時に容器１０から漏れたりすることを低減することができる。 Thereby, it is possible to reduce the specimen 210 and the nucleic acid extraction reagent 310 injected into the container 10 from being evaporated from the container 10 or leaking from the container 10 during stirring.
　また、本実施の形態において、捕捉部４０は、検体２１０に含まれる核酸抽出対象物２２０を捕捉して保持するための本体部４１と、本体部４１を支持する支持部４２とを有している。 In the present embodiment, the capturing unit 40 includes a main body 41 for capturing and holding the nucleic acid extraction target 220 contained in the specimen 210, and a support 42 for supporting the main body 41. Yes.
　これにより、本体部４１において検体２１０に含まれる核酸抽出対象物２２０を捕捉することができる。 Thereby, the nucleic acid extraction target 220 contained in the specimen 210 can be captured in the main body 41.
　この場合、支持部４２の少なくとも一部は、金属によって構成されているとよい。あるいは、支持部４２の少なくとも一部は、高熱伝導性樹脂によって構成されていてもよい。 In this case, at least a part of the support portion 42 may be made of metal. Or at least one part of the support part 42 may be comprised with highly heat conductive resin.
　これにより、支持部４２に接続された加熱冷却ユニット７によって、本体部４１上の検体２１０、核酸抽出対象物２２０又は核酸抽出試薬３１０を効率良く加熱したり冷却したりすることができる。 Thereby, the heating / cooling unit 7 connected to the support portion 42 can efficiently heat or cool the specimen 210, the nucleic acid extraction target 220 or the nucleic acid extraction reagent 310 on the main body portion 41.
　また、支持部４２が高熱伝導性樹脂によって構成されていて金属によって構成されていない場合には、金属そのもの又は金属の腐食によって検査が阻害されることを低減することができる。 Further, when the support portion 42 is made of a high thermal conductive resin and is not made of a metal, it is possible to reduce that the inspection is hindered by the metal itself or metal corrosion.
　また、捕捉部４０の支持部４２の表面は、親水性であるとよい。 Moreover, the surface of the support part 42 of the capturing part 40 may be hydrophilic.
　これにより、検体２１０から核酸抽出対象物２２０を捕捉する速度を向上させることができる。つまり、検体２１０から核酸抽出対象物２２０を捕捉するのに要する時間を短縮することができる。 Thereby, the speed at which the nucleic acid extraction target 220 is captured from the specimen 210 can be improved. That is, the time required to capture the nucleic acid extraction target 220 from the specimen 210 can be shortened.
　また、本実施の形態において、容器１０の少なくとも一部は、高熱伝導性樹脂によって構成されているとよい。 Further, in the present embodiment, at least a part of the container 10 may be made of a high thermal conductive resin.
　これにより、容器１０に接続された加熱冷却ユニット７によって、本体部４１上の検体２１０、核酸抽出対象物２２０又は核酸抽出試薬３１０を効率良く加熱したり冷却したりすることができる。また、容器１０として金属を用いないことで、金属そのもの又は金属の腐食によって検査が阻害されることを低減することができる。 Thereby, the sample 210, the nucleic acid extraction target 220 or the nucleic acid extraction reagent 310 on the main body 41 can be efficiently heated or cooled by the heating / cooling unit 7 connected to the container 10. Further, by not using a metal as the container 10, it is possible to reduce that the inspection is hindered by the metal itself or metal corrosion.
　また、本実施の形態において、第１容器部１１の内部空間の壁面における角部分の角度は、９０度以上であるとよい。 Moreover, in this Embodiment, the angle of the corner | angular part in the wall surface of the internal space of the 1st container part 11 is good in it being 90 degree | times or more.
　これにより、検体２１０及び核酸抽出試薬３１０を第１容器部１１に注入したときに、第１容器部１１の内部空間の壁面に検体２１０及び核酸抽出試薬３１０が留まることを抑制することができる。つまり、検体２１０及び核酸抽出試薬３１０の液ロスを低減できる。 Thereby, when the specimen 210 and the nucleic acid extraction reagent 310 are injected into the first container section 11, it is possible to suppress the specimen 210 and the nucleic acid extraction reagent 310 from staying on the wall surface of the internal space of the first container section 11. That is, the liquid loss of the specimen 210 and the nucleic acid extraction reagent 310 can be reduced.
　また、本実施の形態において、容器１０の内面は、非親水性であるとよい。 In the present embodiment, the inner surface of the container 10 may be non-hydrophilic.
　これにより、検体２１０及び核酸抽出試薬３１０を第１容器部１１に注入したときに、第１容器部１１の内部空間の壁面に検体２１０及び核酸抽出試薬３１０が付着することを抑制することができる。つまり、検体２１０及び核酸抽出試薬３１０の液ロスを低減できる。 Thereby, when the specimen 210 and the nucleic acid extraction reagent 310 are injected into the first container part 11, it is possible to suppress the specimen 210 and the nucleic acid extraction reagent 310 from adhering to the wall surface of the internal space of the first container part 11. . That is, the liquid loss of the specimen 210 and the nucleic acid extraction reagent 310 can be reduced.
　また、本実施の形態において、核酸抽出装置１００は、さらに、容器１０を振動させるための振動装置３を備えている。 Further, in the present embodiment, the nucleic acid extraction device 100 further includes a vibration device 3 for vibrating the container 10.
　これにより、核酸抽出試薬３１０と核酸抽出対象物２２０とを撹拌混合させることができるので核酸２３０の抽出を効率良く行うことができるとともに、第１容器部１１の内部空間の壁面に検体２１０及び核酸抽出試薬３１０が付着することを抑制することができる。 Thereby, since the nucleic acid extraction reagent 310 and the nucleic acid extraction target object 220 can be stirred and mixed, the nucleic acid 230 can be extracted efficiently, and the sample 210 and the nucleic acid are placed on the wall surface of the internal space of the first container portion 11. It can suppress that the extraction reagent 310 adheres.
　また、本実施の形態における核酸抽出方法は、捕捉部４０に検体２１０を通過させることによって検体２１０に含まれる核酸抽出対象物２２０を捕捉部４０で捕捉する捕捉工程Ｓ１と、捕捉部４０に核酸抽出試薬３１０を導入することによって捕捉部４０で捕捉した核酸抽出対象物２２０から核酸２３０を抽出する核酸抽出工程Ｓ２と、抽出した核酸２３０を含む核酸抽出液３２０を捕捉部４０を通過させて回収する回収工程Ｓ３とを含む。 In addition, the nucleic acid extraction method according to the present embodiment includes a capture step S1 in which the nucleic acid extraction target 220 contained in the specimen 210 is captured by the capture section 40 by passing the specimen 210 through the capture section 40, and a nucleic acid in the capture section 40. A nucleic acid extraction step S2 for extracting the nucleic acid 230 from the nucleic acid extraction target 220 captured by the capture unit 40 by introducing the extraction reagent 310, and a nucleic acid extract 320 containing the extracted nucleic acid 230 are passed through the capture unit 40 and recovered. Recovery step S3.
　これにより、作業性が簡便で、核酸抽出液３２０に不純物が混入するリスクを大幅に軽減することができる。したがって、高感度及び高精度で核酸２３０を抽出することができる。 Thereby, the workability is simple, and the risk of impurities being mixed into the nucleic acid extract 320 can be greatly reduced. Therefore, the nucleic acid 230 can be extracted with high sensitivity and high accuracy.
　（変形例）
　以上、本発明に係る核酸抽出装置１００、核酸抽出ユニット１及び核酸抽出方法等について、実施の形態に基づいて説明したが、本発明は、上記実施の形態に限定されるものではない。 (Modification)
As mentioned above, although the nucleic acid extraction apparatus 100, the nucleic acid extraction unit 1, the nucleic acid extraction method, etc. which concern on this invention were demonstrated based on embodiment, this invention is not limited to the said embodiment.
　例えば、上記実施の形態において、第２排出口３２を構成する管の第２容器部１２側の端部は、第２容器部１２の内壁と面一であったが、第２排出口３２を構成する管の第２容器部１２側の少なくとも一部を、第２容器部１２の内方に向かって第２容器部１２の内壁よりも突出させてもよい。 For example, in the above embodiment, the end of the tube constituting the second discharge port 32 on the second container part 12 side is flush with the inner wall of the second container part 12. At least a part of the constituting tube on the second container part 12 side may protrude from the inner wall of the second container part 12 toward the inside of the second container part 12.
　この場合、図７に示すように、第２排出口３２を構成する管の第２容器部１２側の端部の全周を第２容器部１２の内壁から突出させるように突出部１２ａを設けてもよいし、図８に示すように、第２排出口３２を構成する管の第２容器部１２側の端部の上側部分のみを第２容器部１２の内壁から突出させるように突出部１２ｂを設けてもよい。 In this case, as shown in FIG. 7, a projecting portion 12 a is provided so that the entire circumference of the end portion on the second container portion 12 side of the tube constituting the second discharge port 32 projects from the inner wall of the second container portion 12. Alternatively, as shown in FIG. 8, the protruding portion is formed so that only the upper portion of the end portion on the second container portion 12 side of the tube constituting the second discharge port 32 protrudes from the inner wall of the second container portion 12. 12b may be provided.
　このように、第２排出口３２を構成する管の第２容器部１２側の少なくとも一部を、第２容器部１２の内方に向かって第２容器部１２の内壁よりも突出させることで、第１容器部１１から第２容器部１２に核酸抽出液３２０を移動させるときに、核酸抽出液３２０が第２排出口３２に浸入することを抑制できる。つまり、突出部１２ａ及び１２ｂがひさしとして機能し、核酸抽出液３２０が第２排出口３２の管内に浸入することを抑制できる。したがって、核酸抽出液３２０を少ないロスで回収することができる。 In this way, by projecting at least a part of the pipe constituting the second discharge port 32 on the second container part 12 side toward the inside of the second container part 12 from the inner wall of the second container part 12. The nucleic acid extract 320 can be prevented from entering the second outlet 32 when the nucleic acid extract 320 is moved from the first container 11 to the second container 12. That is, the protrusions 12 a and 12 b function as eaves, and the nucleic acid extract 320 can be prevented from entering the tube of the second outlet 32. Therefore, the nucleic acid extract 320 can be recovered with little loss.
　また、上記実施の形態における核酸抽出方法において、検体に含まれる死菌を不活性化して除去する死菌除去工程を行ってもよい。死菌除去工程は、死菌を除去するための死菌除去試薬（例えば死菌の核酸の増幅を阻害する試薬）を捕捉部４０に導入することで死菌の核酸を反応不活化にする工程であり、捕捉工程Ｓ１の後に行われる。具体的には、死菌除去工程は、捕捉工程Ｓ１と核酸抽出工程Ｓ２との間に行われる。 In addition, in the nucleic acid extraction method in the above embodiment, a dead bacteria removing step of inactivating and removing dead bacteria contained in the specimen may be performed. The dead bacteria removing step is a step of inactivating the dead bacteria nucleic acid by introducing a dead bacteria removing reagent (for example, a reagent that inhibits amplification of dead bacteria nucleic acid) into the capturing unit 40 to remove the dead bacteria. And is performed after the capturing step S1. Specifically, the dead bacteria removal step is performed between the capture step S1 and the nucleic acid extraction step S2.
　この場合、核酸抽出対象物を捕捉部４０に捕捉した後、第１注入口２１又は第２注入口２２から死菌除去試薬を容器１０内に投入することで核酸抽出対象物微生物を捕捉した捕捉部４０上に死菌除去試薬を導入し、冷却しながら５分～２０分程度静置する。これにより、死菌を除去して生菌のみを捕捉部４０に集菌することができる。死菌除去試薬としては、例えば、タカラバイオ株式会社製の「Ｖｉａｂｌｅ　Ｂａｃｔｅｒｉａ　Ｓｅｌｅｃｔｉｏｎ　Ｋｉｔ　ｆｏｒ　ＰＣＲ」等を用いることができる。また、死菌除去試薬を捕捉部４０の本体部４１（フィルタ）内に浸透させるために、真空ポンプ２によって軽く吸引する工程や撹拌する工程を適宜追加してもよい。さらに、必要に応じて、死菌除去工程は数回繰り返してもよい。なお、死菌除去試薬は、死菌除去工程が終了した後に、本体部４１を通過させて第２排出口３２等から排出してもよい。このように、捕捉工程Ｓ１の後に死菌除去工程を行うことで、生菌の核酸のみを抽出することができる。 In this case, after the nucleic acid extraction target is captured by the capturing unit 40, the killing microorganism removing reagent is introduced into the container 10 from the first injection port 21 or the second injection port 22 to capture the nucleic acid extraction target microorganism. The dead bacteria removing reagent is introduced onto the part 40 and allowed to stand for 5 to 20 minutes while cooling. Thereby, dead bacteria can be removed and only living bacteria can be collected in the capturing part 40. As the dead bacteria removing reagent, for example, “Viable Bacteria Selection Kit for PCR” manufactured by Takara Bio Inc. can be used. Moreover, in order to infiltrate the dead bacteria removing reagent into the main body 41 (filter) of the capturing unit 40, a step of lightly sucking or stirring with the vacuum pump 2 may be added as appropriate. Furthermore, the dead bacteria removing step may be repeated several times as necessary. Note that the dead bacteria removing reagent may be discharged from the second discharge port 32 or the like through the main body 41 after the dead bacteria removing step is completed. Thus, only the nucleic acid of a living microbe can be extracted by performing a dead bacteria removal process after capture process S1.
　また、上記実施の形態において、容器１０は遮光部材によって構成されていたが、これに限るものではない。例えば、図９に示すように、容器１０の少なくとも一部が透明材料によって構成されていてもよい。 In the above embodiment, the container 10 is formed of a light shielding member, but is not limited thereto. For example, as shown in FIG. 9, at least a part of the container 10 may be made of a transparent material.
　このように、容器１０の一部を透明材料にすることによって、容器１０の内部を観察することが可能となったり、容器１０の内部に光を照射することが可能となったりする。この場合、例えば容器１０の内部に光を照射するための光照射ユニット８を反応ボックス４内又は反応ボックス４の周辺に設けることができる。光照射ユニット８は、例えば、上記の死菌除去工程で用いられる。具体的には、容器１０内に死菌除去試薬を導入して静置した後に光照射ユニット８によって光を照射することで、死菌と死菌除去試薬との反応を促進することができる。これにより、死菌の除去を効率良く行うことができる。 Thus, by making a part of the container 10 a transparent material, the inside of the container 10 can be observed, or the inside of the container 10 can be irradiated with light. In this case, for example, a light irradiation unit 8 for irradiating light inside the container 10 can be provided in the reaction box 4 or around the reaction box 4. The light irradiation unit 8 is used, for example, in the above killed bacteria removal step. Specifically, the reaction between the dead bacteria and the dead bacteria removing reagent can be promoted by irradiating the dead light with the light irradiation unit 8 after introducing the dead bacteria removing reagent into the container 10 and allowing it to stand. Thereby, dead bacteria can be removed efficiently.
　また、上記実施の形態では、容器１０に検体を注入するための第１注入口２１と容器１０に核酸抽出試薬を注入するための第２注入口２２とを別々に設けたが、これに限るものではない。また、核酸抽出液を容器１０から排出するための第１排出口３１と検体を容器１０から排出するための第２排出口３２とについても別々に設けたが、これに限るものではない。 In the above embodiment, the first injection port 21 for injecting the specimen into the container 10 and the second injection port 22 for injecting the nucleic acid extraction reagent into the container 10 are separately provided. It is not a thing. Moreover, although the 1st discharge port 31 for discharging | emitting a nucleic acid extract from the container 10 and the 2nd discharge port 32 for discharging | emitting a test substance from the container 10 were provided separately, it does not restrict to this.
　例えば、第１注入口２１と第２注入口２２とは、図１０に示すように、１つの注入口２３として構成されていてもよい。つまり、第１注入口２１と第２注入口２２とを兼用する１つの注入口２３が容器１０（第１容器部１１）に設けられていてもよい。注入口２３からは、検体が注入されたり核酸抽出試薬が注入されたりする。なお、検体２１０及び核酸抽出試薬３１０以外に、死菌除去試薬等のその他の液体を注入口２３から注入してもよい。 For example, the first inlet 21 and the second inlet 22 may be configured as one inlet 23 as shown in FIG. That is, one inlet 23 that serves as both the first inlet 21 and the second inlet 22 may be provided in the container 10 (first container portion 11). From the injection port 23, a specimen is injected or a nucleic acid extraction reagent is injected. In addition to the specimen 210 and the nucleic acid extraction reagent 310, other liquids such as a dead bacteria removal reagent may be injected from the injection port 23.
　また、第１排出口３１と第２排出口３２とについても、図１０に示すように、１つの排出口３３として構成されていてもよい。つまり、第１排出口３１と第２排出口３２とを兼用する１つの排出口３３が容器１０（第２容器部１２）に設けられていてもよい。排出口３３からは、核酸抽出液が排出されたり検体が排出されたりする。なお、核酸抽出液及び検体以外に、容器１０に導入されたその他の液体が排出口３３から排出されてもよい。 Also, the first outlet 31 and the second outlet 32 may be configured as one outlet 33 as shown in FIG. That is, one discharge port 33 that doubles as the first discharge port 31 and the second discharge port 32 may be provided in the container 10 (second container portion 12). From the outlet 33, the nucleic acid extract is discharged or the sample is discharged. In addition to the nucleic acid extract and the sample, other liquids introduced into the container 10 may be discharged from the discharge port 33.
　なお、図１０では、注入口２３及び排出口３３をいずれも１つとしたが、これに限るものではなく、どちらか一方を図６Ａ等に示すように２つに分けてもよい。 In addition, in FIG. 10, although the injection port 23 and the discharge port 33 were both one, it is not restricted to this, You may divide any one into two as shown to FIG. 6A etc.
　さらに、注入口２３及び排出口３３の各々が１つのみである場合、図１１に示すように、捕捉部４０の上流側に設けられた注入口２３には、バルブ６００を介して、検体投入カップ２００及び核酸抽出試薬容器３００が接続されていてもよい。これにより、バルブ６００を制御することで検体投入カップ２００からの検体と核酸抽出試薬容器３００からの核酸抽出試薬との流れを切り替えることができる。また、捕捉部４０の下流側に設けられた排出口３３には、バルブ７００を介して、回収容器４００（不図示）で核酸抽出液を回収するための配管５ｂと回収容器５００で検体の廃液（ろ液）を回収するための配管２ｂとが接続されていてもよい。これにより、バルブ７００を制御することで核酸抽出液と検体の廃液との流れを切り替えることができる。 Further, when each of the injection port 23 and the discharge port 33 is only one, as shown in FIG. 11, the sample is input to the injection port 23 provided on the upstream side of the capturing unit 40 via the valve 600. The cup 200 and the nucleic acid extraction reagent container 300 may be connected. Thus, by controlling the valve 600, the flow of the sample from the sample insertion cup 200 and the nucleic acid extraction reagent from the nucleic acid extraction reagent container 300 can be switched. In addition, the discharge port 33 provided on the downstream side of the capturing unit 40 is connected to a pipe 5b for collecting a nucleic acid extract in a collection container 400 (not shown) via a valve 700 and a sample waste liquid in the collection container 500. A pipe 2b for collecting (filtrate) may be connected. Thereby, by controlling the valve 700, the flow of the nucleic acid extract and the sample waste liquid can be switched.
　また、上記実施の形態では、検体２１０は捕捉部４０に直接通過させたが、これに限らない。例えば、検体２１０を捕捉部４０に通過させる前に、前処理として検体２１０をプレフィルタに通過させてもよい。一例として、検体投入カップ２００と第１注入口２１との間にプレフィルタを有する配管を挿入すればよい。このように、検体２１０を捕捉部４０に通過させる前にプレフィルタに通過させることで、サイズの大きい不要な成分を除去することができる。これにより、検体２１０からの核酸抽出対象物２２０の捕捉を効率的に行うことができる。 In the above embodiment, the specimen 210 is directly passed through the capturing unit 40, but the present invention is not limited to this. For example, the specimen 210 may be passed through a prefilter as a pretreatment before the specimen 210 is passed through the capturing unit 40. As an example, a pipe having a prefilter may be inserted between the specimen insertion cup 200 and the first inlet 21. In this manner, by passing the specimen 210 through the prefilter before passing through the capturing unit 40, unnecessary components having a large size can be removed. Thereby, capture of the nucleic acid extraction target 220 from the specimen 210 can be performed efficiently.
　その他、各実施の形態及び変形例に対して当業者が思いつく各種変形を施して得られる形態や、本発明の趣旨を逸脱しない範囲で実施の形態における構成要素及び機能を任意に組み合わせることで実現される形態も本発明に含まれる。 In addition, a form obtained by making various modifications conceived by those skilled in the art with respect to each embodiment and modification, and any combination of components and functions in the embodiment without departing from the spirit of the present invention Forms to be made are also included in the invention.
　１　核酸抽出ユニット
　２　真空ポンプ（圧力調整部）
　１０　容器
　１１　第１容器部
　１２　第２容器部
　２１　第１注入口
　２２　第２注入口
　２３　注入口（第１注入口、第２注入口）
　２２ａ　ゴム栓
　３１　第１排出口
　３２　第２排出口
　３３　排出口（第１排出口、第２排出口）
　４０　捕捉部
　４１　本体部
　４２　支持部
　１００　核酸抽出装置
　２１０　検体
　２２０　核酸抽出対象物
　２３０　核酸
　３１０　核酸抽出試薬
　３２０　核酸抽出液 1 Nucleic acid extraction unit 2 Vacuum pump (pressure adjustment unit)
DESCRIPTION OF SYMBOLS 10 Container 11 1st container part 12 2nd container part 21 1st inlet 22 2nd inlet 23 Injector (1st inlet, 2nd inlet)
22a Rubber plug 31 First discharge port 32 Second discharge port 33 Discharge port (first discharge port, second discharge port)
40 Nucleic Acid Extraction Device 230 Nucleic Acid Extraction Object 230 Nucleic Acid 310 Nucleic Acid Extraction Reagent 320 Nucleic Acid Extraction Solution

Claims (18)

  1. 　検体から核酸を抽出するための核酸抽出ユニットを備え、
    　前記核酸抽出ユニットは、
    　容器と、
    　前記容器内に検体を注入するための第１注入口と、
    　少なくとも一部が前記容器内に配置され、前記第１注入口から注入された検体に含まれる核酸抽出対象物を捕捉する捕捉部と、
    　前記捕捉部で捕捉された前記核酸抽出対象物から核酸を抽出するための核酸抽出試薬を前記容器内に注入するための第２注入口と、
    　前記核酸抽出試薬によって前記核酸抽出対象物から抽出された核酸が含まれる核酸抽出液を前記容器から排出するための第１排出口と、
    　前記検体を前記容器から排出するための第２排出口とを有し、
    　前記容器は、前記捕捉部を境界として当該容器の内部空間を分割する第１容器部と第２容器部とを有し、
    　前記第１注入口及び前記第２注入口は、前記第１容器部に設けられており、
    　前記第１排出口及び前記第２排出口は、前記第２容器部に設けられている
    　核酸抽出装置。     A nucleic acid extraction unit for extracting nucleic acid from a specimen;
    The nucleic acid extraction unit comprises:
    A container,
    A first inlet for injecting a specimen into the container;
    A capture unit that captures a nucleic acid extraction target contained in a sample that is at least partially disposed in the container and is injected from the first injection port;
    A second injection port for injecting a nucleic acid extraction reagent for extracting nucleic acid from the nucleic acid extraction target captured by the capturing unit into the container;
    A first outlet for discharging the nucleic acid extract containing the nucleic acid extracted from the nucleic acid extraction target by the nucleic acid extraction reagent from the container;
    A second outlet for discharging the specimen from the container;
    The container has a first container part and a second container part that divide the internal space of the container with the capture part as a boundary,
    The first inlet and the second inlet are provided in the first container part,
    The nucleic acid extraction apparatus, wherein the first discharge port and the second discharge port are provided in the second container part.
  2. 　さらに、前記第２容器部内の圧力を調整するための圧力調整部を備え、
    　前記捕捉部は、前記核酸抽出対象物より小さい孔を複数有するフィルタを有し、
    　前記第２排出口は、前記第１排出口よりも前記捕捉部に近い位置に設けられており、かつ、前記圧力調整部の減圧口を兼ねている
    　請求項１に記載の核酸抽出装置。     Furthermore, a pressure adjusting part for adjusting the pressure in the second container part is provided,
    The capture unit has a filter having a plurality of holes smaller than the nucleic acid extraction target,
    The nucleic acid extraction apparatus according to claim 1, wherein the second discharge port is provided at a position closer to the capturing unit than the first discharge port, and also serves as a decompression port of the pressure adjusting unit.
  3. 　前記圧力調整部は、前記第２容器部内を減圧して前記第１容器部内の圧力を前記第２容器部内の圧力よりも高くすることで、注入された前記検体を前記第１容器部から前記捕捉部を通過させて前記第２容器部に移動させる検体排出モードと、前記第２容器部内を減圧して前記第１容器部内の圧力を前記第２容器部内の圧力よりも高くすることで、前記捕捉部における前記核酸抽出液を前記捕捉部を介して前記第２容器部に移動させる核酸抽出液移動モードとで前記第２容器部内の圧力を調整し、
    　前記核酸抽出液移動モードにおける前記圧力調整部による減圧度は、前記検体排出モードにおける前記圧力調整部による減圧度よりも低い
    　請求項２に記載の核酸抽出装置。     The pressure adjusting unit depressurizes the inside of the second container part to make the pressure in the first container part higher than the pressure in the second container part, so that the injected specimen is removed from the first container part. A specimen discharge mode for passing through the capturing part and moving to the second container part, and reducing the pressure in the second container part to make the pressure in the first container part higher than the pressure in the second container part, Adjusting the pressure in the second container part in the nucleic acid extract moving mode in which the nucleic acid extract in the capturing part is moved to the second container part through the capturing part;
    The nucleic acid extraction device according to claim 2, wherein a degree of decompression by the pressure adjusting unit in the nucleic acid extract moving mode is lower than a degree of decompression by the pressure adjusting unit in the specimen discharge mode.
  4. 　前記圧力調整部は、前記第２容器部内を加圧して前記第２容器部内の圧力を前記第１容器部内の圧力よりも高くすることで、前記容器に注入された前記核酸抽出試薬を前記捕捉部上に保持させる核酸抽出試薬保持モードで前記第２容器部内の圧力を調整する
    　請求項２に記載の核酸抽出装置。     The pressure adjusting unit pressurizes the second container part to make the pressure in the second container part higher than the pressure in the first container part, thereby capturing the nucleic acid extraction reagent injected into the container The nucleic acid extraction device according to claim 2, wherein the pressure in the second container part is adjusted in a nucleic acid extraction reagent holding mode to be held on the part.
  5. 　前記第２排出口を構成する管の前記第２容器部側の少なくとも一部は、前記第２容器部の内方に向かって前記第２容器部の内壁よりも突出している
    　請求項２～４のいずれか１項に記載の核酸抽出装置。     5. At least a part of the pipe constituting the second discharge port on the second container part side protrudes inward of the second container part from the inner wall of the second container part. The nucleic acid extraction apparatus according to any one of the above.
  6. 　前記第２注入口は、前記第１容器部に設けられた貫通孔であり、
    　前記貫通孔には、当該貫通孔を塞ぐゴム栓が設けられており、
    　前記ゴム栓は、前記核酸抽出試薬を前記第１容器部内に注入するための針が貫通可能なゴム材料によって構成されている
    　請求項１～５のいずれか１項に記載の核酸抽出装置。     The second injection port is a through hole provided in the first container part,
    The through hole is provided with a rubber plug for closing the through hole,
    The nucleic acid extraction device according to any one of claims 1 to 5, wherein the rubber stopper is made of a rubber material that can be penetrated by a needle for injecting the nucleic acid extraction reagent into the first container portion.
  7. 　前記容器の少なくとも一部は、透明材料によって構成されている
    　請求項１～６のいずれか１項に記載の核酸抽出装置。     The nucleic acid extraction apparatus according to any one of claims 1 to 6, wherein at least a part of the container is made of a transparent material.
  8. 　前記捕捉部は、前記核酸抽出対象物を捕捉して保持するための本体部と、前記本体部を支持する支持部とを有する
    　請求項１～７のいずれか１項に記載の核酸抽出装置。     The nucleic acid extraction apparatus according to any one of claims 1 to 7, wherein the capturing unit includes a main body unit for capturing and holding the nucleic acid extraction target and a support unit for supporting the main body unit.
  9. 　前記支持部の少なくとも一部は、金属によって構成されている
    　請求項８に記載の核酸抽出装置。     The nucleic acid extraction device according to claim 8, wherein at least a part of the support portion is made of metal.
  10. 　前記支持部の少なくとも一部は、高熱伝導性樹脂によって構成されている
    　請求項８に記載の核酸抽出装置。     The nucleic acid extraction device according to claim 8, wherein at least a part of the support portion is made of a highly heat conductive resin.
  11. 　前記支持部の表面は、親水性である
    　請求項８～１０のいずれか１項に記載の核酸抽出装置。     The nucleic acid extraction device according to any one of claims 8 to 10, wherein a surface of the support portion is hydrophilic.
  12. 　前記容器の少なくとも一部は、高熱伝導性樹脂によって構成されている
    　請求項１～１１のいずれか１項に記載の核酸抽出装置。     The nucleic acid extraction apparatus according to any one of claims 1 to 11, wherein at least a part of the container is made of a high thermal conductive resin.
  13. 　前記第１容器部の内部空間の壁面における角部分の角度は、９０度以上である
    　請求項１～１２のいずれか１項に記載の核酸抽出装置。     The nucleic acid extraction apparatus according to any one of claims 1 to 12, wherein an angle of a corner portion of the wall surface of the internal space of the first container portion is 90 degrees or more.
  14. 　前記容器の内面は、非親水性である
    　請求項１～１３のいずれか１項に記載の核酸抽出装置。     The nucleic acid extraction apparatus according to any one of claims 1 to 13, wherein an inner surface of the container is non-hydrophilic.
  15. 　さらに、前記容器を振動させるための振動装置を備える
    　請求項１～１４のいずれか１項に記載の核酸抽出装置。     The nucleic acid extraction apparatus according to any one of claims 1 to 14, further comprising a vibration device for vibrating the container.
  16. 　検体から核酸を抽出するための核酸抽出装置に用いられる核酸抽出ユニットであって、
    　容器と、
    　前記容器内に検体を注入するための第１注入口と、
    　前記容器内に配置され、前記第１注入口から注入された検体に含まれる核酸抽出対象物を捕捉する捕捉部と、
    　前記捕捉部で捕捉された前記核酸抽出対象物から核酸を抽出するための核酸抽出試薬を前記容器内に注入するための第２注入口と、
    　前記核酸抽出試薬によって前記核酸抽出対象物から抽出された核酸が含まれる核酸抽出液を前記容器から排出するための第１排出口と、
    　前記検体を前記容器から排出するための第２排出口とを備え、
    　前記容器は、前記捕捉部を境界として当該容器の内部空間を分割するための第１容器部と第２容器部とを有し、
    　前記第１注入口及び前記第２注入口は、前記第１容器部に設けられており、
    　前記第１排出口及び前記第２排出口は、前記第２容器部に設けられている
    　核酸抽出ユニット。     A nucleic acid extraction unit used in a nucleic acid extraction apparatus for extracting nucleic acid from a specimen,
    A container,
    A first inlet for injecting a specimen into the container;
    A capture unit that is disposed in the container and captures a nucleic acid extraction target contained in a sample injected from the first injection port;
    A second injection port for injecting a nucleic acid extraction reagent for extracting nucleic acid from the nucleic acid extraction target captured by the capturing unit into the container;
    A first outlet for discharging the nucleic acid extract containing the nucleic acid extracted from the nucleic acid extraction target by the nucleic acid extraction reagent from the container;
    A second outlet for discharging the specimen from the container;
    The container has a first container part and a second container part for dividing the internal space of the container with the capture part as a boundary,
    The first inlet and the second inlet are provided in the first container part,
    The nucleic acid extraction unit, wherein the first discharge port and the second discharge port are provided in the second container part.
  17. 　捕捉部に検体を通過させることによって前記検体に含まれる核酸抽出対象物を前記捕捉部で捕捉する捕捉工程と、
    　前記捕捉部に核酸抽出試薬を導入することによって前記捕捉部で捕捉した前記核酸抽出対象物から核酸を抽出する核酸抽出工程と、
    　抽出した前記核酸を含む核酸抽出液を前記捕捉部を通過させて回収する回収工程とを含む
    　核酸抽出方法。     A capturing step of capturing the nucleic acid extraction target contained in the sample by the capturing unit by allowing the sample to pass through the capturing unit;
    A nucleic acid extraction step of extracting a nucleic acid from the nucleic acid extraction target captured by the capture unit by introducing a nucleic acid extraction reagent into the capture unit;
    And a recovery step of recovering the nucleic acid extract containing the extracted nucleic acid through the capture unit.
  18. 　さらに、前記捕捉工程の後に、前記捕捉部に死菌除去試薬を導入することで死菌の核酸を反応不活化する死菌除去工程を含む
    　請求項１７に記載の核酸抽出方法。     The nucleic acid extraction method according to claim 17, further comprising a dead bacteria removing step of inactivating the dead bacteria nucleic acid by introducing a dead bacteria removing reagent into the capturing section after the capturing step.
PCT/JP2017/001164 2016-03-10 2017-01-16 Nucleic acid extraction device, nucleic acid extraction unit, and nucleic acid extraction method WO2017154349A1 (en)

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