WO2019043779A1 - Nucleic acid extraction method and nucleic acid extraction device - Google Patents

Nucleic acid extraction method and nucleic acid extraction device Download PDF

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Publication number
WO2019043779A1
WO2019043779A1 PCT/JP2017/030906 JP2017030906W WO2019043779A1 WO 2019043779 A1 WO2019043779 A1 WO 2019043779A1 JP 2017030906 W JP2017030906 W JP 2017030906W WO 2019043779 A1 WO2019043779 A1 WO 2019043779A1
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Prior art keywords
nucleic acid
container
sample
acid extraction
internal space
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PCT/JP2017/030906
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French (fr)
Japanese (ja)
Inventor
裕示 三森
朋之 田口
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横河電機株式会社
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Priority to PCT/JP2017/030906 priority Critical patent/WO2019043779A1/en
Publication of WO2019043779A1 publication Critical patent/WO2019043779A1/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
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to a nucleic acid extraction method and a nucleic acid extraction apparatus.
  • nucleic acid In order to extract nucleic acid, it is necessary to collect a sample having nucleic acid including microorganisms, to break up (dissolve) the membrane structure of the sample, and release the contents of the sample to the outside of the sample.
  • Non-Patent Document 1 rice seeds are put in sterile water and treated with an ultrasonic cleaner, and pathogenic bacteria in a treatment solution are collected by a membrane filter method.
  • the filter is collected in sterile water in a test tube, treated again with an ultrasonic cleaner and separated.
  • the obtained sample solution is smeared on a selective medium and cultured to detect and recover pathogenic bacteria.
  • Toru Otani one other person, "Effective recovery method of viable bacteria and brown-row disease bacteria from rice seeds," [online], fiscal 2005, Kanto Tokai Hokuriku Agriculture Kanto Tokai, pests section, [Heisei June 28, '29 Search], Internet ⁇ URL: http: // www. naro. affrc. go. jp / org / narc / seika / kanto17 / 13 / 17_13_11. html>
  • Non-Patent Document 1 has a problem that it takes time and effort.
  • the present invention has been made in view of such circumstances, and an object thereof is to provide a nucleic acid extraction method and a nucleic acid extraction apparatus capable of efficiently extracting a nucleic acid from a sample having a nucleic acid such as a microorganism.
  • one mode of the present invention collects the sample which has nucleic acid using a membrane filter, the process of storing the membrane filter by which the sample was collected in the interior space of a container, membrane
  • a method for nucleic acid extraction comprising the steps of: sealing a container containing a filter; heating the container; and extracting nucleic acid from a sample.
  • a liquid containing water may be injected into the internal space of the heated container to extract nucleic acid into the liquid.
  • a liquid containing water may be contained in the inner space in the step of containing.
  • the liquid may contain at least one cell lysis promoter selected from the group consisting of alkalis, acids, enzymes, surfactants, redox agents and protein denaturants.
  • the surfactant may be sodium dodecyl sulfate and / or octylphenol ethoxylate.
  • the membrane filter may be hydrophilic.
  • the container after heating may be cooled in a sealed state to extract nucleic acid.
  • the container after heating in the extraction step, may be opened in a state where the pressure in the internal space is equal to or higher than the atmospheric pressure to extract the nucleic acid.
  • the container after cooling may be reheated to extract nucleic acids.
  • the liquid in the step of containing, may be injected after containing the membrane filter in which the sample is collected in the inner space.
  • the liquid in the extraction step, may be filled in the space between the container and the membrane filter on which the sample is collected.
  • the liquid comprises a first liquid and a second liquid, wherein the first liquid comprises water and a cell lysis promoter, and the second liquid is a first liquid. It is good also as a method which is higher boiling point.
  • One embodiment of the present invention includes a collection unit that collects a sample having a nucleic acid using a membrane filter, and an extraction unit that extracts a nucleic acid from the collected sample, and the extraction unit is the membrane filter.
  • a nucleic acid extraction device having a container having an internal space capable of being accommodated, means for accommodating a membrane filter in which a sample is collected, in the internal space, and a heating mechanism for heating the container in a sealed state.
  • the means for containing may have a means for injecting a liquid containing water into the internal space.
  • the material of the membrane filter may be hydrophilic.
  • the extraction unit may have a cooling mechanism, and the cooling mechanism may be configured to cool the container after heating in a sealed state.
  • the container may have an opening communicating with the internal space, and the extraction unit may have a closing member capable of closing the opening.
  • a detection unit that detects at least one of the temperature of the container and the pressure in the internal space, and a unit that determines opening / closing of the opening based on the detection result of the detection unit, It is good also as composition provided.
  • At least the drive unit is controlled based on the detection result of the drive unit that drives the closing member, the detection unit that detects at least one of the temperature of the container, and the pressure of the internal space And a control unit.
  • a nucleic acid extraction method and a nucleic acid extraction apparatus capable of efficiently extracting a nucleic acid from a sample having a nucleic acid such as a microorganism are provided.
  • FIG. 1 is a flowchart showing the nucleic acid extraction method of the first embodiment.
  • FIG. 2 is a schematic cross-sectional view showing the nucleic acid extraction device 101 of the first embodiment.
  • FIG. 3 is a schematic cross-sectional view showing the nucleic acid extraction device 102 of the second embodiment.
  • FIG. 4 is a schematic cross-sectional view showing the nucleic acid extraction device 103 of the third embodiment.
  • FIG. 5 is a schematic cross-sectional view showing the nucleic acid extraction device 104 of the fourth embodiment.
  • FIG. 6 is a schematic cross-sectional view showing the nucleic acid extraction device 105 of the fifth embodiment.
  • FIG. 1 is a flowchart showing the nucleic acid extraction method of the first embodiment.
  • a step S1 of culturing a sample having a nucleic acid and preparing a culture solution a step S2 of collecting the sample using a membrane filter, and a sample It has the process S3 which stores the collected membrane filter in the interior space of a container, the process S4 which seals the container in which the membrane filter was stored, and the process S5 which heats a container and extracts nucleic acid from a sample.
  • FIG. 2 is a schematic cross-sectional view showing the nucleic acid extraction device 101 of the first embodiment.
  • the nucleic acid extraction apparatus 101 includes a collection unit 10, an extraction unit 20, and a main unit 100 that accommodates the collection unit 10 and the extraction unit 20.
  • the nucleic acid extraction apparatus 101 of the present embodiment collects a sample B having a nucleic acid from the culture solution BR, and extracts a nucleic acid N from the collected sample B.
  • Examples of the nucleic acid N extracted from the sample B include genomic DNA, ribosomal RNA, and plasmid DNA.
  • the collection unit 10 of the present embodiment uses the membrane filter 11 to collect the sample B from the culture solution BR.
  • the collection unit 10 includes, for example, a funnel and a filter bottle.
  • the culture solution BR used in the nucleic acid extraction apparatus 101 of the present embodiment can be obtained by culturing a sample B having a nucleic acid.
  • the culture method of the sample B is not particularly limited, and examples thereof include a method (solid phase culture) in which the filter on which the sample B is collected is directly placed on a solid medium and the sample B is cultured via the filter.
  • a method (liquid phase culture) in which the sample B is cultured in the presence of a liquid medium or a solution in which a solid medium is dissolved in water is mentioned.
  • the type of liquid medium or solid medium to be used is selected according to the type of sample B to be cultured and physiological conditions.
  • the sample B to be treated is not particularly limited, and examples thereof include microorganisms, animal cells other than microorganisms (for example, insect cells), plant cells, mycoplasma, viruses and the like.
  • microorganism examples include Acinetobacter species, Actinomyces species, Aerococcus species, Aeromonas species, Aerogenes species, Alcaligenes species, Bacillus species, Bacteriodes Species, Bordetella species, Branhamella species, Brevibacterium species, Campylobacter species, Candida species, Capnocytophaga species, Chromobacterium species , Clostridium Clostridium species, Corynebacterium species, Cryptococcus species, Deinococcus species, Deinococcus species, Enterococcus species, Elysipelothrix species, Escherichia species, Flabobacterium (Flavobacterium species) Species, Gemella species, Haemophilus species, Klebsiella species, Lactobacillus species, Lactobacillus species, Lactococcus species, Legionella species, Leuconostoc species, Lico Terrier (Listeria) species, Micrococcus (Micrococcus) species, My
  • Species, b At least one member selected from the group consisting of: Dosophilum (Rhodospirillum) species, Staphylococcus (Staphylococcus) species, Streptomyces (Streptomyces) species, Streptococcus (Streptococcus) species, Vibrio (Vibrio) species, and Yersinia species .
  • the form of the sample B to be treated is not particularly limited.
  • the sample B to be treated may be one type or two or more types.
  • the membrane filter 11 of the present embodiment preferably has a pore diameter capable of capturing the sample B to be treated.
  • the membrane filter 11 preferably has a pore size of 0.45 ⁇ m or less.
  • the material of the membrane filter 11 is not particularly limited as long as it does not inhibit nucleic acid extraction in the extraction unit 20 described later and the extracted nucleic acid N is difficult to adsorb.
  • the material of the membrane filter 11 is, for example, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyethersulfone (PES), cellulose mixed ester, polycarbonate (PC), nylon, polyvinyl chloride (PVC) And sterling silver.
  • PTFE polytetrafluoroethylene
  • PVDF polyvinylidene fluoride
  • PES polyethersulfone
  • PC polycarbonate
  • nylon polyvinyl chloride
  • PVC polyvinyl chloride
  • the "cellulose mixed ester” is a material composed of a biologically inert mixture of cellulose acetate and cellulose nitrate.
  • the material of the membrane filter 11 is preferably hydrophilic.
  • hydrophilic PTFE, hydrophilic PVDF, hydrophilic PES, and hydrophilic PC are preferable.
  • hydrophilic PTFE, hydrophilic PVDF, hydrophilic PES, and hydrophilic PC are preferable.
  • hydrophilic PC is more preferable as the material of the membrane filter 11.
  • the material of the membrane filter 11 is hydrophilic PC, it is easy to obtain a filter having a constant pore size and pore size distribution.
  • the sample B can be collected stably by using a filter having a constant pore size or pore size distribution.
  • the contact angle of the material of the membrane filter 11 can be mentioned.
  • the contact angle of the material of the membrane filter 11 of the present embodiment is preferably 90 ° C. or less.
  • the extraction unit 20 of the present embodiment extracts the nucleic acid N from the collected sample B.
  • the extraction unit 20 of the present embodiment has a container 21, means 22 for containing, and a heating mechanism 23.
  • the container 21 of the present embodiment has an internal space 24 in which the membrane filter 11 can be accommodated.
  • the container 21 according to the present embodiment may be any container capable of withstanding heating and an increase in pressure of the internal space 24 associated with heating, and examples thereof include the following (E-1).
  • E-1) heat sealable bag
  • the above (E-1) can be sealed by heat-sealing the above (E-1).
  • volume of the internal space 24 which the container 21 of this embodiment has 2 ml or less is preferable, 0.6 ml or less is more preferable, 0.2 ml or less is more preferable. If the volume of the internal space 24 is 2 ml or less, the internal space 24 of the container 21 can be uniformly heated in a short time.
  • a volume of the internal space 24 which the container 21 of this embodiment has 0.1 ml or more is preferable, and 0.15 ml or more is more preferable.
  • the container 21 can be easily handled, and nucleic acids N can be extracted from a sufficient amount of sample B at one time.
  • the upper limit value and the lower limit value can be arbitrarily combined.
  • the means 22 for accommodating the present embodiment accommodates the membrane filter 11 in which the sample B is collected in the internal space 24.
  • the membrane filter 11 in which the sample B is collected is obtained by the collection unit 10 described above.
  • the means 22 for containing in the present embodiment comprises means 22A for injecting.
  • the injecting means 22A of the present embodiment injects the liquid L into the internal space 24.
  • the liquid L of the present embodiment contains water.
  • the internal space 24 of the present embodiment together with the membrane filter 11 in which the sample B is collected, when the container 21 is heated by the heating mechanism 23 described later, the internal space 24 of the container 21 is pressurized. It is easy to When the internal space 24 is pressurized, the sample B collected by the membrane filter 11 is also pressurized. As a result, nucleic acid N can be efficiently extracted from sample B.
  • the sample B is broken and the nucleic acid N is released and at the same time the nucleic acid N is extracted into water. be able to.
  • Cell lysis promoter The liquid L of the present embodiment exerts the above effect even with water alone, but for the purpose of extracting the nucleic acid N more efficiently from the sample B, in addition to water, alkali, acid, enzyme, surfactant, redox agent It is preferable to include at least one cell lysis promoter selected from the group consisting of and protein denaturants.
  • the cytolytic promoter has the ability to lyse the membrane structure of sample B.
  • the action of the cell lysis promoter on the membrane structure of the sample B makes it easy to destroy the sample B, and the nucleic acid N can be extracted more efficiently from the sample B.
  • alkali examples include sodium hydroxide (NaOH), potassium hydroxide (KOH) and the like.
  • Examples of the acid include hydrochloric acid (HCl) or sulfuric acid (H 2 SO 4 ).
  • enzymes examples include proteolytic enzymes such as Proteinase K, and polysaccharide degrading enzymes such as chitinase, lysozyme and zymolyase.
  • the surfactant may be, for example, ionic or non-ionic.
  • nonionic surfactants include octylphenol ethoxylate (C 14 H 22 O (C 2 H 4 O) n ) and the like.
  • octylphenol ethoxylate C 14 H 22 O (C 2 H 4 O) n
  • the ionic surfactant may be anionic, cationic or zwitterionic.
  • anionic surfactants include sodium dodecyl sulfate (SDS) and the like.
  • cationic surfactants examples include cetyltrimethylammonium bromide (CTAB) and the like.
  • amphoteric surfactants examples include betaine and the like.
  • betaine has a positive charge and a negative charge at positions not adjacent to each other in the same molecule, and an atom having a positive charge has no dissociable hydrogen atom bonded thereto, and the molecule as a whole is It is a generic term for compounds without charge.
  • a representative example of betaine is trimethylglycine.
  • Examples of the above-mentioned redox agent include hydrogen peroxide water, ⁇ -mercaptoethanol, dithiothreitol and the like.
  • protein modifying agent examples include guanidine hydrochloride, urea and the like.
  • chelating agent examples include ethylenediaminetetraacetic acid (EDTA) and the like.
  • the liquid L of this embodiment contains the said surfactant among the cytolysis promoter mentioned above, and it is more preferable that one or both of SDS and an octyl phenol ethoxylate are included.
  • the nucleic acid N extracted by the nucleic acid extraction apparatus 101 of the present embodiment when it is desired to detect the nucleic acid N extracted by the nucleic acid extraction apparatus 101 of the present embodiment with high sensitivity, it is preferable to use SDS.
  • the nucleic acid N extracted by the nucleic acid extraction apparatus 101 of the present embodiment is used for an enzyme reaction inhibited by SDS, the octylphenol ethoxylate which acts milder than SDS on the membrane structure of the sample B It is good to use
  • the liquid L of the present embodiment may contain a buffer as needed as long as the effects of the present invention are not impaired.
  • the buffer include tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) and the like.
  • Heating mechanism 23 of the present embodiment heats the container 21 in a closed state.
  • the heating mechanism 23 according to the present embodiment is not particularly limited as long as the internal space 24 of the container 21 can heat the container 21 so that the temperature at which the sample B can be destroyed and the nucleic acid N can be extracted.
  • the heating mechanism 23 of this embodiment is an apparatus which can heat the container 21 from room temperature to about 200 degreeC.
  • a heat block, an oil bath, etc. are mentioned, for example.
  • the above nucleic acid extraction method using the nucleic acid extraction apparatus 101 is a crude extract of nucleic acid purification such as silica membrane method, charged microparticle method, phenol chloroform method, etc., a template for nucleic acid amplification such as PCR, RT-PCR, LAMP, NASBA, etc.
  • the present invention can be applied to targets for nucleic acid detection such as PCR detection, microarray detection, hybridization protection assay, nucleic acid sequence and the like.
  • a nucleic acid extraction method using the above-described nucleic acid extraction apparatus 101 will be described.
  • Step S1 of producing In step S1 of the present embodiment, a sample B having a nucleic acid is cultured using a liquid medium or a solution obtained by dissolving a solid medium in water, to prepare a culture solution BR.
  • the type of liquid medium or solid medium is selected according to the type of sample B to be cultured and physiological conditions.
  • Step S2 of collecting In step S2 of collecting the present embodiment, the sample B is collected from the culture solution BR in the collecting unit 10 using the membrane filter 11.
  • Step S3 of containing In step S3 of storing the present embodiment, the membrane filter 11 on which the sample B is collected and the liquid L are stored in the internal space 24 of the container 21 using the storing means 22.
  • step S3 of the present embodiment it is preferable to inject the liquid L into the internal space 24 using the injecting means 22A after accommodating the membrane filter 11 in which the sample B is collected in the internal space 24. .
  • Step S4 of sealing At sealing process S4 of this embodiment, the membrane filter 11 by which the sample B was collected, and the container 21 in which the liquid L was accommodated are sealed.
  • the above (E-1) can be sealed by heat-sealing the above (E-1) as the container 21.
  • Step S5 of extracting In the extraction step S5 of the present embodiment, the container 21 is heated using the heating mechanism 23, and the nucleic acid N is extracted from the sample B.
  • the liquid L containing water is heated together with the membrane filter 11 on which the sample B is collected.
  • the internal space 24 of the container 21 is likely to be pressurized.
  • the sample B collected by the membrane filter 11 is also pressurized.
  • nucleic acid N can be efficiently extracted from sample B.
  • the liquid L be filled in the space between the container 21 and the membrane filter 11 in which the sample B is collected. Thereby, the internal space 24 of the container 21 tends to be pressurized in a short time.
  • the cell lysis promoter acts on the membrane structure of the sample B, thereby making it easy to destroy the sample B, and extracting the nucleic acid N more efficiently from the sample B can do. Furthermore, it is thought that the above-mentioned effect can be exhibited more by the liquid L containing a cell lysis promoter being heated with the membrane filter 11 in which the sample B was collected. Therefore, this method is effective when the amount of sample B to be treated is small and it is desired to extract nucleic acid N more reliably under a single condition.
  • the heating conditions such as the heating temperature and the heating time are determined according to the type of the sample B, the presence or absence of the cell lysis promoter, and the solubility of the cell lysis promoter.
  • SDS acts sufficiently on the membrane structure of sample B even under mild heating conditions than in the case of using octylphenol ethoxylate having a lower dissolving capacity than SDS, and nucleic acid N is extracted from sample B It is considered possible.
  • the container 21 after heating may be gradually cooled at room temperature in a sealed state, and the nucleic acid N may be extracted from the sample B.
  • a rapid temperature change does not easily occur in the internal space 24, and the destruction of the nucleic acid N extracted from the sample B can be suppressed. Therefore, according to this method, it is possible to extract long-chain nucleic acid N that is easily broken from sample B.
  • the container 21 after cooling may be reheated to extract the nucleic acid N from the sample B.
  • this method since the sample B is easily destroyed by the temperature change of the internal space 24, the nucleic acid N can be efficiently extracted from the sample B. Therefore, this method is effective for sample B having a rigid structure such as gram positive bacteria and fungi.
  • the nucleic acid extraction method of the present embodiment is performed.
  • a nucleic acid extraction method and a nucleic acid extraction apparatus 101 capable of efficiently extracting a nucleic acid N from a sample B having a nucleic acid such as a microorganism are provided.
  • FIG. 3 is a schematic cross-sectional view showing the nucleic acid extraction device 102 of the second embodiment.
  • the nucleic acid extraction device 102 according to the second embodiment includes a collection unit 10, an extraction unit 120, and a main unit 100. Therefore, in the second embodiment, the components common to the first embodiment are denoted by the same reference numerals, and the detailed description is omitted.
  • the extraction unit 120 of the present embodiment has a container 21, means 22 for containing, a lid member 25 and a heating mechanism 23.
  • the "lid member 25" corresponds to the "closing member” in the claims.
  • the container 21 of the present embodiment has an opening 26 communicating with the internal space 24.
  • the container 21 of the present embodiment may be any container capable of withstanding heating and an increase in pressure of the internal space 24 associated with heating, and examples thereof include the following (E-2) to (E-4).
  • E-2) Plastic tube for reaction
  • E-3) Glass test tube
  • E-4) Micro TAS chip
  • the heat resistance is high, the volume of the internal space 24 is easily maintained even at high temperature, and the internal space 24 tends to be pressurized. Is preferred.
  • the lid member 25 of the present embodiment can close the opening 26.
  • the lid member 25 is not particularly limited as long as it is a member having a mechanical structure capable of closing the opening 26.
  • the lid member 25 may be a member having a portion fitted with the container 21.
  • a forming material of the lid member 25 for example, a material which can follow the shape of the opening 26 such as rubber or silicone resin is used.
  • the lid member 25 of the present embodiment may be integral with or separate from the container 21.
  • nucleic acid extraction method a nucleic acid extraction method using the above-described nucleic acid extraction apparatus 102 will be described.
  • the step S1 of producing, the step S2 of collecting, and the step S3 of holding can be performed in the same manner as the nucleic acid extraction method of the first embodiment.
  • Step S4 of sealing In the sealing step S4 of the present embodiment, the lid 26 is used to close the opening 26 to seal the membrane filter 11 in which the sample B is collected and the container 21 in which the liquid L is stored.
  • Step S5 of extracting In the extraction step S5 of the present embodiment, the container 21 is heated. As in the first embodiment, the container 21 after heating may be gradually cooled at room temperature in a sealed state, and the nucleic acid N may be extracted from the sample B. Further, as in the first embodiment, the container 21 after cooling may be reheated to extract the nucleic acid N from the sample B.
  • the container 21 may be opened by manually removing the lid member 25 of the container 21 after heating or cooling.
  • this method is effective in extracting nucleic acid N from sample B having a rigid structure such as gram positive bacteria and fungi.
  • the open state and the closed state of the container 21 can be repeated according to the extraction condition of the nucleic acid N.
  • the nucleic acid extraction method of the present embodiment is performed.
  • a nucleic acid extraction method and a nucleic acid extraction apparatus 102 capable of efficiently extracting the nucleic acid N from the sample B are provided.
  • the nucleic acid extraction method and the nucleic acid extraction apparatus 102 of the second embodiment can repeat the open state and the closed state of the container 21 according to the extraction condition of the nucleic acid N by using the lid member 25.
  • FIG. 4 is a schematic cross-sectional view showing the nucleic acid extraction device 103 of the third embodiment.
  • the nucleic acid extraction apparatus 103 of the third embodiment includes a collection unit 10, an extraction unit 220, and a main unit 100. Therefore, in the third embodiment, the same components as in the second embodiment are denoted by the same reference numerals, and the detailed description is omitted.
  • the extraction unit 220 of the present embodiment has a container 21, means 22 for containing, a lid member 25, a heating mechanism 23, and a cooling mechanism 27.
  • the cooling mechanism 27 of the present embodiment cools the container 21 after heating in a sealed state.
  • a Peltier device As a cooling mechanism 27 of this embodiment, a Peltier device etc. are mentioned, for example.
  • nucleic acid extraction method a nucleic acid extraction method using the above-described nucleic acid extraction apparatus 103 will be described.
  • the step S1 of producing, the step S2 of collecting, the step S3 of containing, and the step S4 of sealing can be performed in the same manner as the nucleic acid extraction method of the second embodiment.
  • Step S5 of extracting In step S5 of extracting in the present embodiment, the container 21 is heated. Next, the container 21 after heating is cooled using the cooling mechanism 27 in a sealed state, and the nucleic acid N is extracted from the sample B. Thereby, a rapid temperature change is likely to occur in the internal space 24. As a result, since the sample B is easily destroyed, the nucleic acid N can be efficiently extracted from the sample B. Therefore, this method is effective for sample B having a rigid structure such as gram positive bacteria and fungi.
  • the nucleic acid extraction method of the present embodiment is performed.
  • a nucleic acid extraction method and a nucleic acid extraction apparatus 103 capable of efficiently extracting the nucleic acid N from the sample B are provided.
  • the nucleic acid extraction method and the nucleic acid extraction apparatus 103 according to the third embodiment is effective for the sample B having a rigid structure such as gram positive bacteria and fungi because the sample B is easily destroyed due to the rapid temperature change of the internal space 24. is there.
  • FIG. 5 is a schematic cross-sectional view showing the nucleic acid extraction device 104 of the fourth embodiment.
  • the nucleic acid extraction device 104 of the fourth embodiment includes a collection unit 10, an extraction unit 120, a main unit 100, a detection unit 28, and a means 29 for notifying. Therefore, in the fourth embodiment, the components common to the second embodiment are denoted by the same reference numerals, and the detailed description is omitted.
  • the detection unit 28 of the present embodiment detects at least one of the temperature of the container 21 and the pressure of the internal space 24.
  • the detection unit 28 of the present embodiment has a device capable of detecting at least one of the temperature of the container 21 and the pressure of the internal space 24.
  • the detection unit 28 detects the temperature of the container 21, the detection unit 28 has a thermocouple as the above-described device.
  • the detection result of the detection unit 28 of the present embodiment is output to a notifying unit 29 described later.
  • Means 29 for notifying the present embodiment determines opening and closing of the opening 26 based on the detection result of the detection unit 28 and notifies the worker. The operator manually attaches and detaches the lid member 25 when notified by the notifying unit 29 and opens and closes the opening 26.
  • nucleic acid extraction method a nucleic acid extraction method using the above-described nucleic acid extraction device 104 will be described.
  • the step S1 of producing, the step S2 of collecting, the step S3 of containing, and the step S4 of sealing can be performed in the same manner as the nucleic acid extraction method of the second embodiment.
  • Step S5 of extracting In the extraction step S5 of the present embodiment, the container 21 is heated.
  • the detection unit 28 is used to detect at least one of the temperature of the container 21 and the pressure of the internal space 24 after heating.
  • the opening and closing of the opening 26 is determined based on the detection result of the detecting unit 28, and the operator is notified.
  • the notification of the notifying means 29 will be described by taking a specific example.
  • the operator is notified that the opening 26 is open.
  • the operator manually removes the lid member 25 when notified by the notifying means 29, and opens the opening 26.
  • a rapid pressure change occurs in the inner space 24 and shear stress is generated in the sample B accommodated in the inner space 24.
  • the sample B can be easily destroyed, and the nucleic acid N can be extracted more efficiently from the sample B.
  • the means 29 for notifying the worker when the temperature of the container 21 is less than 100 ° C. or the pressure of the internal space 24 is atmospheric pressure is opened.
  • the release of the part 26 is notified.
  • the operator manually removes the lid member 25 when notified by the notifying means 29, and opens the opening 26.
  • the nucleic acid extraction method of the present embodiment is performed.
  • a nucleic acid extraction method and a nucleic acid extraction device 104 capable of efficiently extracting the nucleic acid N from the sample B are provided.
  • the nucleic acid extraction method and the nucleic acid extraction apparatus 104 of the fourth embodiment can control the pressure change of the internal space 24 according to the type of the sample B to be processed and the type of the nucleic acid N to be extracted.
  • FIG. 6 is a schematic cross-sectional view showing the nucleic acid extraction device 105 of the fifth embodiment.
  • the nucleic acid extraction apparatus 105 of the fifth embodiment includes the collection unit 10, the extraction unit 320, the main unit 100, the detection unit 28A, the drive unit 30, and the control unit 31.
  • the components common to the second embodiment are denoted by the same reference numerals, and the detailed description is omitted.
  • the extraction unit 320 of the present embodiment has a container 21, means 22 for containing, a plug member 25 A, and a heating mechanism 23.
  • the plug member 25A of the present embodiment can close the opening 26.
  • the plug member 25A is not particularly limited as long as it is a member having a mechanical structure capable of closing the opening 26.
  • the plug member 25A may be a rubber plug or a silicone plug having an inner diameter slightly smaller than the inner diameter of the container 21.
  • the plug member 25A of the present embodiment may be integral with or separate from the container 21.
  • the plug member 25A of the present embodiment may have a pressing member (not shown) for suppressing the drop of the plug member 25A from the container 21 when the pressure in the internal space 24 of the container 21 rises. .
  • the detection unit 28A of the present embodiment detects at least one of the temperature of the container 21 and the pressure of the internal space 24.
  • the detection unit 28A of the present embodiment has a device capable of detecting at least one of the temperature of the container 21 and the pressure of the internal space 24.
  • the detection result of the detection unit 28A of the present embodiment is output to the control unit 31 described later.
  • the drive unit 30 of the present embodiment drives the plug member 25A.
  • the control unit 31 of the present embodiment controls at least the drive of the drive unit 30 based on the detection result of the detection unit 28A.
  • the control unit 31 of the present embodiment may control the heating mechanism 23 in addition to the drive unit 30.
  • nucleic acid extraction method a nucleic acid extraction method using the above-described nucleic acid extraction apparatus 105 will be described.
  • the step S1 of producing, the step S2 of collecting, and the step S3 of holding can be performed in the same manner as the nucleic acid extraction method of the second embodiment.
  • Step S4 of sealing In the sealing step S4 of the present embodiment, by closing the opening 26 using the plug member 25A, the membrane filter 11 in which the sample B is collected and the container 21 in which the liquid L is stored are sealed.
  • Step S5 of extracting In the extraction step S5 of the present embodiment, the container 21 is heated.
  • control unit 31 controls at least the drive of the drive unit 30 based on the detection result of the detection unit 28A.
  • control of the control unit 31 will be described by taking a specific example.
  • the driving unit 30 is driven when the temperature of the container 21 is 100 ° C. or higher, or the pressure of the internal space 24 is atmospheric pressure or higher.
  • the plug member 25A is automatically removed by the drive of the drive unit 30, and the container 21 is opened.
  • a rapid pressure change occurs in the inner space 24 and shear stress is generated in the sample B accommodated in the inner space 24.
  • the sample B can be easily destroyed, and the nucleic acid N can be extracted more efficiently from the sample B.
  • the driving unit 30 When extracting long-chain nucleic acid from sample B, the driving unit 30 is driven when the temperature of the container 21 is less than 100 ° C. or the pressure of the internal space 24 is atmospheric pressure. The plug member 25A is automatically removed by the drive of the drive unit 30, and the container 21 is opened. As a result, a rapid pressure change does not easily occur in the internal space 24, and shear stress does not easily occur in the sample B accommodated in the internal space 24. As a result, destruction of the nucleic acid N extracted from the sample B can be suppressed.
  • the nucleic acid extraction method of the present embodiment is performed.
  • a nucleic acid extraction method and a nucleic acid extraction apparatus 105 capable of automatically extracting the nucleic acid N from the sample B are provided.
  • the nucleic acid extraction method and the nucleic acid extraction apparatus 105 of the fifth embodiment can control the pressure change of the internal space 24 according to the type of the sample B to be processed and the type of the nucleic acid N to be extracted.
  • the liquid of the present embodiment includes a first liquid and a second liquid.
  • the first liquid contains water, the above-mentioned cytolytic promoter, and the above-mentioned buffer solution which is optionally used.
  • the second liquid has a boiling point higher than that of the first liquid.
  • a 2nd liquid mineral oil etc. are mentioned, for example.
  • nucleic acid extraction method of the present embodiment even when the amount of the sample B is extremely small relative to the volume of the inner space 24, the void portion of the inner space 24 is filled with the second liquid. As a result, even when the amount of the sample B is extremely small relative to the volume of the internal space 24, when heating the container 21, the internal space 24 of the container 21 tends to be pressurized. When the internal space 24 is pressurized, the sample B collected by the membrane filter 11 is also pressurized. As a result, nucleic acid N can be efficiently extracted from sample B.
  • a nucleic acid extraction method and a nucleic acid extraction apparatus capable of efficiently extracting the nucleic acid N from the sample B are provided.
  • the nucleic acid extraction method of the sixth embodiment is effective even when the amount of the sample B is extremely small relative to the volume of the internal space 24.
  • the liquid L may be injected into the internal space 24 of the container 21 after heating in the extracting step S5. Good.
  • the nucleic acid N can be extracted to the liquid L also by this method.
  • the cell lysis promoter acts on the membrane structure of the sample B, thereby making it easy to destroy the sample B. As a result, the nucleic acid N can be extracted more efficiently from the sample B.
  • the nucleic acid was extracted from the sample having the nucleic acid using a nucleic acid extraction apparatus configured as shown in FIG.
  • Example 1 First, E. coli strain (NBRC3972) and S. aureus strain (NBRC12732) and concentrated SCD medium were added to commercially available mineral water, and then cultured at 37 ° C. for 48 hours to prepare a culture solution (prepared Process).
  • a filtration apparatus (collection unit) equipped with a membrane filter (diameter 13 mm, pore diameter 0.45 ⁇ m, manufactured by hydrophilic PC), 1 mL of culture solution was filtered to collect bacteria (sample) on the membrane filter (Step of collecting).
  • a membrane filter in which bacteria were collected and 200 ⁇ L of a liquid (a mixed solution of 40 mM Tris-HCl and 0.5% SDS) prepared in advance were accommodated in a container (a step of accommodating).
  • the container was brought into contact with a heat block (heating mechanism), heated at 140 ° C. for 45 seconds, and quenched after heating to extract DNA (nucleic acid) from E. coli strains and S. aureus strains (extraction step).
  • a heat block heating mechanism
  • DNA nucleic acid
  • the amount (detected value) of DNA capable of amplification by PCR extracted in the above solution calculated by real time PCR was respectively determined.
  • the ratio of each detection value to the amount of E. coli strain or S. aureus strain used in the step of producing was expressed as a percentage, and these were regarded as extraction efficiency. The results are shown in Table 1.
  • Example 1 As shown in Table 1, in Example 1 to which one aspect of the present invention was applied, it was found that DNA can be efficiently extracted from E. coli strains and S. aureus strains. Moreover, the extracted DNA was able to be amplified by PCR. From this, it was confirmed that the nucleic acid extraction method of one embodiment of the present invention can be applied to real-time PCR detection.
  • SYMBOLS 10 Collection part, 11 ... Membrane filter, 20, 120, 220, 320 ... Extraction part, 21 ... Container, 22 ... Means to accommodate, 22A ... Means to inject, 23 ... Heating mechanism, 24 ... Internal space, 25 ... Lid member, 25A: plug member, 26: opening, 27: cooling mechanism, 28, 28A: detection unit, 29: means for notifying, 30: drive unit, 31: control unit, 100: main unit, 101, 102, 103, 104, 105: nucleic acid extraction apparatus, B: sample, BR: culture solution, L: liquid, N: nucleic acid

Abstract

A nucleic acid extraction method which comprises: a step for capturing a nucleic acid-containing sample using a membrane filter; a step for housing the membrane filter, which holds the sample captured thereby, in the inner space of a container; a step for sealing the container housing the membrane filter; and a step for heating the container and thus extracting the nucleic acid from the sample.

Description

核酸抽出方法および核酸抽出装置Nucleic acid extraction method and nucleic acid extraction apparatus
　本発明は、核酸抽出方法および核酸抽出装置に関する。 The present invention relates to a nucleic acid extraction method and a nucleic acid extraction apparatus.
　核酸を抽出するためには、微生物をはじめとする核酸を有する試料を捕集し、試料の膜構造を破壊（溶解）し、試料の内容物を試料外に放出させる必要がある。 In order to extract nucleic acid, it is necessary to collect a sample having nucleic acid including microorganisms, to break up (dissolve) the membrane structure of the sample, and release the contents of the sample to the outside of the sample.
　従来、上記試料を捕集する方法としては、メンブレンフィルター法を利用した方法が知られている（例えば、非特許文献１）。 Conventionally, as a method of collecting the above-mentioned sample, a method using a membrane filter method is known (for example, non-patent document 1).
　非特許文献１に記載の方法では、イネ種子を滅菌水中に入れて超音波洗浄機で処理し、処理液中の病原細菌をメンブレンフィルター法により捕集する。このフィルターを試験管内の滅菌水中に回収し、再び超音波洗浄機で処理し、分離する。得られた試料液を選択培地上に塗抹し、培養することで、病原細菌を検出し、回収する。 In the method described in Non-Patent Document 1, rice seeds are put in sterile water and treated with an ultrasonic cleaner, and pathogenic bacteria in a treatment solution are collected by a membrane filter method. The filter is collected in sterile water in a test tube, treated again with an ultrasonic cleaner and separated. The obtained sample solution is smeared on a selective medium and cultured to detect and recover pathogenic bacteria.
　しかしながら、非特許文献１に記載されているような従来の方法は、手間や時間がかかるという問題があった。 However, the conventional method as described in Non-Patent Document 1 has a problem that it takes time and effort.
　本発明はこのような事情に鑑みてなされたものであって、微生物等の核酸を有する試料から核酸を効率的に抽出できる核酸抽出方法および核酸抽出装置を提供することを目的とする。 The present invention has been made in view of such circumstances, and an object thereof is to provide a nucleic acid extraction method and a nucleic acid extraction apparatus capable of efficiently extracting a nucleic acid from a sample having a nucleic acid such as a microorganism.
　上記課題を解決するため、本発明の一態様は、メンブレンフィルターを用いて核酸を有する試料を捕集する工程と、試料が捕集されたメンブレンフィルターを容器の内部空間に収容する工程と、メンブレンフィルターが収容された容器を密閉する工程と、容器を加熱し、試料から核酸を抽出する工程と、を有する核酸抽出方法を提供する。 In order to solve the above-mentioned subject, one mode of the present invention collects the sample which has nucleic acid using a membrane filter, the process of storing the membrane filter by which the sample was collected in the interior space of a container, membrane A method for nucleic acid extraction is provided, comprising the steps of: sealing a container containing a filter; heating the container; and extracting nucleic acid from a sample.
　本発明の一態様においては、抽出する工程において、加熱後の容器の内部空間に水を含有する液体を注入し、液体に核酸を抽出する方法としてもよい。 In one embodiment of the present invention, in the extraction step, a liquid containing water may be injected into the internal space of the heated container to extract nucleic acid into the liquid.
　本発明の一態様においては、収容する工程において、水を含有する液体を内部空間に収容する方法としてもよい。 In one aspect of the present invention, a liquid containing water may be contained in the inner space in the step of containing.
　本発明の一態様においては、液体は、アルカリ、酸、酵素、界面活性剤、酸化還元剤およびタンパク変性剤からなる群から選ばれる少なくとも一種の細胞溶解促進剤を含む方法としてもよい。 In one aspect of the present invention, the liquid may contain at least one cell lysis promoter selected from the group consisting of alkalis, acids, enzymes, surfactants, redox agents and protein denaturants.
　本発明の一態様においては、界面活性剤が、ドデシル硫酸ナトリウムと、オクチルフェノールエトキシレートとのいずれか一方または両方である方法としてもよい。 In one embodiment of the present invention, the surfactant may be sodium dodecyl sulfate and / or octylphenol ethoxylate.
　本発明の一態様においては、メンブレンフィルターは、親水性である方法としてもよい。 In one aspect of the invention, the membrane filter may be hydrophilic.
　本発明の一態様においては、抽出する工程において、加熱後の容器を、密閉状態のまま冷却し、核酸を抽出する方法としてもよい。 In one embodiment of the present invention, in the extraction step, the container after heating may be cooled in a sealed state to extract nucleic acid.
　本発明の一態様においては、抽出する工程において、加熱後の容器を、内部空間の圧力が大気圧以上である状態で開放して、核酸を抽出する方法としてもよい。 In one embodiment of the present invention, in the extraction step, the container after heating may be opened in a state where the pressure in the internal space is equal to or higher than the atmospheric pressure to extract the nucleic acid.
　本発明の一態様においては、抽出する工程において、冷却後の容器を再加熱して、核酸を抽出する方法としてもよい。 In one embodiment of the present invention, in the extracting step, the container after cooling may be reheated to extract nucleic acids.
　本発明の一態様においては、収容する工程において、内部空間に試料が捕集されたメンブレンフィルターを収容した後、液体を注入する方法としてもよい。 In one aspect of the present invention, in the step of containing, the liquid may be injected after containing the membrane filter in which the sample is collected in the inner space.
　本発明の一態様においては、抽出する工程において、液体が、容器と、試料が捕集されたメンブレンフィルターとの空隙に充填されている方法としてもよい。 In one embodiment of the present invention, in the extraction step, the liquid may be filled in the space between the container and the membrane filter on which the sample is collected.
　本発明の一態様においては、液体は、第１液体と、第２液体と、を有し、第１液体は、水と、細胞溶解促進剤と、を含み、第２液体は、第１液体よりも高沸点である方法としてもよい。 In one aspect of the present invention, the liquid comprises a first liquid and a second liquid, wherein the first liquid comprises water and a cell lysis promoter, and the second liquid is a first liquid. It is good also as a method which is higher boiling point.
　本発明の一態様は、メンブレンフィルターを用いて核酸を有する試料を捕集する捕集部と、捕集された試料から核酸を抽出する抽出部と、を備え、抽出部は、前記メンブレンフィルターを収容可能な内部空間を有する容器と、試料が捕集されたメンブレンフィルターを、内部空間に収容する手段と、容器を密閉状態で加熱する加熱機構と、を有する核酸抽出装置を提供する。 One embodiment of the present invention includes a collection unit that collects a sample having a nucleic acid using a membrane filter, and an extraction unit that extracts a nucleic acid from the collected sample, and the extraction unit is the membrane filter. Provided is a nucleic acid extraction device having a container having an internal space capable of being accommodated, means for accommodating a membrane filter in which a sample is collected, in the internal space, and a heating mechanism for heating the container in a sealed state.
　本発明の一態様においては、収容する手段は、内部空間に水を含有する液体を注入する手段を有する構成としてもよい。 In one aspect of the present invention, the means for containing may have a means for injecting a liquid containing water into the internal space.
　本発明の一態様においては、メンブレンフィルターの材質は、親水性である構成としてもよい。 In one aspect of the present invention, the material of the membrane filter may be hydrophilic.
　本発明の一態様においては、抽出部は、冷却機構を有し、冷却機構は、加熱後の容器を、密閉状態のまま冷却する構成としてもよい。 In one aspect of the present invention, the extraction unit may have a cooling mechanism, and the cooling mechanism may be configured to cool the container after heating in a sealed state.
　本発明の一態様においては、容器は、内部空間と連通する開口部を有し、抽出部は、開口部を塞ぐことができる閉塞部材を有する構成としてもよい。 In one aspect of the present invention, the container may have an opening communicating with the internal space, and the extraction unit may have a closing member capable of closing the opening.
　本発明の一態様においては、容器の温度、および内部空間の圧力の少なくとも一方を検出する検出部と、検出部の検出結果に基づいて、開口部の開閉を決定し、通知する手段と、を備える構成としてもよい。 In one aspect of the present invention, a detection unit that detects at least one of the temperature of the container and the pressure in the internal space, and a unit that determines opening / closing of the opening based on the detection result of the detection unit, It is good also as composition provided.
　本発明の一態様においては、閉塞部材を駆動させる駆動部と、容器の温度、および内部空間の圧力の少なくとも一方を検出する検出部と、検出部の検出結果に基づいて、少なくとも駆動部を制御する制御部と、を備える構成としてもよい。 In one aspect of the present invention, at least the drive unit is controlled based on the detection result of the drive unit that drives the closing member, the detection unit that detects at least one of the temperature of the container, and the pressure of the internal space And a control unit.
　本発明の一態様によれば、微生物等の核酸を有する試料から核酸を効率的に抽出できる核酸抽出方法および核酸抽出装置が提供される。 According to one aspect of the present invention, a nucleic acid extraction method and a nucleic acid extraction apparatus capable of efficiently extracting a nucleic acid from a sample having a nucleic acid such as a microorganism are provided.
図１は、第１実施形態の核酸抽出方法を示すフローチャートである。FIG. 1 is a flowchart showing the nucleic acid extraction method of the first embodiment. 図２は、第１実施形態の核酸抽出装置１０１を示す断面模式図である。FIG. 2 is a schematic cross-sectional view showing the nucleic acid extraction device 101 of the first embodiment. 図３は、第２実施形態の核酸抽出装置１０２を示す断面模式図である。FIG. 3 is a schematic cross-sectional view showing the nucleic acid extraction device 102 of the second embodiment. 図４は、第３実施形態の核酸抽出装置１０３を示す断面模式図である。FIG. 4 is a schematic cross-sectional view showing the nucleic acid extraction device 103 of the third embodiment. 図５は、第４実施形態の核酸抽出装置１０４を示す断面模式図である。FIG. 5 is a schematic cross-sectional view showing the nucleic acid extraction device 104 of the fourth embodiment. 図６は、第５実施形態の核酸抽出装置１０５を示す断面模式図である。FIG. 6 is a schematic cross-sectional view showing the nucleic acid extraction device 105 of the fifth embodiment.
＜第１実施形態＞
　以下、本発明の第１実施形態について、図面を参照しながら説明する。なお、以下の全ての図面においては、図面を見やすくするため、各構成要素の寸法や比率などは適宜異ならせてある。 First Embodiment
Hereinafter, a first embodiment of the present invention will be described with reference to the drawings. In addition, in all the following drawings, in order to make a drawing intelligible, the dimension, the ratio, etc. of each component are suitably varied.
　図１は、第１実施形態の核酸抽出方法を示すフローチャートである。図１に示すように、本実施形態の核酸抽出方法は、核酸を有する試料を培養し、培養液を作製する工程Ｓ１と、メンブレンフィルターを用いて上記試料を捕集する工程Ｓ２と、試料が捕集されたメンブレンフィルターを容器の内部空間に収容する工程Ｓ３と、メンブレンフィルターが収容された容器を密閉する工程Ｓ４と、容器を加熱し、試料から核酸を抽出する工程Ｓ５と、を有する。 FIG. 1 is a flowchart showing the nucleic acid extraction method of the first embodiment. As shown in FIG. 1, in the nucleic acid extraction method of the present embodiment, a step S1 of culturing a sample having a nucleic acid and preparing a culture solution, a step S2 of collecting the sample using a membrane filter, and a sample It has the process S3 which stores the collected membrane filter in the interior space of a container, the process S4 which seals the container in which the membrane filter was stored, and the process S5 which heats a container and extracts nucleic acid from a sample.
［核酸抽出装置］
　図２は、第１実施形態の核酸抽出装置１０１を示す断面模式図である。図２に示すように、第１実施形態の核酸抽出装置１０１は、捕集部１０と、抽出部２０と、捕集部１０および抽出部２０を収容する本体部１００と、を備える。 [Nucleic acid extraction device]
FIG. 2 is a schematic cross-sectional view showing the nucleic acid extraction device 101 of the first embodiment. As shown in FIG. 2, the nucleic acid extraction apparatus 101 according to the first embodiment includes a collection unit 10, an extraction unit 20, and a main unit 100 that accommodates the collection unit 10 and the extraction unit 20.
　本実施形態の核酸抽出装置１０１は、培養液ＢＲから核酸を有する試料Ｂを捕集し、捕集した試料Ｂから核酸Ｎを抽出する。試料Ｂから抽出される核酸Ｎとしては、例えばゲノムＤＮＡ、リボソーマルＲＮＡ、プラスミドＤＮＡが挙げられる。 The nucleic acid extraction apparatus 101 of the present embodiment collects a sample B having a nucleic acid from the culture solution BR, and extracts a nucleic acid N from the collected sample B. Examples of the nucleic acid N extracted from the sample B include genomic DNA, ribosomal RNA, and plasmid DNA.
（捕集部）
　本実施形態の捕集部１０は、メンブレンフィルター１１を用いて、培養液ＢＲから試料Ｂを捕集する。捕集部１０は、例えばファンネルおよび濾過瓶を含む。 (Collection unit)
The collection unit 10 of the present embodiment uses the membrane filter 11 to collect the sample B from the culture solution BR. The collection unit 10 includes, for example, a funnel and a filter bottle.
（培養液）
　本実施形態の核酸抽出装置１０１で用いられる培養液ＢＲは、核酸を有する試料Ｂを培養することにより得られる。試料Ｂの培養方法は、特に限定されず、例えば、試料Ｂを捕集したフィルターを直接固体培地上に置き、フィルターを介して試料Ｂを培養する方法（固相培養）が挙げられる。また、試料Ｂの別の培養方法としては、例えば液体培地または固体培地を水に溶解させた溶液の存在下で試料Ｂを培養する方法（液相培養）が挙げられる。用いる液体培地または固体培地の種類は、培養する試料Ｂの種類や生理的条件によって選択される。 (Culture fluid)
The culture solution BR used in the nucleic acid extraction apparatus 101 of the present embodiment can be obtained by culturing a sample B having a nucleic acid. The culture method of the sample B is not particularly limited, and examples thereof include a method (solid phase culture) in which the filter on which the sample B is collected is directly placed on a solid medium and the sample B is cultured via the filter. Moreover, as another culture method of the sample B, for example, a method (liquid phase culture) in which the sample B is cultured in the presence of a liquid medium or a solution in which a solid medium is dissolved in water is mentioned. The type of liquid medium or solid medium to be used is selected according to the type of sample B to be cultured and physiological conditions.
　本実施形態の核酸抽出装置１０１において、処理対象である試料Ｂとしては、特に限定されず、微生物、微生物以外の動物細胞（例えば、昆虫細胞など）、植物細胞、マイコプラズマ、ウィルスなどが挙げられる。 In the nucleic acid extraction device 101 of the present embodiment, the sample B to be treated is not particularly limited, and examples thereof include microorganisms, animal cells other than microorganisms (for example, insect cells), plant cells, mycoplasma, viruses and the like.
　上記微生物としては、例えば、アシネトバクター（Ａｃｉｎｅｔｏｂａｃｔｅｒ）種、アクチノミセス（Ａｃｔｉｎｏｍｙｃｅｓ）種、アエロコッカス（Ａｅｒｏｃｏｃｃｕｓ）種、アエロモナス（Ａｅｒｏｍｏｎａｓ）種、アルカリゲネス（Ａｌｃａｌｉｇｅｎｅｓ）種、バチルス（Ｂａｃｉｌｌｕｓ）種、バクテリオデス（Ｂａｃｔｅｒｉｏｄｅｓ）種、ボルデテラ（Ｂｏｒｄｅｔｅｌｌａ）種、ブランハメラ（Ｂｒａｎｈａｍｅｌｌａ）種、ブレビバクテリウム（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ）種、カンピロバクター（Ｃａｍｐｙｌｏｂａｃｔｅｒ）種、カンジダ（Ｃａｎｄｉｄａ）種、カプノシトファギア（Ｃａｐｎｏｃｙｔｏｐｈａｇｉａ）種、クロモバクテリウム（Ｃｈｒｏｍｏｂａｃｔｅｒｉｕｍ）種、クロストリジウム（Ｃｌｏｓｔｒｉｄｉｕｍ）種、コリネバクテリウム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ）種、クリプトコッカス（Ｃｒｙｐｔｏｃｏｃｃｕｓ）種、デイノコッカス（Ｄｅｉｎｏｃｏｃｃｕｓ）種、エンテロコッカス（Ｅｎｔｅｒｏｃｏｃｃｕｓ）種、エリジペロスリックス（Ｅｒｙｓｉｐｅｌｏｔｈｒｉｘ）種、エシェリシア（Ｅｓｃｈｅｒｉｃｈｉａ）種、フラボバクテリウム（Ｆｌａｖｏｂａｃｔｅｒｉｕｍ）種、ゲメラ（Ｇｅｍｅｌｌａ）種、ヘモフィルス（Ｈａｅｍｏｐｈｉｌｕｓ）種、クレブシエラ（Ｋｌｅｂｓｉｅｌｌａ）種、ラクトバチルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ）種、ラクトコッカス（Ｌａｃｔｏｃｏｃｃｕｓ）種、レジオネラ（Ｌｅｇｉｏｎｅｌｌａ）種、ロイコノストック（Ｌｅｕｃｏｎｏｓｔｏｃ）種、リステリア（Ｌｉｓｔｅｒｉａ）種、ミクロコッカス（Ｍｉｃｒｏｃｏｃｃｕｓ）種、マイコバクテリウム（Ｍｙｃｏｂａｃｔｅｒｉｕｍ）種、ナイセリア（Ｎｅｉｓｓｅｒｉａ）種、クリプトスポリジウム（Ｃｒｙｐｔｏｓｐｏｒｉｄｉｕｍ）種、ノカルディア（Ｎｏｃａｒｄｉａ）種、オエルスコビア（Ｏｅｒｓｋｏｖｉａ）種、パラコッカス（Ｐａｒａｃｏｃｃｕｓ）種、ペディオコッカス（Ｐｅｄｉｏｃｏｃｃｕｓ）種、ペプトストレプトコッカス（Ｐｅｐｔｏｓｔｒｅｐｔｏｃｏｃｃｕｓ）種、プロピオニバクテリウム（Ｐｒｏｐｉｏｎｉｂａｃｔｅｒｉｕｍ）種、プロテウス（Ｐｒｏｔｅｕｓ）種、シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）種、ラーネラ（Ｒａｈｎｅｌｌａ）種、ロドコッカス（Ｒｈｏｄｏｃｏｃｃｕｓ）種、ロドスピリルム（Ｒｈｏｄｏｓｐｉｒｉｌｌｕｍ）種、スタフィロコッカス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ）種、ストレプトミセス（Ｓｔｒｅｐｔｏｍｙｃｅｓ）種、ストレプトコッカス（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ）種、ビブリオ（Ｖｉｂｒｉｏ）種、およびイェルシニア（Ｙｅｒｓｉｎｉａ）種からなる群より選ばれる少なくとも１種である。 Examples of the microorganism include Acinetobacter species, Actinomyces species, Aerococcus species, Aeromonas species, Aerogenes species, Alcaligenes species, Bacillus species, Bacteriodes Species, Bordetella species, Branhamella species, Brevibacterium species, Campylobacter species, Candida species, Capnocytophaga species, Chromobacterium species , Clostridium Clostridium species, Corynebacterium species, Cryptococcus species, Deinococcus species, Deinococcus species, Enterococcus species, Elysipelothrix species, Escherichia species, Flabobacterium (Flavobacterium species) Species, Gemella species, Haemophilus species, Klebsiella species, Lactobacillus species, Lactobacillus species, Lactococcus species, Legionella species, Leuconostoc species, Lico Terrier (Listeria) species, Micrococcus (Micrococcus) species, Mycobacterium (Mycobacterium) species, Neisseria (Neisseria) species, Cryptosporidium (Cryptosporidium) species, Nocardia (Nocardia) species, Oerskovia (Oerskovia) species, Paracoccus (Paracoccus) Species), Pediococcus species, Peptostreptococcus species, Propionibacterium species, Proteus species, Proteus species, Pseudomonas species, Ranella species, Rhodococcus species. Species, b At least one member selected from the group consisting of: Dosophilum (Rhodospirillum) species, Staphylococcus (Staphylococcus) species, Streptomyces (Streptomyces) species, Streptococcus (Streptococcus) species, Vibrio (Vibrio) species, and Yersinia species .
　上述した微生物の中には、生育状態によって芽胞や胞子といった形態をとる微生物が存在する。本実施形態の核酸抽出装置１０１において、処理対象である試料Ｂの形態は、特に限定されない。 Among the above-described microorganisms, there are microorganisms that take the form of spores and spores depending on the growth state. In the nucleic acid extraction apparatus 101 of the present embodiment, the form of the sample B to be treated is not particularly limited.
　本実施形態の核酸抽出装置１０１において、処理対象である試料Ｂは、１種類であってもよく、２種類以上であってもよい。 In the nucleic acid extraction apparatus 101 of the present embodiment, the sample B to be treated may be one type or two or more types.
（メンブレンフィルター）
　本実施形態のメンブレンフィルター１１は、処理対象である試料Ｂを捕捉可能な孔径を有することが好ましい。例えば、メンブレンフィルター１１は、０．４５μｍ以下の孔径を有することが好ましい。 (Membrane filter)
The membrane filter 11 of the present embodiment preferably has a pore diameter capable of capturing the sample B to be treated. For example, the membrane filter 11 preferably has a pore size of 0.45 μm or less.
　メンブレンフィルター１１の材質は、後述する抽出部２０での核酸抽出を阻害せず、抽出した核酸Ｎが吸着しにくい材質であれば特に限定されない。 The material of the membrane filter 11 is not particularly limited as long as it does not inhibit nucleic acid extraction in the extraction unit 20 described later and the extracted nucleic acid N is difficult to adsorb.
　メンブレンフィルター１１の材質としては、例えば、ポリテトラフルオロエチレン（ＰＴＦＥ）、ポリビニリデンフロライド（ＰＶＤＦ）、ポリエーテルスルホン（ＰＥＳ）、セルロース混合エステル、ポリカーボネート（ＰＣ）、ナイロン、ポリ塩化ビニル（ＰＶＣ）、純銀などが挙げられる。 The material of the membrane filter 11 is, for example, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyethersulfone (PES), cellulose mixed ester, polycarbonate (PC), nylon, polyvinyl chloride (PVC) And sterling silver.
　なお、「セルロース混合エステル」とは、生物学的に不活性な、酢酸セルロースと硝酸セルロースとの混合物から構成される材料である。 The "cellulose mixed ester" is a material composed of a biologically inert mixture of cellulose acetate and cellulose nitrate.
　メンブレンフィルター１１の材質は、親水性であることが好ましく、例えば、親水性ＰＴＦＥ、親水性ＰＶＤＦ、親水性ＰＥＳ、親水性ＰＣが好ましい。メンブレンフィルター１１の材質が親水性であると、後述する抽出部２０で抽出した核酸Ｎがメンブレンフィルター１１により吸着しにくい。 The material of the membrane filter 11 is preferably hydrophilic. For example, hydrophilic PTFE, hydrophilic PVDF, hydrophilic PES, and hydrophilic PC are preferable. When the material of the membrane filter 11 is hydrophilic, it is difficult for the membrane filter 11 to adsorb the nucleic acid N extracted by the extraction unit 20 described later.
　なかでも、メンブレンフィルター１１の材質としては、親水性ＰＣがより好ましい。メンブレンフィルター１１の材質が親水性ＰＣであると、孔径や孔径分布が一定であるフィルターが得られやすい。孔径や孔径分布が一定であるフィルターを用いることにより、試料Ｂを安定的に捕集することができる。 Among them, hydrophilic PC is more preferable as the material of the membrane filter 11. When the material of the membrane filter 11 is hydrophilic PC, it is easy to obtain a filter having a constant pore size and pore size distribution. The sample B can be collected stably by using a filter having a constant pore size or pore size distribution.
　メンブレンフィルター１１の材質が親水性であることを示す一つの指標として、メンブレンフィルター１１の材質の接触角を挙げることができる。 As one index indicating that the material of the membrane filter 11 is hydrophilic, the contact angle of the material of the membrane filter 11 can be mentioned.
　メンブレンフィルター１１の材質の接触角が小さいほど、メンブレンフィルター１１は濡れやすくなり、メンブレンフィルター１１の材質は親水性が高くなると言える。本実施形態のメンブレンフィルター１１の材質の接触角は、９０℃以下であることが好ましい。 It can be said that the smaller the contact angle of the material of the membrane filter 11, the easier the membrane filter 11 gets wet, and the material of the membrane filter 11 becomes more hydrophilic. The contact angle of the material of the membrane filter 11 of the present embodiment is preferably 90 ° C. or less.
（抽出部）
　本実施形態の抽出部２０は、捕集された試料Ｂから核酸Ｎを抽出する。本実施形態の抽出部２０は、容器２１と、収容する手段２２と、加熱機構２３と、を有する。 (Extraction unit)
The extraction unit 20 of the present embodiment extracts the nucleic acid N from the collected sample B. The extraction unit 20 of the present embodiment has a container 21, means 22 for containing, and a heating mechanism 23.
（容器）
　本実施形態の容器２１は、メンブレンフィルター１１を収容可能な内部空間２４を有する。 (container)
The container 21 of the present embodiment has an internal space 24 in which the membrane filter 11 can be accommodated.
　本実施形態の容器２１は、加熱や、加熱に伴う内部空間２４の圧力の上昇に耐え得る容器であればよく、例えば下記（Ｅ－１）が挙げられる。
　（Ｅ－１）：熱融着可能な袋 The container 21 according to the present embodiment may be any container capable of withstanding heating and an increase in pressure of the internal space 24 associated with heating, and examples thereof include the following (E-1).
(E-1): heat sealable bag
　本実施形態の核酸抽出装置１０１においては、上記（Ｅ－１）を熱融着することで、上記（Ｅ－１）を密閉することができる。 In the nucleic acid extraction apparatus 101 of the present embodiment, the above (E-1) can be sealed by heat-sealing the above (E-1).
　本実施形態の容器２１が有する内部空間２４の容積としては、２ｍｌ以下が好ましく、０．６ｍｌ以下がより好ましく、０．２ｍｌ以下がさらに好ましい。内部空間２４の容積が２ｍｌ以下であると、短時間で容器２１の内部空間２４を均一に加熱することができる。 As a volume of the internal space 24 which the container 21 of this embodiment has, 2 ml or less is preferable, 0.6 ml or less is more preferable, 0.2 ml or less is more preferable. If the volume of the internal space 24 is 2 ml or less, the internal space 24 of the container 21 can be uniformly heated in a short time.
　本実施形態の容器２１が有する内部空間２４の容積としては、０．１ｍｌ以上が好ましく、０．１５ｍｌ以上がより好ましい。内部空間２４の容積が０．１ｍｌ以上であると、容器２１を取り扱いやすく、かつ、一度に十分な量の試料Ｂから核酸Ｎを抽出することができる。 As a volume of the internal space 24 which the container 21 of this embodiment has, 0.1 ml or more is preferable, and 0.15 ml or more is more preferable. When the volume of the internal space 24 is 0.1 ml or more, the container 21 can be easily handled, and nucleic acids N can be extracted from a sufficient amount of sample B at one time.
　上記上限値および下限値は任意に組み合わせることができる。 The upper limit value and the lower limit value can be arbitrarily combined.
（収容する手段）
　本実施形態の収容する手段２２は、試料Ｂが捕集されたメンブレンフィルター１１を、内部空間２４に収容する。 (Means to accommodate)
The means 22 for accommodating the present embodiment accommodates the membrane filter 11 in which the sample B is collected in the internal space 24.
　試料Ｂが捕集されたメンブレンフィルター１１は、上述した捕集部１０で得られる。 The membrane filter 11 in which the sample B is collected is obtained by the collection unit 10 described above.
（注入する手段）
　本実施形態の収容する手段２２は、注入する手段２２Ａを有する。本実施形態の注入する手段２２Ａは、内部空間２４に液体Ｌを注入する。 (Means to inject)
The means 22 for containing in the present embodiment comprises means 22A for injecting. The injecting means 22A of the present embodiment injects the liquid L into the internal space 24.
（液体）
　本実施形態の液体Ｌは、水を含有する。本実施形態の内部空間２４に、試料Ｂが捕集されたメンブレンフィルター１１と共に水を収容することにより、後述する加熱機構２３により容器２１を加熱する際、容器２１の内部空間２４が加圧された状態となりやすい。内部空間２４が加圧された状態となることで、メンブレンフィルター１１に捕集された試料Ｂも加圧される。その結果、試料Ｂから核酸Ｎを効率的に抽出することができる。 (liquid)
The liquid L of the present embodiment contains water. By storing water in the internal space 24 of the present embodiment together with the membrane filter 11 in which the sample B is collected, when the container 21 is heated by the heating mechanism 23 described later, the internal space 24 of the container 21 is pressurized. It is easy to When the internal space 24 is pressurized, the sample B collected by the membrane filter 11 is also pressurized. As a result, nucleic acid N can be efficiently extracted from sample B.
　また、本実施形態の内部空間２４に、試料Ｂが捕集されたメンブレンフィルター１１と共に水を収容することにより、試料Ｂが破壊されて核酸Ｎが放出されると同時に核酸Ｎを水に抽出することができる。 Further, by storing water in the internal space 24 of the present embodiment together with the membrane filter 11 in which the sample B is collected, the sample B is broken and the nucleic acid N is released and at the same time the nucleic acid N is extracted into water. be able to.
（細胞溶解促進剤）
　本実施形態の液体Ｌは、水のみでも上記効果を発揮するが、試料Ｂから核酸Ｎをより効率的に抽出する目的で、水以外に、アルカリ、酸、酵素、界面活性剤、酸化還元剤およびタンパク変性剤からなる群から選ばれる少なくとも一種の細胞溶解促進剤を含むことが好ましい。 (Cell lysis promoter)
The liquid L of the present embodiment exerts the above effect even with water alone, but for the purpose of extracting the nucleic acid N more efficiently from the sample B, in addition to water, alkali, acid, enzyme, surfactant, redox agent It is preferable to include at least one cell lysis promoter selected from the group consisting of and protein denaturants.
　細胞溶解促進剤は、試料Ｂの膜構造を溶解させる能力を有する。細胞溶解促進剤が試料Ｂの膜構造に作用することで、試料Ｂを破壊しやすくなり、試料Ｂから核酸Ｎをより効率的に抽出することができる。 The cytolytic promoter has the ability to lyse the membrane structure of sample B. The action of the cell lysis promoter on the membrane structure of the sample B makes it easy to destroy the sample B, and the nucleic acid N can be extracted more efficiently from the sample B.
　上記アルカリとしては、例えば水酸化ナトリウム（ＮａＯＨ）、または水酸化カリウム（ＫＯＨ）などが挙げられる。 Examples of the alkali include sodium hydroxide (NaOH), potassium hydroxide (KOH) and the like.
　上記酸としては、例えば塩酸（ＨＣｌ）、または硫酸（Ｈ２ＳＯ４）などが挙げられる。 Examples of the acid include hydrochloric acid (HCl) or sulfuric acid (H 2 SO 4 ).
　上記酵素としては、例えばＰｒｏｔｅｉｎａｓｅ　Ｋなどのタンパク分解酵素、またはｃｈｉｔｉｎａｓｅ、ｌｙｓｏｚｙｍｅ、ｚｙｍｏｌｙａｓｅなどの多糖分解酵素が挙げられる。 Examples of the enzyme include proteolytic enzymes such as Proteinase K, and polysaccharide degrading enzymes such as chitinase, lysozyme and zymolyase.
　上記界面活性剤は、例えばイオン性であってもよく、非イオン性であってもよい。 The surfactant may be, for example, ionic or non-ionic.
　非イオン性の界面活性剤としては、例えばオクチルフェノールエトキシレート（Ｃ１４Ｈ２２Ｏ（Ｃ２Ｈ４Ｏ）ｎ）などが挙げられる。
　本実施形態の核酸抽出装置１０１は、市販品のオクチルフェノールエトキシレートを用いることができ、例えばＳＩＧＭＡ社製のＴｒｉｔｏｎＸ－１００（Ｃ１４Ｈ２２Ｏ（Ｃ２Ｈ４Ｏ）ｎ，ｎ＝１００）などが挙げられる。 Examples of nonionic surfactants include octylphenol ethoxylate (C 14 H 22 O (C 2 H 4 O) n ) and the like.
A commercially available octylphenol ethoxylate can be used as the nucleic acid extraction apparatus 101 of this embodiment, and for example, Triton X-100 (C 14 H 22 O (C 2 H 4 O) n , n = 100) manufactured by SIGMA, etc. Can be mentioned.
　また、イオン性の界面活性剤は、陰イオン性であってもよく、陽イオン性であってもよく、両イオン性であってもよい。 The ionic surfactant may be anionic, cationic or zwitterionic.
　陰イオン性の界面活性剤としては、例えばドデシル硫酸ナトリウム（Ｓｏｄｉｕｍ　ｄｏｄｅｃｙｌ　ｓｕｌｆａｔｅ，ＳＤＳ）などが挙げられる。 Examples of anionic surfactants include sodium dodecyl sulfate (SDS) and the like.
　陽イオン性の界面活性剤としては、例えば臭化セチルトリメチルアンモニウム（Ｃｅｔｙｌｔｒｉｍｅｔｈｙｌａｍｍｏｎｉｕｍ　ｂｒｏｍｉｄｅ，ＣＴＡＢ）などが挙げられる。 Examples of cationic surfactants include cetyltrimethylammonium bromide (CTAB) and the like.
　両イオン性の界面活性剤としては、例えばベタインなどが挙げられる。ここで、「ベタイン」とは、正電荷と負電荷とを同一分子内の隣り合わない位置に持ち、正電荷をもつ原子には解離しうる水素原子が結合しておらず、分子全体としては電荷を持たない化合物の総称のことである。
ベタインの代表例としては、トリメチルグリシンが挙げられる。 Examples of amphoteric surfactants include betaine and the like. Here, "betaine" has a positive charge and a negative charge at positions not adjacent to each other in the same molecule, and an atom having a positive charge has no dissociable hydrogen atom bonded thereto, and the molecule as a whole is It is a generic term for compounds without charge.
A representative example of betaine is trimethylglycine.
　上記酸化還元剤としては、例えば過酸化水素水、β-メルカプトエタノール、ジチオトレイトールなどが挙げられる。 Examples of the above-mentioned redox agent include hydrogen peroxide water, β-mercaptoethanol, dithiothreitol and the like.
　上記タンパク変性剤としては、例えばグアニジン塩酸塩、尿素などが挙げられる。 Examples of the protein modifying agent include guanidine hydrochloride, urea and the like.
　上記キレート剤としては、例えばエチレンジアミン四酢酸（Ｅｔｈｙｌｅｎｅｄｉａｍｉｎｅｔｅｔｒａａｃｅｔｉｃ　ａｃｉｄ，ＥＤＴＡ）などが挙げられる。 Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA) and the like.
　上述した細胞溶解促進剤の中でも、本実施形態の液体Ｌは、上記界面活性剤を含むことが好ましく、ＳＤＳと、オクチルフェノールエトキシレートとのいずれか一方または両方を含むことがより好ましい。 It is preferable that the liquid L of this embodiment contains the said surfactant among the cytolysis promoter mentioned above, and it is more preferable that one or both of SDS and an octyl phenol ethoxylate are included.
　例えば、本実施形態の核酸抽出装置１０１で抽出した核酸Ｎを高感度で検出したい場合にはＳＤＳを用いるとよい。これに対し、本実施形態の核酸抽出装置１０１で抽出した核酸Ｎを、ＳＤＳによって阻害される酵素反応に用いる場合には、試料Ｂの膜構造に対してＳＤＳよりも温和に作用するオクチルフェノールエトキシレートを用いるとよい。 For example, when it is desired to detect the nucleic acid N extracted by the nucleic acid extraction apparatus 101 of the present embodiment with high sensitivity, it is preferable to use SDS. On the other hand, when the nucleic acid N extracted by the nucleic acid extraction apparatus 101 of the present embodiment is used for an enzyme reaction inhibited by SDS, the octylphenol ethoxylate which acts milder than SDS on the membrane structure of the sample B It is good to use
　本実施形態の液体Ｌは、本発明の効果を損なわない範囲において、必要に応じて緩衝液を含んでもよい。緩衝液としては、例えばトリス（ヒドロキシメチル）アミノメタン塩酸塩（Ｔｒｉｓ－ＨＣｌ）などが挙げられる。 The liquid L of the present embodiment may contain a buffer as needed as long as the effects of the present invention are not impaired. Examples of the buffer include tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) and the like.
（加熱機構）
　本実施形態の加熱機構２３は、容器２１を密閉状態で加熱する。 (Heating mechanism)
The heating mechanism 23 of the present embodiment heats the container 21 in a closed state.
　本実施形態の加熱機構２３は、容器２１の内部空間２４が、試料Ｂを破壊して核酸Ｎを抽出可能な温度となるように、容器２１を加熱できる装置であれば、特に限定されない。例えば、本実施形態の加熱機構２３は、室温から２００℃程度まで容器２１を加熱できる装置であることが好ましい。このような装置としては、例えばヒートブロック、またはオイルバスなどが挙げられる。 The heating mechanism 23 according to the present embodiment is not particularly limited as long as the internal space 24 of the container 21 can heat the container 21 so that the temperature at which the sample B can be destroyed and the nucleic acid N can be extracted. For example, it is preferable that the heating mechanism 23 of this embodiment is an apparatus which can heat the container 21 from room temperature to about 200 degreeC. As such an apparatus, a heat block, an oil bath, etc. are mentioned, for example.
［核酸抽出方法］
　上述の核酸抽出装置１０１を用いる核酸抽出方法は、シリカメンブレン法、荷電微粒子法、フェノールクロロフォルム法などの核酸精製の粗抽出液、ＰＣＲ、ＲＴ－ＰＣＲ、ＬＡＭＰ、ＮＡＳＢＡなどの核酸増幅の鋳型、リアルタイムＰＣＲ検出、マイクロアレイ検出、ハイブリダイゼーションプロテクトアッセイ、核酸配列シーケンスなどの核酸検出の標的などに適用することができる。
　以下、上述の核酸抽出装置１０１を用いる核酸抽出方法について説明する。 [Nucleic acid extraction method]
The above nucleic acid extraction method using the nucleic acid extraction apparatus 101 is a crude extract of nucleic acid purification such as silica membrane method, charged microparticle method, phenol chloroform method, etc., a template for nucleic acid amplification such as PCR, RT-PCR, LAMP, NASBA, etc. The present invention can be applied to targets for nucleic acid detection such as PCR detection, microarray detection, hybridization protection assay, nucleic acid sequence and the like.
Hereinafter, a nucleic acid extraction method using the above-described nucleic acid extraction apparatus 101 will be described.
（作製する工程Ｓ１）
　本実施形態の作製する工程Ｓ１では、液体培地、または固体培地を水に溶解させた溶液を用いて、核酸を有する試料Ｂを培養し、培養液ＢＲを作製する。液体培地または固体培地の種類は、培養する試料Ｂの種類や生理的条件によって選択される。 (Step S1 of producing)
In step S1 of the present embodiment, a sample B having a nucleic acid is cultured using a liquid medium or a solution obtained by dissolving a solid medium in water, to prepare a culture solution BR. The type of liquid medium or solid medium is selected according to the type of sample B to be cultured and physiological conditions.
（捕集する工程Ｓ２）
　本実施形態の捕集する工程Ｓ２では、捕集部１０において、メンブレンフィルター１１を用いて、培養液ＢＲから試料Ｂを捕集する。 (Step S2 of collecting)
In step S2 of collecting the present embodiment, the sample B is collected from the culture solution BR in the collecting unit 10 using the membrane filter 11.
（収容する工程Ｓ３）
　本実施形態の収容する工程Ｓ３では、収容する手段２２を用いて、試料Ｂが捕集されたメンブレンフィルター１１、および液体Ｌを、容器２１の内部空間２４に収容する。 (Step S3 of containing)
In step S3 of storing the present embodiment, the membrane filter 11 on which the sample B is collected and the liquid L are stored in the internal space 24 of the container 21 using the storing means 22.
　本実施形態の収容する工程Ｓ３においては、容器２１として下記（Ｅ－１）を用いることができる。
　（Ｅ－１）：熱融着可能な袋 In the step S3 of the present embodiment, the following (E-1) can be used as the container 21.
(E-1): heat sealable bag
　本実施形態の収容する工程Ｓ３においては、内部空間２４に試料Ｂが捕集されたメンブレンフィルター１１を収容した後、注入する手段２２Ａを用いて、内部空間２４に液体Ｌを注入することが好ましい。 In step S3 of the present embodiment, it is preferable to inject the liquid L into the internal space 24 using the injecting means 22A after accommodating the membrane filter 11 in which the sample B is collected in the internal space 24. .
（密閉する工程Ｓ４）
　本実施形態の密閉する工程Ｓ４では、試料Ｂが捕集されたメンブレンフィルター１１、および液体Ｌが収容された容器２１を密閉する。 (Step S4 of sealing)
At sealing process S4 of this embodiment, the membrane filter 11 by which the sample B was collected, and the container 21 in which the liquid L was accommodated are sealed.
　本実施形態の密閉する工程Ｓ４では、容器２１である上記（Ｅ－１）を熱融着することで、上記（Ｅ－１）を密閉することができる。 In the sealing step S4 of the present embodiment, the above (E-1) can be sealed by heat-sealing the above (E-1) as the container 21.
（抽出する工程Ｓ５）
　本実施形態の抽出する工程Ｓ５では、加熱機構２３を用いて、容器２１を加熱し、試料Ｂから核酸Ｎを抽出する。 (Step S5 of extracting)
In the extraction step S5 of the present embodiment, the container 21 is heated using the heating mechanism 23, and the nucleic acid N is extracted from the sample B.
　本実施形態の抽出する工程Ｓ５においては、試料Ｂが捕集されたメンブレンフィルター１１と共に、水を含有する液体Ｌが加熱される。これにより、容器２１の内部空間２４が加圧された状態となりやすい。内部空間２４が加圧された状態となることで、メンブレンフィルター１１に捕集された試料Ｂも加圧される。その結果、試料Ｂから核酸Ｎを効率的に抽出することができる。 In the extraction step S5 of the present embodiment, the liquid L containing water is heated together with the membrane filter 11 on which the sample B is collected. As a result, the internal space 24 of the container 21 is likely to be pressurized. When the internal space 24 is pressurized, the sample B collected by the membrane filter 11 is also pressurized. As a result, nucleic acid N can be efficiently extracted from sample B.
　本実施形態の抽出する工程Ｓ５においては、液体Ｌが、容器２１と、試料Ｂが捕集されたメンブレンフィルター１１との空隙に充填されていることが好ましい。これにより、短時間で容器２１の内部空間２４が加圧された状態となりやすい。 In the extraction step S5 of the present embodiment, it is preferable that the liquid L be filled in the space between the container 21 and the membrane filter 11 in which the sample B is collected. Thereby, the internal space 24 of the container 21 tends to be pressurized in a short time.
　また、使用する液体Ｌが細胞溶解促進剤を含む場合、細胞溶解促進剤が試料Ｂの膜構造に作用することで、試料Ｂを破壊しやすくなり、試料Ｂから核酸Ｎをより効率的に抽出することができる。さらに、細胞溶解促進剤を含む液体Ｌが、試料Ｂが捕集されたメンブレンフィルター１１と共に加熱されることで、上記作用をより発揮させることができると考えられる。したがって、この方法は、処理対象である試料Ｂの量が少なく、より確実に単一条件で核酸Ｎを抽出したい場合に有効である。 In addition, when the liquid L to be used contains a cell lysis promoter, the cell lysis promoter acts on the membrane structure of the sample B, thereby making it easy to destroy the sample B, and extracting the nucleic acid N more efficiently from the sample B can do. Furthermore, it is thought that the above-mentioned effect can be exhibited more by the liquid L containing a cell lysis promoter being heated with the membrane filter 11 in which the sample B was collected. Therefore, this method is effective when the amount of sample B to be treated is small and it is desired to extract nucleic acid N more reliably under a single condition.
　本実施形態の抽出する工程Ｓ５において、加熱温度や加熱時間などの加熱条件は、試料Ｂの種類、細胞溶解促進剤の有無、細胞溶解促進剤の溶解能力に応じて決定される。例えば、ＳＤＳを用いる場合、ＳＤＳよりも溶解能力が低いオクチルフェノールエトキシレートを用いる場合よりも温和な加熱条件であっても、ＳＤＳが試料Ｂの膜構造に十分作用し、試料Ｂから核酸Ｎが抽出できると考えられる。 In the extraction step S5 of the present embodiment, the heating conditions such as the heating temperature and the heating time are determined according to the type of the sample B, the presence or absence of the cell lysis promoter, and the solubility of the cell lysis promoter. For example, in the case of using SDS, SDS acts sufficiently on the membrane structure of sample B even under mild heating conditions than in the case of using octylphenol ethoxylate having a lower dissolving capacity than SDS, and nucleic acid N is extracted from sample B It is considered possible.
（冷却）
　本実施形態の抽出する工程Ｓ５において、加熱後の容器２１を、密閉状態のまま室温で徐々に冷却し、試料Ｂから核酸Ｎを抽出してもよい。これにより、内部空間２４に急激な温度変化が生じにくく、試料Ｂから抽出した核酸Ｎが破壊されるのを抑制できる。したがって、この方法によれば、試料Ｂから破壊されやすい長鎖の核酸Ｎを抽出することができる。 (cooling)
In the extraction step S5 of the present embodiment, the container 21 after heating may be gradually cooled at room temperature in a sealed state, and the nucleic acid N may be extracted from the sample B. As a result, a rapid temperature change does not easily occur in the internal space 24, and the destruction of the nucleic acid N extracted from the sample B can be suppressed. Therefore, according to this method, it is possible to extract long-chain nucleic acid N that is easily broken from sample B.
（再加熱）
　本実施形態の抽出する工程Ｓ５において、冷却後の容器２１を再加熱し、試料Ｂから核酸Ｎを抽出してもよい。この場合、加熱（再加熱を含む）と冷却とを少なくとも２回実施することが好ましい。この方法では、内部空間２４の温度変化により試料Ｂを破壊しやすいので、試料Ｂから核酸Ｎを効率的に抽出することができる。したがって、この方法は、グラム陽性菌や真菌など堅い構造を有する試料Ｂに対し有効である。 (Reheating)
In the extraction step S5 of the present embodiment, the container 21 after cooling may be reheated to extract the nucleic acid N from the sample B. In this case, it is preferable to carry out heating (including reheating) and cooling at least twice. In this method, since the sample B is easily destroyed by the temperature change of the internal space 24, the nucleic acid N can be efficiently extracted from the sample B. Therefore, this method is effective for sample B having a rigid structure such as gram positive bacteria and fungi.
　このようにして、本実施形態の核酸抽出方法が行われる。 Thus, the nucleic acid extraction method of the present embodiment is performed.
　第１実施形態によれば、微生物等の核酸を有する試料Ｂから核酸Ｎを効率的に抽出できる核酸抽出方法および核酸抽出装置１０１が提供される。 According to the first embodiment, a nucleic acid extraction method and a nucleic acid extraction apparatus 101 capable of efficiently extracting a nucleic acid N from a sample B having a nucleic acid such as a microorganism are provided.
＜第２実施形態＞
　以下、本発明の第２実施形態における核酸抽出装置および核酸抽出方法について、図面に基づき説明する。 Second Embodiment
Hereinafter, a nucleic acid extraction apparatus and a nucleic acid extraction method according to a second embodiment of the present invention will be described based on the drawings.
［核酸抽出装置］
　図３は、第２実施形態の核酸抽出装置１０２を示す断面模式図である。図３に示すように、第２実施形態の核酸抽出装置１０２は、捕集部１０と、抽出部１２０と、本体部１００と、を備える。したがって、第２実施形態において第１実施形態と共通する構成要素については同じ符号を付し、詳細な説明は省略する。 [Nucleic acid extraction device]
FIG. 3 is a schematic cross-sectional view showing the nucleic acid extraction device 102 of the second embodiment. As shown in FIG. 3, the nucleic acid extraction device 102 according to the second embodiment includes a collection unit 10, an extraction unit 120, and a main unit 100. Therefore, in the second embodiment, the components common to the first embodiment are denoted by the same reference numerals, and the detailed description is omitted.
（抽出部）
　本実施形態の抽出部１２０は、容器２１と、収容する手段２２と、蓋部材２５と、加熱機構２３と、を有する。 (Extraction unit)
The extraction unit 120 of the present embodiment has a container 21, means 22 for containing, a lid member 25 and a heating mechanism 23.
　本明細書において、「蓋部材２５」は請求の範囲における「閉塞部材」に相当する。 In the present specification, the "lid member 25" corresponds to the "closing member" in the claims.
（容器）
　本実施形態の容器２１は、内部空間２４と連通する開口部２６を有する。本実施形態の容器２１は、加熱や、加熱に伴う内部空間２４の圧力の上昇に耐え得る容器であればよく、例えば下記（Ｅ－２）～（Ｅ－４）が挙げられる。
　（Ｅ－２）：反応用プラスチックチューブ
　（Ｅ－３）：ガラス試験管
　（Ｅ－４）：マイクロＴＡＳチップ (container)
The container 21 of the present embodiment has an opening 26 communicating with the internal space 24. The container 21 of the present embodiment may be any container capable of withstanding heating and an increase in pressure of the internal space 24 associated with heating, and examples thereof include the following (E-2) to (E-4).
(E-2): Plastic tube for reaction (E-3): Glass test tube (E-4): Micro TAS chip
　なかでも、本実施形態の容器２１としては、耐熱性が高く、かつ、高温でも内部空間２４の容積が維持されやすく、内部空間２４が加圧された状態となりやすいことから、上記（Ｅ－３）が好ましい。 Above all, as the container 21 of the present embodiment, the heat resistance is high, the volume of the internal space 24 is easily maintained even at high temperature, and the internal space 24 tends to be pressurized. Is preferred.
（蓋部材）
　本実施形態の蓋部材２５は、開口部２６を塞ぐことができる。蓋部材２５は、開口部２６を塞ぐことができる機械的な構造を有する部材であれば、特に限定されない。例えば、蓋部材２５は、容器２１と嵌合する部分を有する部材などが挙げられる。蓋部材２５の形成材料として、例えばゴムまたはシリコーン樹脂などの開口部２６の形状に追従可能な材料が用いられる。 (Lid member)
The lid member 25 of the present embodiment can close the opening 26. The lid member 25 is not particularly limited as long as it is a member having a mechanical structure capable of closing the opening 26. For example, the lid member 25 may be a member having a portion fitted with the container 21. As a forming material of the lid member 25, for example, a material which can follow the shape of the opening 26 such as rubber or silicone resin is used.
　本実施形態の蓋部材２５は、容器２１と一体であってもよく、別体であってもよい。 The lid member 25 of the present embodiment may be integral with or separate from the container 21.
［核酸抽出方法］
　以下、上述の核酸抽出装置１０２を用いる核酸抽出方法について説明する。第２実施形態の核酸抽出方法では、作製する工程Ｓ１、捕集する工程Ｓ２、および収容する工程Ｓ３を、第１実施形態の核酸抽出方法と同様に行うことができる。 [Nucleic acid extraction method]
Hereinafter, a nucleic acid extraction method using the above-described nucleic acid extraction apparatus 102 will be described. In the nucleic acid extraction method of the second embodiment, the step S1 of producing, the step S2 of collecting, and the step S3 of holding can be performed in the same manner as the nucleic acid extraction method of the first embodiment.
（密閉する工程Ｓ４）
　本実施形態の密閉する工程Ｓ４では、蓋部材２５を用いて、開口部２６を塞ぐことにより、試料Ｂが捕集されたメンブレンフィルター１１、および液体Ｌが収容された容器２１を密閉する。 (Step S4 of sealing)
In the sealing step S4 of the present embodiment, the lid 26 is used to close the opening 26 to seal the membrane filter 11 in which the sample B is collected and the container 21 in which the liquid L is stored.
（抽出する工程Ｓ５）
　本実施形態の抽出する工程Ｓ５において、容器２１を加熱する。第１実施形態と同様に、加熱後の容器２１を、密閉状態のまま室温で徐々に冷却し、試料Ｂから核酸Ｎを抽出してもよい。また、第１実施形態と同様に、冷却後の容器２１を再加熱し、試料Ｂから核酸Ｎを抽出してもよい。 (Step S5 of extracting)
In the extraction step S5 of the present embodiment, the container 21 is heated. As in the first embodiment, the container 21 after heating may be gradually cooled at room temperature in a sealed state, and the nucleic acid N may be extracted from the sample B. Further, as in the first embodiment, the container 21 after cooling may be reheated to extract the nucleic acid N from the sample B.
（開放）
　本実施形態の抽出する工程Ｓ５において、加熱後または冷却後の容器２１の蓋部材２５を手動で取り外すことにより、容器２１を開放してもよい。 (Open)
In the extraction step S5 of the present embodiment, the container 21 may be opened by manually removing the lid member 25 of the container 21 after heating or cooling.
　加熱後の容器２１を開放する場合、内部空間２４に急激な圧力変化が生じる可能性がある。このとき、内部空間２４に収容された試料Ｂにせん断応力が発生する。その結果、試料Ｂを破壊しやすくなり、試料Ｂから核酸Ｎをより効率的に抽出することができる。したがって、この方法は、グラム陽性菌や真菌など堅い構造を有する試料Ｂから核酸Ｎを抽出する場合に有効である。 When opening the container 21 after heating, a sudden pressure change may occur in the internal space 24. At this time, shear stress is generated in the sample B accommodated in the internal space 24. As a result, the sample B can be easily destroyed, and the nucleic acid N can be extracted more efficiently from the sample B. Therefore, this method is effective in extracting nucleic acid N from sample B having a rigid structure such as gram positive bacteria and fungi.
　これに対し、冷却後の容器２１を開放する場合、内部空間２４に急激な圧力変化が生じにくい可能性がある。このとき、内部空間２４に収容された試料Ｂにせん断応力が発生しにくい。その結果、試料Ｂから抽出した核酸Ｎが破壊されるのを抑制できる。したがって、この方法は、試料Ｂから破壊されやすい長鎖の核酸Ｎを抽出する場合に有効である。 On the other hand, when opening the container 21 after cooling, there is a possibility that a sudden pressure change may not easily occur in the internal space 24. At this time, shear stress is less likely to occur in the sample B accommodated in the internal space 24. As a result, destruction of the nucleic acid N extracted from the sample B can be suppressed. Therefore, this method is effective in extracting long-chain nucleic acid N that is easily broken from sample B.
　また、本実施形態の抽出する工程Ｓ５においては、蓋部材２５を手動で着脱することにより、核酸Ｎの抽出状況に応じて、容器２１の開放状態と密閉状態を繰り返すことができる。 In addition, in the extraction step S5 of the present embodiment, by manually attaching and detaching the lid member 25, the open state and the closed state of the container 21 can be repeated according to the extraction condition of the nucleic acid N.
　このようにして、本実施形態の核酸抽出方法が行われる。 Thus, the nucleic acid extraction method of the present embodiment is performed.
　第２実施形態によれば、試料Ｂから核酸Ｎを効率的に抽出できる核酸抽出方法および核酸抽出装置１０２が提供される。特に、第２実施形態の核酸抽出方法および核酸抽出装置１０２は、蓋部材２５を用いることにより、核酸Ｎの抽出状況に応じて、容器２１の開放状態と密閉状態を繰り返すことができる。 According to the second embodiment, a nucleic acid extraction method and a nucleic acid extraction apparatus 102 capable of efficiently extracting the nucleic acid N from the sample B are provided. In particular, the nucleic acid extraction method and the nucleic acid extraction apparatus 102 of the second embodiment can repeat the open state and the closed state of the container 21 according to the extraction condition of the nucleic acid N by using the lid member 25.
＜第３実施形態＞
　以下、本発明の第３実施形態における核酸抽出装置および核酸抽出方法について、図面に基づき説明する。 Third Embodiment
Hereinafter, a nucleic acid extraction apparatus and a nucleic acid extraction method according to a third embodiment of the present invention will be described based on the drawings.
［核酸抽出装置］
　図４は、第３実施形態の核酸抽出装置１０３を示す断面模式図である。図４に示すように、第３実施形態の核酸抽出装置１０３は、捕集部１０と、抽出部２２０と、本体部１００と、を備える。したがって、第３実施形態において第２実施形態と共通する構成要素については同じ符号を付し、詳細な説明は省略する。 [Nucleic acid extraction device]
FIG. 4 is a schematic cross-sectional view showing the nucleic acid extraction device 103 of the third embodiment. As shown in FIG. 4, the nucleic acid extraction apparatus 103 of the third embodiment includes a collection unit 10, an extraction unit 220, and a main unit 100. Therefore, in the third embodiment, the same components as in the second embodiment are denoted by the same reference numerals, and the detailed description is omitted.
（抽出部）
　本実施形態の抽出部２２０は、容器２１と、収容する手段２２と、蓋部材２５と、加熱機構２３と、冷却機構２７と、を有する。 (Extraction unit)
The extraction unit 220 of the present embodiment has a container 21, means 22 for containing, a lid member 25, a heating mechanism 23, and a cooling mechanism 27.
（冷却機構）
　本実施形態の冷却機構２７は、加熱後の容器２１を、密閉状態のまま冷却する。 (Cooling mechanism)
The cooling mechanism 27 of the present embodiment cools the container 21 after heating in a sealed state.
　本実施形態の冷却機構２７としては、例えばペルチェ素子などが挙げられる。 As a cooling mechanism 27 of this embodiment, a Peltier device etc. are mentioned, for example.
［核酸抽出方法］
　以下、上述の核酸抽出装置１０３を用いる核酸抽出方法について説明する。第３実施形態の核酸抽出方法では、作製する工程Ｓ１、捕集する工程Ｓ２、収容する工程Ｓ３、および密閉する工程Ｓ４を、第２実施形態の核酸抽出方法と同様に行うことができる。 [Nucleic acid extraction method]
Hereinafter, a nucleic acid extraction method using the above-described nucleic acid extraction apparatus 103 will be described. In the nucleic acid extraction method of the third embodiment, the step S1 of producing, the step S2 of collecting, the step S3 of containing, and the step S4 of sealing can be performed in the same manner as the nucleic acid extraction method of the second embodiment.
（抽出する工程Ｓ５）
　本実施形態の抽出する工程Ｓ５においては、容器２１を加熱する。次に、加熱後の容器２１を、密閉状態のまま冷却機構２７を用いて冷却し、試料Ｂから核酸Ｎを抽出する。これにより、内部空間２４に急激な温度変化が生じやすい。その結果、試料Ｂを破壊しやすいので、試料Ｂから核酸Ｎを効率的に抽出することができる。したがって、この方法は、グラム陽性菌や真菌など堅い構造を有する試料Ｂに対し有効である。 (Step S5 of extracting)
In step S5 of extracting in the present embodiment, the container 21 is heated. Next, the container 21 after heating is cooled using the cooling mechanism 27 in a sealed state, and the nucleic acid N is extracted from the sample B. Thereby, a rapid temperature change is likely to occur in the internal space 24. As a result, since the sample B is easily destroyed, the nucleic acid N can be efficiently extracted from the sample B. Therefore, this method is effective for sample B having a rigid structure such as gram positive bacteria and fungi.
　このようにして、本実施形態の核酸抽出方法が行われる。 Thus, the nucleic acid extraction method of the present embodiment is performed.
　第３実施形態によれば、試料Ｂから核酸Ｎを効率的に抽出できる核酸抽出方法および核酸抽出装置１０３が提供される。特に、第３実施形態の核酸抽出方法および核酸抽出装置１０３は、内部空間２４の急激な温度変化により試料Ｂを破壊しやすいので、グラム陽性菌や真菌など堅い構造を有する試料Ｂに対し有効である。 According to the third embodiment, a nucleic acid extraction method and a nucleic acid extraction apparatus 103 capable of efficiently extracting the nucleic acid N from the sample B are provided. In particular, the nucleic acid extraction method and the nucleic acid extraction apparatus 103 according to the third embodiment is effective for the sample B having a rigid structure such as gram positive bacteria and fungi because the sample B is easily destroyed due to the rapid temperature change of the internal space 24. is there.
＜第４実施形態＞
　以下、本発明の第４実施形態における核酸抽出装置および核酸抽出方法について、図面に基づき説明する。 Fourth Embodiment
Hereinafter, a nucleic acid extraction apparatus and a nucleic acid extraction method according to a fourth embodiment of the present invention will be described based on the drawings.
［核酸抽出装置］
　図５は、第４実施形態の核酸抽出装置１０４を示す断面模式図である。図５に示すように、第４実施形態の核酸抽出装置１０４は、捕集部１０と、抽出部１２０と、本体部１００と、検出部２８と、通知する手段２９と、を備える。したがって、第４実施形態において第２実施形態と共通する構成要素については同じ符号を付し、詳細な説明は省略する。 [Nucleic acid extraction device]
FIG. 5 is a schematic cross-sectional view showing the nucleic acid extraction device 104 of the fourth embodiment. As shown in FIG. 5, the nucleic acid extraction device 104 of the fourth embodiment includes a collection unit 10, an extraction unit 120, a main unit 100, a detection unit 28, and a means 29 for notifying. Therefore, in the fourth embodiment, the components common to the second embodiment are denoted by the same reference numerals, and the detailed description is omitted.
（検出部）
　本実施形態の検出部２８は、容器２１の温度、および内部空間２４の圧力の少なくとも一方を検出する。本実施形態の検出部２８は、容器２１の温度、および内部空間２４の圧力の少なくとも一方を検出可能な装置を有する。例えば、本実施形態の検出部２８が容器２１の温度を検出する場合、検出部２８は上記装置として熱電対を有する。本実施形態の検出部２８の検出結果は、後述の通知する手段２９に出力される。 (Detection unit)
The detection unit 28 of the present embodiment detects at least one of the temperature of the container 21 and the pressure of the internal space 24. The detection unit 28 of the present embodiment has a device capable of detecting at least one of the temperature of the container 21 and the pressure of the internal space 24. For example, when the detection unit 28 of the present embodiment detects the temperature of the container 21, the detection unit 28 has a thermocouple as the above-described device. The detection result of the detection unit 28 of the present embodiment is output to a notifying unit 29 described later.
（通知する手段）
　本実施形態の通知する手段２９は、検出部２８の検出結果に基づいて、開口部２６の開閉を決定し、作業者に通知する。作業者は、通知する手段２９により通知された時点で蓋部材２５を手動で着脱し、開口部２６を開閉する。 (Means to notify)
Means 29 for notifying the present embodiment determines opening and closing of the opening 26 based on the detection result of the detection unit 28 and notifies the worker. The operator manually attaches and detaches the lid member 25 when notified by the notifying unit 29 and opens and closes the opening 26.
［核酸抽出方法］
　以下、上述の核酸抽出装置１０４を用いる核酸抽出方法について説明する。第４実施形態の核酸抽出方法では、作製する工程Ｓ１、捕集する工程Ｓ２、収容する工程Ｓ３、および密閉する工程Ｓ４を、第２実施形態の核酸抽出方法と同様に行うことができる。 [Nucleic acid extraction method]
Hereinafter, a nucleic acid extraction method using the above-described nucleic acid extraction device 104 will be described. In the nucleic acid extraction method of the fourth embodiment, the step S1 of producing, the step S2 of collecting, the step S3 of containing, and the step S4 of sealing can be performed in the same manner as the nucleic acid extraction method of the second embodiment.
（抽出する工程Ｓ５）
　本実施形態の抽出する工程Ｓ５において、容器２１を加熱する。 (Step S5 of extracting)
In the extraction step S5 of the present embodiment, the container 21 is heated.
　次に、検出部２８を用いて、加熱後の容器２１の温度および内部空間２４の圧力の少なくとも一方を検出する。 Next, the detection unit 28 is used to detect at least one of the temperature of the container 21 and the pressure of the internal space 24 after heating.
　次に、通知する手段２９を用いて、検出部２８の検出結果に基づいて、開口部２６の開閉を決定し、作業者に通知する。以下、通知する手段２９の通知について、具体例を挙げて説明する。 Next, using the notifying means 29, the opening and closing of the opening 26 is determined based on the detection result of the detecting unit 28, and the operator is notified. Hereinafter, the notification of the notifying means 29 will be described by taking a specific example.
（堅い構造を有する試料Ｂから核酸を抽出する場合）
　グラム陽性菌や真菌など堅い構造を有する試料Ｂから核酸Ｎを抽出する場合には、容器２１の温度が１００℃以上、または内部空間２４の圧力が大気圧以上であるとき、通知する手段２９が作業者に、開口部２６の開放を通知する。作業者は、通知する手段２９により通知された時点で蓋部材２５を手動で取り外し、開口部２６を開放する。これにより、内部空間２４に急激な圧力変化が生じ、内部空間２４に収容された試料Ｂにせん断応力が発生する。その結果、試料Ｂを破壊しやすくなり、試料Ｂから核酸Ｎをより効率的に抽出することができる。 (When extracting nucleic acid from sample B having a rigid structure)
When extracting nucleic acid N from sample B which has a rigid structure such as gram positive bacteria and fungi, the means 29 for notifying when the temperature of the container 21 is 100 ° C. or higher or the pressure of the internal space 24 is atmospheric pressure or higher. The operator is notified that the opening 26 is open. The operator manually removes the lid member 25 when notified by the notifying means 29, and opens the opening 26. As a result, a rapid pressure change occurs in the inner space 24 and shear stress is generated in the sample B accommodated in the inner space 24. As a result, the sample B can be easily destroyed, and the nucleic acid N can be extracted more efficiently from the sample B.
（試料Ｂから長鎖の核酸を抽出する場合）
　試料Ｂから破壊されやすい長鎖の核酸Ｎを抽出する場合には、容器２１の温度が１００℃未満、または内部空間２４の圧力が大気圧であるとき、通知する手段２９が作業者に、開口部２６の開放を通知する。作業者は、通知する手段２９により通知された時点で蓋部材２５を手動で取り外し、開口部２６を開放する。これにより、内部空間２４に急激な圧力変化が生じにくく、内部空間２４に収容された試料Ｂにせん断応力が発生しにくい。その結果、試料Ｂから抽出した核酸Ｎが破壊されるのを抑制できる。 (When extracting long-chain nucleic acid from sample B)
When extracting a long-chain nucleic acid N which is easily broken from the sample B, the means 29 for notifying the worker when the temperature of the container 21 is less than 100 ° C. or the pressure of the internal space 24 is atmospheric pressure is opened. The release of the part 26 is notified. The operator manually removes the lid member 25 when notified by the notifying means 29, and opens the opening 26. As a result, a rapid pressure change does not easily occur in the internal space 24, and shear stress does not easily occur in the sample B accommodated in the internal space 24. As a result, destruction of the nucleic acid N extracted from the sample B can be suppressed.
　このようにして、本実施形態の核酸抽出方法が行われる。 Thus, the nucleic acid extraction method of the present embodiment is performed.
　第４実施形態によれば、試料Ｂから核酸Ｎを効率的に抽出できる核酸抽出方法および核酸抽出装置１０４が提供される。特に、第４実施形態の核酸抽出方法および核酸抽出装置１０４は、処理対象である試料Ｂの種類や、抽出する核酸Ｎの種類に応じて、内部空間２４の圧力変化を制御することができる。 According to the fourth embodiment, a nucleic acid extraction method and a nucleic acid extraction device 104 capable of efficiently extracting the nucleic acid N from the sample B are provided. In particular, the nucleic acid extraction method and the nucleic acid extraction apparatus 104 of the fourth embodiment can control the pressure change of the internal space 24 according to the type of the sample B to be processed and the type of the nucleic acid N to be extracted.
＜第５実施形態＞
　以下、本発明の第５実施形態における核酸抽出方法について、説明する。 Fifth Embodiment
Hereinafter, the nucleic acid extraction method in the fifth embodiment of the present invention will be described.
［核酸抽出装置］
　図６は、第５実施形態の核酸抽出装置１０５を示す断面模式図である。図６に示すように、第５実施形態の核酸抽出装置１０５は、捕集部１０と、抽出部３２０と、本体部１００と、検出部２８Ａと、駆動部３０と、制御部３１と、を備える。したがって、第４実施形態において第２実施形態と共通する構成要素については同じ符号を付し、詳細な説明は省略する。 [Nucleic acid extraction device]
FIG. 6 is a schematic cross-sectional view showing the nucleic acid extraction device 105 of the fifth embodiment. As shown in FIG. 6, the nucleic acid extraction apparatus 105 of the fifth embodiment includes the collection unit 10, the extraction unit 320, the main unit 100, the detection unit 28A, the drive unit 30, and the control unit 31. Prepare. Therefore, in the fourth embodiment, the components common to the second embodiment are denoted by the same reference numerals, and the detailed description is omitted.
（抽出部）
　本実施形態の抽出部３２０は、容器２１と、収容する手段２２と、栓部材２５Ａと、加熱機構２３と、を有する。 (Extraction unit)
The extraction unit 320 of the present embodiment has a container 21, means 22 for containing, a plug member 25 A, and a heating mechanism 23.
　本明細書において、「栓部材２５Ａ」は請求の範囲における「閉塞部材」に相当する。 In the present specification, the "plug member 25A" corresponds to the "closing member" in the claims.
（栓部材）
　本実施形態の栓部材２５Ａは、開口部２６を塞ぐことができる。栓部材２５Ａは、開口部２６を塞ぐことができる機械的な構造を有する部材であれば、特に限定されない。例えば、栓部材２５Ａは、容器２１の内径より僅かに小さい内径を有するゴム栓またはシリコーン栓などが挙げられる。 (Plug member)
The plug member 25A of the present embodiment can close the opening 26. The plug member 25A is not particularly limited as long as it is a member having a mechanical structure capable of closing the opening 26. For example, the plug member 25A may be a rubber plug or a silicone plug having an inner diameter slightly smaller than the inner diameter of the container 21.
　本実施形態の栓部材２５Ａは、容器２１と一体であってもよく、別体であってもよい。 The plug member 25A of the present embodiment may be integral with or separate from the container 21.
　また、本実施形態の栓部材２５Ａは、容器２１の内部空間２４の圧力が上昇する際、容器２１から栓部材２５Ａが脱落するのを抑制する押さえ部材（図示なし）を有していてもよい。 Further, the plug member 25A of the present embodiment may have a pressing member (not shown) for suppressing the drop of the plug member 25A from the container 21 when the pressure in the internal space 24 of the container 21 rises. .
（検出部）
　本実施形態の検出部２８Ａは、容器２１の温度、および内部空間２４の圧力の少なくとも一方を検出する。本実施形態の検出部２８Ａは、容器２１の温度、および内部空間２４の圧力の少なくとも一方を検出可能な装置を有する。本実施形態の検出部２８Ａの検出結果は、後述の制御部３１に出力される。 (Detection unit)
The detection unit 28A of the present embodiment detects at least one of the temperature of the container 21 and the pressure of the internal space 24. The detection unit 28A of the present embodiment has a device capable of detecting at least one of the temperature of the container 21 and the pressure of the internal space 24. The detection result of the detection unit 28A of the present embodiment is output to the control unit 31 described later.
（駆動部）
　本実施形態の駆動部３０は、栓部材２５Ａを駆動させる。 (Drive part)
The drive unit 30 of the present embodiment drives the plug member 25A.
（制御部）
　本実施形態の制御部３１は、検出部２８Ａの検出結果に基づいて、少なくとも駆動部３０の駆動を制御する。本実施形態の制御部３１は、駆動部３０以外に加熱機構２３を制御してもよい。 (Control unit)
The control unit 31 of the present embodiment controls at least the drive of the drive unit 30 based on the detection result of the detection unit 28A. The control unit 31 of the present embodiment may control the heating mechanism 23 in addition to the drive unit 30.
［核酸抽出方法］
　以下、上述の核酸抽出装置１０５を用いる核酸抽出方法について説明する。第５実施形態の核酸抽出方法では、作製する工程Ｓ１、捕集する工程Ｓ２、および収容する工程Ｓ３を、第２実施形態の核酸抽出方法と同様に行うことができる。 [Nucleic acid extraction method]
Hereinafter, a nucleic acid extraction method using the above-described nucleic acid extraction apparatus 105 will be described. In the nucleic acid extraction method of the fifth embodiment, the step S1 of producing, the step S2 of collecting, and the step S3 of holding can be performed in the same manner as the nucleic acid extraction method of the second embodiment.
（密閉する工程Ｓ４）
　本実施形態の密閉する工程Ｓ４では、栓部材２５Ａを用いて、開口部２６を塞ぐことにより、試料Ｂが捕集されたメンブレンフィルター１１、および液体Ｌが収容された容器２１を密閉する。 (Step S4 of sealing)
In the sealing step S4 of the present embodiment, by closing the opening 26 using the plug member 25A, the membrane filter 11 in which the sample B is collected and the container 21 in which the liquid L is stored are sealed.
（抽出する工程Ｓ５）
　本実施形態の抽出する工程Ｓ５において、容器２１を加熱する。 (Step S5 of extracting)
In the extraction step S5 of the present embodiment, the container 21 is heated.
　次に、検出部２８Ａを用いて、加熱後の容器２１の温度および内部空間２４の圧力の少なくとも一方を検出する。 Next, at least one of the temperature of the heated container 21 and the pressure of the internal space 24 is detected using the detection unit 28A.
　次に、制御部３１を用いて、検出部２８Ａの検出結果に基づいて、少なくとも駆動部３０の駆動を制御する。以下、制御部３１の制御について、具体例を挙げて説明する。 Next, the control unit 31 controls at least the drive of the drive unit 30 based on the detection result of the detection unit 28A. Hereinafter, control of the control unit 31 will be described by taking a specific example.
（堅い構造を有する試料Ｂから核酸を抽出する場合）
　グラム陽性菌や真菌など堅い構造を有する試料Ｂから核酸Ｎを抽出する場合には、容器２１の温度が１００℃以上、または内部空間２４の圧力が大気圧以上であるとき、駆動部３０を駆動させる。駆動部３０の駆動により栓部材２５Ａを自動で取り外し、容器２１を開放する。これにより、内部空間２４に急激な圧力変化が生じ、内部空間２４に収容された試料Ｂにせん断応力が発生する。その結果、試料Ｂを破壊しやすくなり、試料Ｂから核酸Ｎをより効率的に抽出することができる。 (When extracting nucleic acid from sample B having a rigid structure)
In the case of extracting nucleic acid N from sample B having a rigid structure such as gram positive bacteria or fungus, the driving unit 30 is driven when the temperature of the container 21 is 100 ° C. or higher, or the pressure of the internal space 24 is atmospheric pressure or higher. Let The plug member 25A is automatically removed by the drive of the drive unit 30, and the container 21 is opened. As a result, a rapid pressure change occurs in the inner space 24 and shear stress is generated in the sample B accommodated in the inner space 24. As a result, the sample B can be easily destroyed, and the nucleic acid N can be extracted more efficiently from the sample B.
（試料Ｂから長鎖の核酸を抽出する場合）
　試料Ｂから破壊されやすい長鎖の核酸Ｎを抽出する場合には、容器２１の温度が１００℃未満、または内部空間２４の圧力が大気圧であるとき、駆動部３０を駆動させる。駆動部３０の駆動により栓部材２５Ａを自動で取り外し、容器２１を開放する。これにより、内部空間２４に急激な圧力変化が生じにくく、内部空間２４に収容された試料Ｂにせん断応力が発生しにくい。その結果、試料Ｂから抽出した核酸Ｎが破壊されるのを抑制できる。 (When extracting long-chain nucleic acid from sample B)
When extracting a long-chain nucleic acid N that is easily broken from the sample B, the driving unit 30 is driven when the temperature of the container 21 is less than 100 ° C. or the pressure of the internal space 24 is atmospheric pressure. The plug member 25A is automatically removed by the drive of the drive unit 30, and the container 21 is opened. As a result, a rapid pressure change does not easily occur in the internal space 24, and shear stress does not easily occur in the sample B accommodated in the internal space 24. As a result, destruction of the nucleic acid N extracted from the sample B can be suppressed.
　このようにして、本実施形態の核酸抽出方法が行われる。 Thus, the nucleic acid extraction method of the present embodiment is performed.
　第５実施形態によれば、自動で試料Ｂから核酸Ｎを効率的に抽出できる核酸抽出方法および核酸抽出装置１０５が提供される。特に、第５実施形態の核酸抽出方法および核酸抽出装置１０５は、処理対象である試料Ｂの種類や、抽出する核酸Ｎの種類に応じて、内部空間２４の圧力変化を制御することができる。 According to the fifth embodiment, a nucleic acid extraction method and a nucleic acid extraction apparatus 105 capable of automatically extracting the nucleic acid N from the sample B are provided. In particular, the nucleic acid extraction method and the nucleic acid extraction apparatus 105 of the fifth embodiment can control the pressure change of the internal space 24 according to the type of the sample B to be processed and the type of the nucleic acid N to be extracted.
＜第６実施形態＞
　以下、本発明の第６実施形態における核酸抽出方法について、説明する。 Sixth Embodiment
Hereinafter, the nucleic acid extraction method in the sixth embodiment of the present invention will be described.
　本実施形態の液体は、第１液体と、第２液体と、を有する。 The liquid of the present embodiment includes a first liquid and a second liquid.
　第１液体は、水と、上述の細胞溶解促進剤と、必要に応じて使用される上述の緩衝液と、を含む。 The first liquid contains water, the above-mentioned cytolytic promoter, and the above-mentioned buffer solution which is optionally used.
　一方、第２液体は、第１液体よりも高沸点である。第２液体としては、例えばミネラルオイルなどが挙げられる。 On the other hand, the second liquid has a boiling point higher than that of the first liquid. As a 2nd liquid, mineral oil etc. are mentioned, for example.
　本実施形態の核酸抽出方法においては、試料Ｂの量が内部空間２４の容積に対して極めて少ない場合でも、内部空間２４の空隙部分が第２液体によって充填される。これにより、試料Ｂの量が内部空間２４の容積に対して極めて少ない場合でも、容器２１を加熱する際、容器２１の内部空間２４が加圧された状態となりやすい。内部空間２４が加圧された状態となることで、メンブレンフィルター１１に捕集された試料Ｂも加圧される。その結果、試料Ｂから核酸Ｎを効率的に抽出することができる。 In the nucleic acid extraction method of the present embodiment, even when the amount of the sample B is extremely small relative to the volume of the inner space 24, the void portion of the inner space 24 is filled with the second liquid. As a result, even when the amount of the sample B is extremely small relative to the volume of the internal space 24, when heating the container 21, the internal space 24 of the container 21 tends to be pressurized. When the internal space 24 is pressurized, the sample B collected by the membrane filter 11 is also pressurized. As a result, nucleic acid N can be efficiently extracted from sample B.
　第６実施形態によれば、試料Ｂから核酸Ｎを効率的に抽出できる核酸抽出方法および核酸抽出装置が提供される。特に、第６実施形態の核酸抽出方法は、試料Ｂの量が内部空間２４の容積に対して極めて少ない場合であっても有効である。 According to the sixth embodiment, a nucleic acid extraction method and a nucleic acid extraction apparatus capable of efficiently extracting the nucleic acid N from the sample B are provided. In particular, the nucleic acid extraction method of the sixth embodiment is effective even when the amount of the sample B is extremely small relative to the volume of the internal space 24.
　以上、本発明の実施形態を説明したが、本実施形態における各構成およびそれらの組み合わせ等は一例であり、本発明の趣旨から逸脱しない範囲内で、構成の付加、省略、置換、およびその他の変更が可能である。また、本発明は実施形態によって限定されることはない。 As mentioned above, although embodiment of this invention was described, each structure in this embodiment, those combinations, etc. are an example, In the range which does not deviate from the meaning of this invention, addition of structure, omission, substitution, and others Changes are possible. Further, the present invention is not limited by the embodiments.
　例えば、上記実施形態の収容する工程Ｓ３において、容器２１の内部空間２４に液体Ｌを注入する代わりに、抽出する工程Ｓ５において、加熱後の容器２１の内部空間２４に液体Ｌを注入してもよい。この方法によっても、液体Ｌに核酸Ｎを抽出することができる。さらに、上記液体Ｌが細胞溶解促進剤を含む場合、細胞溶解促進剤が試料Ｂの膜構造に作用することで、試料Ｂを破壊しやすくなる。その結果、試料Ｂから核酸Ｎをより効率的に抽出することができる。 For example, instead of injecting the liquid L into the internal space 24 of the container 21 in the step S3 of the above embodiment, the liquid L may be injected into the internal space 24 of the container 21 after heating in the extracting step S5. Good. The nucleic acid N can be extracted to the liquid L also by this method. Furthermore, when the liquid L contains a cell lysis promoter, the cell lysis promoter acts on the membrane structure of the sample B, thereby making it easy to destroy the sample B. As a result, the nucleic acid N can be extracted more efficiently from the sample B.
　以下に本発明を実施例により説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES The present invention will be described by way of examples, but the present invention is not limited to these examples.
　図３に示すような構成の核酸抽出装置を用いて、核酸を有する試料から核酸を抽出した。 The nucleic acid was extracted from the sample having the nucleic acid using a nucleic acid extraction apparatus configured as shown in FIG.
［実施例１］
　まず、市販のミネラルウォーターに、大腸菌株（ＮＢＲＣ３９７２）および黄色ブドウ球菌株（ＮＢＲＣ１２７３２）と、濃縮したＳＣＤ培地と、を添加した後、３７℃で４８時間培養し、培養液を作製した（作製する工程）。 Example 1
First, E. coli strain (NBRC3972) and S. aureus strain (NBRC12732) and concentrated SCD medium were added to commercially available mineral water, and then cultured at 37 ° C. for 48 hours to prepare a culture solution (prepared Process).
　次に、メンブレンフィルター（直径１３ｍｍ、孔径０．４５μｍ、親水性ＰＣ製）を装着した濾過装置（捕集部）を用い、培養液１ｍＬを濾過し、メンブレンフィルターに菌（試料）を捕集した（捕集する工程）。 Next, using a filtration apparatus (collection unit) equipped with a membrane filter (diameter 13 mm, pore diameter 0.45 μm, manufactured by hydrophilic PC), 1 mL of culture solution was filtered to collect bacteria (sample) on the membrane filter (Step of collecting).
　次に、菌が捕集されたメンブレンフィルターと、予め調製しておいた液体（４０ｍＭ　Ｔｒｉｓ－ＨＣｌと０．５％ＳＤＳとの混合液）２００μＬと、を容器に収容した（収容する工程）。 Next, a membrane filter in which bacteria were collected and 200 μL of a liquid (a mixed solution of 40 mM Tris-HCl and 0.5% SDS) prepared in advance were accommodated in a container (a step of accommodating).
　さらに、容器の開口部を塞ぎ、容器を密閉した（密閉する工程）。 Furthermore, the opening of the container was closed to seal the container (sealing step).
　次に、容器をヒートブロック（加熱機構）に接触させ、１４０℃で４５秒間加熱し、加熱後に急冷し、大腸菌株および黄色ブドウ球菌株からＤＮＡ（核酸）を抽出した（抽出する工程）。 Next, the container was brought into contact with a heat block (heating mechanism), heated at 140 ° C. for 45 seconds, and quenched after heating to extract DNA (nucleic acid) from E. coli strains and S. aureus strains (extraction step).
［評価］
　上記抽出する工程で得られた溶液を１０倍希釈し、ゲノムＤＮＡ中に存在する遺伝子を増幅可能なプライマーを用いて、リアルタイムＰＣＲを行った。 [Evaluation]
The solution obtained in the above extraction step was diluted 10-fold, and a real-time PCR was performed using primers capable of amplifying a gene present in genomic DNA.
　リアルタイムＰＣＲより算出された上記溶液中に抽出されたＰＣＲによる増幅が可能なＤＮＡ量（検出値）をそれぞれ求めた。作製する工程で用いた大腸菌株または黄色ブドウ球菌株の量に対する各検出値の割合を百分率でそれぞれ表し、これらを抽出効率とした。結果を表１に示す。 The amount (detected value) of DNA capable of amplification by PCR extracted in the above solution calculated by real time PCR was respectively determined. The ratio of each detection value to the amount of E. coli strain or S. aureus strain used in the step of producing was expressed as a percentage, and these were regarded as extraction efficiency. The results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表１に示すように、本発明の一態様を適用した実施例１では、大腸菌株および黄色ブドウ球菌株からＤＮＡを効率的に抽出できることがわかった。また、抽出したＤＮＡは、ＰＣＲによる増幅が可能であった。このことから、本発明の一態様の核酸抽出方法は、リアルタイムＰＣＲ検出に適用できることが確認された。 As shown in Table 1, in Example 1 to which one aspect of the present invention was applied, it was found that DNA can be efficiently extracted from E. coli strains and S. aureus strains. Moreover, the extracted DNA was able to be amplified by PCR. From this, it was confirmed that the nucleic acid extraction method of one embodiment of the present invention can be applied to real-time PCR detection.
　以上のことから、本発明が有用であることが示された。 From the above, it is shown that the present invention is useful.
　１０…捕集部、１１…メンブレンフィルター、２０，１２０，２２０，３２０…抽出部、２１…容器、２２…収容する手段、２２Ａ…注入する手段、２３…加熱機構、２４…内部空間、２５…蓋部材、２５Ａ…栓部材、２６…開口部、２７…冷却機構、２８，２８Ａ…検出部、２９…通知する手段、３０…駆動部、３１…制御部、１００…本体部、１０１，１０２，１０３，１０４，１０５…核酸抽出装置、Ｂ…試料、ＢＲ…培養液、Ｌ…液体、Ｎ…核酸 DESCRIPTION OF SYMBOLS 10 ... Collection part, 11 ... Membrane filter, 20, 120, 220, 320 ... Extraction part, 21 ... Container, 22 ... Means to accommodate, 22A ... Means to inject, 23 ... Heating mechanism, 24 ... Internal space, 25 ... Lid member, 25A: plug member, 26: opening, 27: cooling mechanism, 28, 28A: detection unit, 29: means for notifying, 30: drive unit, 31: control unit, 100: main unit, 101, 102, 103, 104, 105: nucleic acid extraction apparatus, B: sample, BR: culture solution, L: liquid, N: nucleic acid

Claims (19)

  1. 　メンブレンフィルターを用いて核酸を有する試料を捕集する工程と、
    　前記試料が捕集された前記メンブレンフィルターを容器の内部空間に収容する工程と、
    　前記メンブレンフィルターが収容された前記容器を密閉する工程と、
    　前記容器を加熱し、前記試料から核酸を抽出する工程と、を有する核酸抽出方法。     Collecting a sample having nucleic acid using a membrane filter;
    Storing the membrane filter in which the sample is collected in an internal space of a container;
    Sealing the container containing the membrane filter;
    And heating the container to extract nucleic acid from the sample.
  2. 　前記抽出する工程において、加熱後の前記容器の前記内部空間に水を含有する液体を注入し、前記液体に前記核酸を抽出する請求項１に記載の核酸抽出方法。 The nucleic acid extraction method according to claim 1, wherein a liquid containing water is injected into the internal space of the container after heating in the extraction step, and the nucleic acid is extracted into the liquid.
  3. 　前記収容する工程において、水を含有する液体を前記内部空間に収容する請求項１に記載の核酸抽出方法。 The nucleic acid extraction method according to claim 1, wherein a liquid containing water is accommodated in the internal space in the accommodating step.
  4. 　前記液体は、アルカリ、酸、酵素、界面活性剤、酸化還元剤およびタンパク変性剤からなる群から選ばれる少なくとも一種の細胞溶解促進剤を含む請求項２または３に記載の核酸抽出方法。 The method for nucleic acid extraction according to claim 2 or 3, wherein the liquid contains at least one cell lysis promoter selected from the group consisting of alkalis, acids, enzymes, surfactants, redox agents and protein denaturants.
  5. 　前記界面活性剤が、ドデシル硫酸ナトリウムと、オクチルフェノールエトキシレートとのいずれか一方または両方である請求項４に記載の核酸抽出方法。 The method for nucleic acid extraction according to claim 4, wherein the surfactant is one or both of sodium dodecyl sulfate and octylphenol ethoxylate.
  6. 　前記メンブレンフィルターは、親水性である請求項１～５のいずれか１項に記載の核酸抽出方法。 The nucleic acid extraction method according to any one of claims 1 to 5, wherein the membrane filter is hydrophilic.
  7. 　前記抽出する工程において、加熱後の前記容器を、密閉状態のまま冷却し、前記核酸を抽出する請求項１～６のいずれか１項に記載の核酸抽出方法。 The nucleic acid extraction method according to any one of claims 1 to 6, wherein in the extraction step, the heated container is cooled in a sealed state to extract the nucleic acid.
  8. 　前記抽出する工程において、加熱後の前記容器を、前記内部空間の圧力が大気圧以上である状態で開放して、前記核酸を抽出する請求項１～７のいずれか１項に記載の核酸抽出方法。 The nucleic acid extraction according to any one of claims 1 to 7, wherein in the extraction step, the container after heating is opened in a state where the pressure in the internal space is equal to or higher than atmospheric pressure, and the nucleic acid is extracted. Method.
  9. 　前記抽出する工程において、冷却後の前記容器を再加熱して、前記核酸を抽出する請求項８に記載の核酸抽出方法。 The nucleic acid extraction method according to claim 8, wherein in the extraction step, the cooled container is reheated to extract the nucleic acid.
  10. 　前記収容する工程において、前記内部空間に前記試料が捕集された前記メンブレンフィルターを収容した後、前記液体を注入する請求項３～９のいずれか１項に記載の核酸抽出方法。 The nucleic acid extraction method according to any one of claims 3 to 9, wherein in the step of containing, the liquid is injected after the membrane filter from which the sample is collected is housed in the internal space.
  11. 　前記抽出する工程において、前記液体が、前記容器と、前記試料が捕集された前記メンブレンフィルターとの空隙に充填されている請求項３～１０のいずれか１項に記載の核酸抽出方法。 The nucleic acid extraction method according to any one of claims 3 to 10, wherein in the extraction step, the liquid is filled in a space between the container and the membrane filter from which the sample is collected.
  12. 　前記液体は、第１液体と、第２液体と、を有し、
    　前記第１液体は、前記水と、前記細胞溶解促進剤と、を含み、
    　前記第２液体は、前記第１液体よりも高沸点である請求項４～１１のいずれか１項に記載の核酸抽出方法。     The liquid comprises a first liquid and a second liquid,
    The first liquid contains the water and the cell lysis promoter.
    The nucleic acid extraction method according to any one of claims 4 to 11, wherein the second liquid has a boiling point higher than that of the first liquid.
  13. 　メンブレンフィルターを用いて核酸を有する試料を捕集する捕集部と、
    　捕集された前記試料から核酸を抽出する抽出部と、を備え、
    　前記抽出部は、
    　前記メンブレンフィルターを収容可能な内部空間を有する容器と、
    　前記試料が捕集された前記メンブレンフィルターを、前記内部空間に収容する手段と、
    　前記容器を密閉状態で加熱する加熱機構と、を有する核酸抽出装置。     A collection unit that collects a sample having nucleic acid using a membrane filter;
    An extraction unit for extracting a nucleic acid from the collected sample;
    The extraction unit
    A container having an internal space capable of accommodating the membrane filter;
    A means for storing the membrane filter in which the sample is collected in the internal space;
    And a heating mechanism for heating the container in a sealed state.
  14. 　前記収容する手段は、前記内部空間に水を含有する液体を注入する手段を有する請求項１３に記載の核酸抽出装置。 The nucleic acid extraction device according to claim 13, wherein the means for containing comprises means for injecting a liquid containing water into the internal space.
  15. 　前記メンブレンフィルターの材質は、親水性である請求項１３または１４に記載の核酸抽出装置。 The nucleic acid extraction device according to claim 13, wherein a material of the membrane filter is hydrophilic.
  16. 　前記抽出部は、冷却機構を有し、
    　前記冷却機構は、加熱後の前記容器を、密閉状態のまま冷却する請求項１３～１５のいずれか１項に記載の核酸抽出装置。     The extraction unit has a cooling mechanism,
    The nucleic acid extraction device according to any one of claims 13 to 15, wherein the cooling mechanism cools the heated container in a sealed state.
  17. 　前記容器は、前記内部空間と連通する開口部を有し、
    　前記抽出部は、前記開口部を塞ぐことができる閉塞部材を有する請求項１３～１６のいずれか１項に記載の核酸抽出装置。     The container has an opening communicating with the internal space,
    The nucleic acid extraction device according to any one of claims 13 to 16, wherein the extraction unit has a closing member capable of closing the opening.
  18. 　前記容器の温度、および前記内部空間の圧力の少なくとも一方を検出する検出部と、
    　前記検出部の検出結果に基づいて、前記開口部の開閉を決定し、通知する手段と、を備える請求項１７に記載の核酸抽出装置。     A detection unit that detects at least one of the temperature of the container and the pressure of the internal space;
    The nucleic acid extraction device according to claim 17, further comprising: means for determining opening / closing of the opening based on the detection result of the detection unit and notifying the opening / closing.
  19. 　前記閉塞部材を駆動させる駆動部と、
    　前記容器の温度、および前記内部空間の圧力の少なくとも一方を検出する検出部と、
    　前記検出部の検出結果に基づいて、少なくとも前記駆動部を制御する制御部と、を備える請求項１８に記載の核酸抽出装置。     A drive unit for driving the closing member;
    A detection unit that detects at least one of the temperature of the container and the pressure of the internal space;
    The nucleic acid extraction device according to claim 18, further comprising: a control unit configured to control at least the drive unit based on a detection result of the detection unit.
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