WO2019043779A1 - Procédé d'extraction d'acide nucléique et dispositif d'extraction d'acide nucléique - Google Patents

Procédé d'extraction d'acide nucléique et dispositif d'extraction d'acide nucléique 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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nucleic acid
container
sample
acid extraction
internal space
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PCT/JP2017/030906
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English (en)
Japanese (ja)
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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/fr
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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

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Abstract

L'invention concerne un procédé d'extraction d'acide nucléique qui comprend : une étape de capture d'un échantillon contenant un acide nucléique à l'aide d'un filtre à membrane ; une étape de logement du filtre à membrane, qui maintient l'échantillon capturé par celui-ci, dans l'espace interne d'un récipient ; une étape consistant à sceller le récipient contenant le filtre à membrane ; et une étape consistant à chauffer le récipient et à extraire ainsi l'acide nucléique de l'échantillon.
PCT/JP2017/030906 2017-08-29 2017-08-29 Procédé d'extraction d'acide nucléique et dispositif d'extraction d'acide nucléique WO2019043779A1 (fr)

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