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WO2020218551A1 - Sequence-screening method from single-cell genome library using gel encapsulation technique - Google Patents

Sequence-screening method from single-cell genome library using gel encapsulation technique Download PDF

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Publication number
WO2020218551A1
WO2020218551A1 PCT/JP2020/017790 JP2020017790W WO2020218551A1 WO 2020218551 A1 WO2020218551 A1 WO 2020218551A1 JP 2020017790 W JP2020017790 W JP 2020017790W WO 2020218551 A1 WO2020218551 A1 WO 2020218551A1
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cell
cells
capsule
amplification
drug
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PCT/JP2020/017790
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French (fr)
Japanese (ja)
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正人 細川
春子 竹山
西川 洋平
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bitBiome株式会社
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions

Definitions

  • the present disclosure relates to the application of individual analysis of single cell or cell-like structures using the encapsulation method.
  • Genome sequence information is basic information for understanding living organisms, and genome decoding enables understanding of the diversity and function of microorganisms related to lineage, evolution, diseases, and biogeochemical cycle. For this reason, whole-genome analysis is indispensable for understanding the function of refractory culture.
  • metagenomic analysis in which various microbial genomes are collectively extracted from a sample and sequenced, has been the mainstream.
  • each cell is first divided and then processing is started. Since only the genetic information derived from a single cell is directly acquired, for example, the calculation difficulty for reconstructing the genomic information is overwhelmingly easier than the metagenomic analysis. However, highly accurate cell manipulation and nucleic acid reaction are required to decipher the extremely small amount of intracellular genome sequence without omission.
  • the steps of 1-cell genome analysis can be roughly divided into (1) separation of 1 cell of microorganism, (2) lysis of microorganism, (3) whole genome amplification, and (4) sequence analysis of amplified genome.
  • the reaction system of several tens of microliters used in the conventional molecular biology experiment is inappropriate for precisely handling a very small sample of one cell according to the above flow.
  • the genome decoding rate remained at about 30%. Since it is difficult to completely prevent contamination from aerosols and the like in an open experimental environment, a clean experimental environment dedicated to single-cell genome analysis experiments is required. Another problem is that the throughput of the reaction system is extremely low with respect to the need to execute the sequence from each cell, and expensive dispensing robots and the like have been used for experimental operations.
  • nucleic acid amplification and detection can be performed as it is, and have completed the present disclosure.
  • Examples of embodiments of the present disclosure include: (Item 1) (A) A step of encapsulating two or more cells or cell-like structures together with a first agent for each cell or cell-like structure.
  • (B) A step of performing a reaction based on the first drug on the cell or cell-like structure in a capsule, and (C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each cell or cell-like structure or a substance derived thereto together with a second agent.
  • (D) A step of carrying out a reaction based on the second drug on the cell or cell-like structure in a capsule, and (E) If necessary, the cell or cell-like structure or a substance derived thereto, or a capsule containing them, is encapsulated for each cell or cell-like structure or a substance derived thereto together with a drug X.
  • X is an integer of 3 or more.
  • (B) A step of performing a reaction based on the first drug on the cell or cell-like structure in a capsule, and (C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each cell or cell-like structure or a substance derived thereto together with a second agent.
  • (D) A step of carrying out a reaction based on the second drug on the cell or cell-like structure in a capsule, and (E) If necessary, the cell or cell-like structure or a substance derived thereto, or a capsule containing them, is encapsulated for each cell or cell-like structure or a substance derived thereto together with a drug X.
  • X is an integer of 3 or more.
  • (D') A method for analyzing a cell or cell-like structure comprising the step of analyzing the cell or cell-like structure with the desired agent.
  • (Item 3) The method according to any one of the above items, wherein (E) to (G) do not exist, and the first drug and the second drug are drugs required for nucleic acid amplification.
  • (Item 4) The method according to any one of the above items, wherein the desired drug is a drug required for nucleic acid amplification.
  • the single cell or single cell-like structure is encapsulated by flowing the suspension of the single cell or single cell-like structure into a microchannel and shearing the suspension with oil.
  • the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, matrigel, collagen or a photocurable resin.
  • the solubilizing reagents are lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride.
  • the first amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
  • the second amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
  • the one cell or cell-like structure and the first label are encapsulated in the droplet.
  • (Item 23) The method according to any one of the above items, wherein the nucleic acid contained in the cell or cell-like structure in A) is genomic DNA.
  • (Item 24) The method according to any one of the above items, which comprises amplifying a species-specific sequence or a sequence stored in a specific species in the analysis of C).
  • (Item 25) A system for analyzing cells or cell-like structures [X] A drug for encapsulating two or more cells or cell-like structures or a substance derived from them, or a capsule containing them, and / or an encapsulating drug storage unit for storing them.
  • [Y] A desired drug used for analysis and / or an analytical drug storage unit for storing them, A system comprising [Z] means for encapsulation and [W] means for performing analysis with the desired agent, if desired.
  • the system of item 25 further comprising the features of any one or more of the above items.
  • (Item 26) A kit for providing capsules for analyzing cells or cell-like structures.
  • [X] An agent for encapsulating two or more cells or cell-like structures or substances derived from them, or capsules containing them.
  • [Y] With the desired drug used for analysis A kit that includes.
  • the kit of item 26 further comprising the features of any one or more of the above items.
  • a method of analyzing cells or cell-like structures (A) A step of encapsulating two or more cells or cell-like structures together with a drug for nucleic acid amplification for each cell or cell-like structure. (B) A step of performing the first nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and (C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each of the cell or cell-like structure or a substance derived thereto together with a drug for nucleic acid amplification.
  • (D) A step of performing a second nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and (E) If necessary, the cell or cell-like structure or a substance derived from the cell or cell-like structure obtained in D), or a capsule containing them, is prepared for each cell or cell-like structure or a substance derived from the cell or cell-like structure.
  • nucleic acid amplification, detection, selection, etc. can be performed while holding individual single biological units (for example, "single cell") as a set, and when it is desired to acquire rare bacterial data, host DNA When a large amount of such substances are mixed, data on only specific bacteria is required, and it can be applied when evaluation of diversity is unnecessary.
  • single biological units for example, "single cell”
  • the probability of catching all rare microbial species is high.
  • the present disclosure can also identify microbial species in advance and select samples for transition to analysis such as whole genome sequences.
  • the present disclosure can also selectively select or eliminate certain microorganisms to perform effective genomic or other analyses by preliminarily examining the presence or absence of specific specific genes.
  • FIG. 1 is a diagram showing a step of encapsulating a single cell or cell-like structure in a droplet and performing primary amplification. After the primary amplification, the nucleic acid in the gel capsule is stained using a fluorescent DNA intercalator such as DAPI.
  • FIG. 2 is a diagram showing a step of reencapsulating a gel capsule in a droplet.
  • the PCR primer and the PCR reaction solution to which the fluorescently labeled probe is added are also re-encapsulated.
  • the PCR reaction solution is an example, and other than this, a LAMP reaction, a SmartAmp method, and an RPA (recombinase polymerase) amplification method are also possible.
  • FIG. 3 is a diagram showing PCR amplification (or other technique) and gene detection.
  • a label such as Taqman primer
  • Gene detection can also be performed by using a DNA-binding fluorescent intercalator and a primer for specific gene detection in combination.
  • FIG. 4 is a diagram showing the stages of secondary amplification. The droplets to be subjected to the secondary amplification can be selected based on the results of staining and gene detection after the primary amplification. Sorting is also possible with flow cytometry, fluorescence microscopy, and microfluidic devices.
  • FIG. 5 is a diagram showing the steps of confirmation by PCR and library preparation.
  • FIG. 6 is a schematic diagram showing the overall flow of the sequence screening method from the 1-cell genome library by the gel capsule method.
  • FIG. 7 is a diagram showing a gel capsule at the time of gene detection.
  • FIG. 7A shows staining of gel capsules with DAPI and
  • FIG. 7B shows staining of gel capsules with FAM.
  • Two types of fluorescence are shown: droplets that do not show fluorescence, droplets that show only DAPI-derived fluorescence (blue) bound to DNA amplified by MDA, and DAPI-derived fluorescence and probe-derived fluorescence (FAM: green). Three types of droplets can be confirmed.
  • an encapsulation technique is used to perform another or the same reaction on a set that has been reacted with respect to a set that is individually separated and generated. And regarding the technology that enables efficient screening. Specifically, it can be applied to gene sequence analysis and can be specifically detected and selected from amplified polynucleotides prepared in parallel from various microorganisms by referring to the gene sequence.
  • cell refers to a particle that contains a molecule that carries the genetic information and is any particle that can be replicated (whether or not it is possible alone).
  • the term “cell” as used herein includes cells of unicellular organisms, bacteria, cells derived from multicellular organisms, fungi and the like.
  • cell-like structure refers to any particle containing a molecule having genetic information.
  • cell-like structures include organelles such as mitochondria, cell nuclei, and chloroplasts, and viruses.
  • gel refers to a colloidal solution (sol) in which a polymer substance or colloidal particles interact with each other to form a network structure as a whole and contain a large amount of a liquid phase as a solvent or a dispersion medium. A state in which fluidity is lost.
  • gelling means changing a solution into a “gel” state.
  • capsule refers to a capsule-like substance capable of holding cells or cell-like structures therein, and when it is made of gel, it is referred to as a gel-like fine particle. It is a shaped structure.
  • encapsulation refers to the process of encapsulating cells or cell-like structures in a capsule.
  • gel encapsulation refers to a process of encapsulating cells or cell-like structures in a gel capsule.
  • the "gel capsule” refers to a gel-like fine particle structure capable of holding a cell or a cell-like structure therein.
  • gene analysis means examining the state of nucleic acids (DNA, RNA, etc.) in a biological sample.
  • the gene analysis can include those that utilize a nucleic acid amplification reaction.
  • Examples of gene analysis including these include sequencing, genotyping / polymorphism analysis (SNP analysis, copy number polymorphism, restriction enzyme fragment length polymorphism, repeat number polymorphism), expression analysis, fluorescence quenching probe ( Quenching Probe: Q-Probe), SYBR green method, melting curve analysis, real-time PCR, quantitative RT-PCR, digital PCR and the like can be mentioned.
  • single cell level refers to genetic information contained in one cell or cell-like structure or information on other biomolecules, as opposed to genetic information contained in another cell or cell-like structure. Or, it means that the processing is performed in a state of being distinguished from the information of other biomolecules. For example, when amplifying a polynucleotide at the "single biological unit level” or “single cell level”, the polynucleotide in one single biological unit or cell or cell-like structure, and another single organism, respectively. Each amplification takes place with the polynucleotides in the unit, or other cell or cell-like structure, distinguishable.
  • single cell analysis refers to genetic information or other biomolecule information contained in one cell or cell-like structure, and genetic information or other information contained in another cell or cell-like structure. It refers to analysis in a state that is distinguished from the information on biomolecules.
  • genetic information refers to information on a nucleic acid encoding a gene or other information contained in one cell or cell-like structure, and refers to the presence or absence of a specific gene sequence, the yield of a specific gene, or the total nucleic acid. Including yield.
  • nucleic acid information refers to information on nucleic acids contained in one cell or cell-like structure, and includes the presence or absence of a specific gene sequence, the yield of a specific gene, or the total nucleic acid yield.
  • identity refers to sequence similarity between two nucleic acid molecules. Identity can be determined by comparing positions in each sequence that can be aligned for comparison.
  • the disclosure comprises (A) encapsulating two or more cells or cell-like structures, together with a first agent, for each cell or cell-like structure, and (B). A step of performing a reaction based on the first drug on the cell or cell-like structure in a capsule, and (C) the cell or cell-like structure or a substance derived from the substance, or a capsule containing them, the cell. Alternatively, a step of encapsulating each cell-like structure or a substance derived thereto together with a second drug, and (D) a step of performing a reaction based on the second drug on the cell or cell-like structure in the capsule.
  • X is an integer of 3 or more
  • F if necessary, a reaction based on the Xth drug is carried out on the cell or cell-like structure in a capsule.
  • a method for analyzing a cell or a cell-like structure including the steps to be performed.
  • X is an integer of 3 or more, and an arbitrary integer is selected.
  • (E) to (G) are optional steps, and if they do not exist, the embodiment is encapsulated twice. It is very difficult to analyze a large number of samples simply by encapsulating two or more cells or cell-like structures with a first drug and performing a reaction based on the first drug on the cells or cell-like structures. Although it may be difficult, the cells or cell-like structures that have undergone a reaction based on the first drug, or substances derived from the cells, or capsules containing them, are encapsulated together with the second drug, and the second drug is used.
  • the reaction of one drug and the second drug is reacted with different types of drugs, the first drug is used for primary screening, which is then used with the second and / or third to X drugs. Further (secondary or higher) screening can be performed.
  • the disclosure provides an aspect that applies once a single cell or cell-like structure has been prepared or processed.
  • (C') two or more cells or cell-like structures or substances derived thereto, or capsules containing them, are desired agents for each cell or cell-like structure or substances derived thereto.
  • a method for analyzing a cell or cell-like structure which comprises a step of encapsulating with and (D') analyzing the cell or cell-like structure using the desired agent.
  • the method of the present disclosure it is possible to select a cell or cell-like structure containing and / or not containing the gene of interest from a large amount of samples and subject it to analysis. For example, if you want to obtain only data on rare bacteria, if a large amount of host DNA is mixed and the proportion of target cells or cell-like structures is low, data on only specific bacteria is required, and evaluation of diversity is required. This disclosure is useful when is not required.
  • each capsule when the capsules are further encapsulated, each capsule may be made of a different material or the same material.
  • the capsule of step (A) may be a gel capsule.
  • the agent may be an agent required for nucleic acid amplification.
  • the drug may be a nucleic acid probe, or a drug that identifies a protein such as an antibody or other antigen.
  • the plurality of (first to X) agents may be independently the same or different.
  • it may contain a specific nucleic acid degrading agent, a modifier, a label, various enzymes, and the like.
  • the analysis in the step (H) is to analyze the capsules, and it may or may not be analyzed for each encapsulation. If it is performed in the middle, it can perform a function such as screening, and if it is finally performed, an analysis that also serves as an evaluation can be performed, but the present invention is not limited to these.
  • the analysis of step (H) may or may not include nucleic acid amplification.
  • the analysis of step (H) may analyze the sequencing of the nucleic acids contained and / or amplified, the yield of total nucleic acids, the presence or absence of specific sequences, the yield of nucleic acids of specific sequences. ..
  • the analysis of step (D) may be an analysis targeting proteins, sugar chains, lipids and the like.
  • a next-generation sequencer (NGS) or the like can be used for sequencing, and a whole genome sequence may be obtained.
  • the desired drug may be a drug required for nucleic acid amplification.
  • the desired agent may be an agent for detecting a particular molecule.
  • agents for detecting a particular molecule include probes, antibodies, intercalators, tags, radioactive substances, fluorescent dye-binding nucleotides, fluorescently labeled proteins and the like.
  • the disclosure is a method of analyzing a cell or cell-like structure.
  • B A step of performing the first nucleic acid amplification reaction on the cell or cell-like structure in a capsule
  • C A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each of the cell or cell-like structure or a substance derived thereto together with a drug for nucleic acid amplification.
  • (D) A step of performing a second nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and (E) If necessary, the cell or cell-like structure or a substance derived from the cell or cell-like structure obtained in D), or a capsule containing them, is prepared for each cell or cell-like structure or a substance derived from the cell or cell-like structure.
  • the disclosure A) selects two or more cells or cell-like structures by a single cell or structure unit and includes the cells or cell-like structures.
  • the step A) is also referred to as primary amplification and can be performed by the method described below.
  • the present disclosure provides a method of amplifying a polynucleotide in a cell.
  • This amplification method uses a sample containing two or more cells or cell-like structures (including, for example, viruses, organs (Mt, Nuc), etc.) and uses the cells or cell-like structures as one cell or structure.
  • a step of encapsulating the droplets unit by unit, a step of gelling the droplets to form gel capsules, and a step of immersing the gel capsules in one or more lysis reagents to lyse the cells or cell-like structures.
  • the amplification method of the present disclosure can individually amplify a genome at the so-called single cell level or a gene assembly similar thereto.
  • the amplification method of the present disclosure realizes individual genome amplification by a very simple method, and therefore, in units of 100 units, 1000 units, 10,000 units, 100,000 units, or more.
  • Genomic information about cells can be obtained at one time, and therefore can be used as a library.
  • the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
  • any embodiment detailed in the following (droplet generation) section or other sections can be adopted.
  • the cells or cell-like structures that can be targeted in the amplification methods of the present disclosure are any number of two or more, eg, 10 or more, 50 or more, 100 or more, 500 or more. , 1000 or more, 5000 or more, 10,000 or more, 50,000 or more, 100,000 or more, 500,000 or more, 1 million or more, 5 million or more, 10 million or more.
  • the amplification method of the present disclosure can target a larger number of cells than using conventional single cell reaction systems, such as 0.2 mL, 1.5 mL microtube reaction systems.
  • any cell or cell-like structure described in the section can be adopted.
  • cells can be targeted.
  • cell-like structures are targeted, among which viruses or organelles such as mitochondria and nuclei can be targeted.
  • the sample containing the provided cells or cell-like structures may be provided in any form.
  • select an appropriate medium including buffers, salts, nutrients, other components, etc.
  • any cell or cell-like structure selected from the section (cells and cell-like structures). can do.
  • any component may be used as long as it is a component suitable for droplet generation. It is preferable that the component is also suitable for gelation. Examples of such components include, but are not limited to, buffer solutions such as PBS, Tris-HCl, TE, and HEPES, as well as sterile water, seawater, artificial seawater, and various liquid media.
  • a detergent-free medium such as water or buffer may be preferred.
  • any embodiment described in the section can be adopted for encapsulation of cells or cell-like structures in droplets of cells or structure units one by one.
  • a microchannel is used to flow a suspension of cells or cell-like structures into the microchannel and shear the suspension to create individual cells or cell-like structures.
  • Encapsulated droplets can be produced, and in addition to the explanation in (Droplet preparation), those skilled in the art can appropriately prepare and carry out components and parameters with reference to the representative examples exemplified in the examples. it can.
  • any embodiment described in the following (gelation) section can be adopted as the step of gelling the droplet to form a gel capsule.
  • gelation cools a droplet made so that the material of the droplet or droplet (eg, a sample containing a cell or cell-like structure) contains the material of a gel capsule.
  • the material of the droplet or droplet eg, a sample containing a cell or cell-like structure
  • gelation can be gelled by giving a stimulus such as light.
  • any material described in the following (gelation) section can be used.
  • the step of lysing a cell or cell-like structure can be realized by immersing a gel capsule in one or more lysing reagents, and any embodiment described in the following (dissolution) item. Can be adopted.
  • the polynucleotide in the cell is eluted into the gel capsule and retained in the gel capsule in a state where the substance binding to the polynucleotide is removed. It is important to be processed in.
  • the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
  • the lysate remaining in the gel capsule and the lysis reagent after the dissolution operation are passed through the gel capsule using an appropriate washing solution, and the inhibitor is transferred to the outside of the gel capsule. It may be desirable to release to.
  • the residual reagent can be diluted while retaining the genetic material. This step can be repeated. By diluting the reagent to a level that does not cause inhibition, downstream operations, such as amplification reactions, can be performed smoothly.
  • the step of amplifying a polynucleotide in a gel capsule can be realized by contacting the polynucleotide with an amplification reagent, and any embodiment defined in the following (amplification) can be adopted. it can.
  • the present disclosure may include encapsulating cells or cell-like structures in droplets, one cell or structural unit at a time, using a sample containing two or more cells or cell-like structures. Further, in the present disclosure, the device may include a droplet making section in which cells or cell-like structures are encapsulated in droplets one by one or in units of structures.
  • Droplet production can be performed using, for example, a microchannel.
  • the droplet making section may include a microchannel.
  • droplets encapsulating each cell or cell-like structure can be made. Shearing can be done at regular intervals. Shearing of the suspension can be done with oil.
  • oil for example, mineral oil (for example, light mineral oil), vegetable oil, silicone oil, fluorinated oil, etc. can be used.
  • the diameter of the droplet may be from about 1 to 250 ⁇ m, more preferably from about 10 to 200 ⁇ m, for example, the diameter of the droplet is about 1 ⁇ m, about 5 ⁇ m, about 10 ⁇ m, about 15 ⁇ m, about 20 ⁇ m, about 25 ⁇ m, about. It may be 30 ⁇ m, about 40 ⁇ m, about 50 ⁇ m, about 80 ⁇ m, about 100 ⁇ m, about 150 ⁇ m, about 200 ⁇ m, or about 250 ⁇ m.
  • the present disclosure may include the step of gelling a droplet to form a gel capsule.
  • the device may include a gel capsule generating unit that gels droplets to form gel capsules.
  • Gelation of the droplets can be performed by configuring the droplets to contain the material of the gel capsule and cooling the prepared droplets. Alternatively, gelation can be performed by giving a stimulus such as light to the droplet.
  • the inclusion of the gel capsule material in the droplets can be done, for example, by including the gel capsule material in a suspension of cells or cell-like structures.
  • the diameter of the gel capsule may be about 1-250 ⁇ m, more preferably about 10-200 ⁇ m, eg, about 1 ⁇ m, about 5 ⁇ m, about 10 ⁇ m, about 15 ⁇ m, about 20 ⁇ m, about 25 ⁇ m, about 30 ⁇ m, about 40 ⁇ m, about. It may be 50 ⁇ m, about 80 ⁇ m, about 100 ⁇ m, about 150 ⁇ m, about 200 ⁇ m, or about 250 ⁇ m.
  • the diameter of the gel capsule may be the same as that of the droplet to be produced, but the diameter may change during gelation.
  • the material of the gel capsule may include agarose, acrylamide, a photocurable resin (for example, PEG-DA), PEG, gelatin, sodium alginate, matrigel, collagen and the like.
  • a photocurable resin for example, PEG-DA
  • the gel capsule may be a hydrogel capsule.
  • hydrogel refers to one in which the solvent or dispersion medium held by the network structure of the polymer substance or colloidal particles is water.
  • DNA When extracting DNA from a large number of cells at once, DNA can be purified by phenol / chloroform extraction and ethanol precipitation.
  • the amount of the genetic substance per cell is very small, and it is necessary to individually convert the genetic substance into a nucleic acid-only state without loss.
  • nucleic acid purification is attempted by a general bulk scale procedure, the result is that no nucleic acid can be extracted or only nucleic acid derived from impurities can be extracted.
  • Loss of contamination and target genetic material is a major problem in single-cell experiments, but by using a gel capsule containing a single cell or cell-like structure, purified genetic material (eg, DNA) can be placed in the gel capsule. It can be retained in the cell, and the possibility of contamination of molecules from the outside can be eliminated. In addition, a large amount of one cell can be processed in parallel with a very simple operation. The steps of centrifuging the test tube containing the gelled droplets, removing the supernatant and replacing it with a cleaning solution can be performed. Alternatively, the gelled droplets can be filtered through a filter to remove the supernatant, the cleaning solution is passed through the mixture, and finally the gel capsules are collected. By using a gel capsule, the residual reagent can be diluted while retaining the genetic material. This step can be repeated. By diluting the reagent to a level that does not cause inhibition, downstream operations, such as amplification reactions, can be performed smoothly.
  • purified genetic material eg, DNA
  • a composition comprising a gel capsule or a material thereof may be provided. From the points described above or below, such compositions may be useful for amplifying nucleic acids in cells at the single cell level. Also, such compositions can be useful for making genomic libraries. In a further embodiment, a composition comprising a gel capsule or a material thereof and cells in a single cell state can be provided. From the points described above or below, such compositions may be useful for amplifying nucleic acids in cells at the single cell level. Also, such compositions can be useful for making genomic libraries. Such compositions can be useful for sequencing intracellular nucleic acids at the single cell level.
  • the present disclosure may include immersing a gel capsule in one or more lysis reagents to lyse the cell or cell-like structure. Further, in the present disclosure, the device may include a dissolution reagent immersion portion for immersing the gel capsule in the dissolution reagent. Upon lysis, the polynucleotide in the cell may be eluted in the gel capsule and retained in the gel capsule with the substance binding to the polynucleotide removed. Dissolving reagents include, for example, enzymes, surfactants, other denaturing agents, reducing agents and pH regulators, and combinations thereof can also be used.
  • a composition comprising a lysing reagent for amplifying nucleic acids in cells at the single cell level may be provided.
  • the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
  • Reagents for lysis include lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride.
  • the lysis reagent may be selected from at least one group consisting of lysozyme, achromopeptidase, protease, sodium dodecyl sulfate and potassium hydroxide.
  • the only purpose is to detect the presence or absence of some sequences in a cell or cell-like structure, it is not always necessary to actively lyse the cell or cell-like structure, and physical stimulation or heat. It is also possible to perform detection based on the leakage of nucleic acid from cells or cell-like structures by stimulus. However, in order to obtain a large amount of information such as the entire genome from a single cell, it is preferable to actively destroy the cell or cell-like structure and isolate the genetic substance in the cell from the cell in a complete state. .. Further, when a gel capsule is used, thermal / mechanical irritation may lead to disintegration of the gel capsule, and it may be preferable to use a dissolving reagent.
  • a lytic reagent or a combination of lysing reagents that is strong to some extent.
  • Gram-positive bacteria have a cell wall with a thick peptidoglycan layer, so mild ones alone may not be sufficient to lyse cells.
  • a strong lysing reagent may inhibit a reaction such as DNA amplification, and is preferably sufficiently removed before the downstream reaction.
  • the gel capsule retains the genetic substance to be analyzed or amplified, so that the lytic reagent can be removed even in a single cell analysis in which the amount of the genetic substance is small. Therefore, it is possible to use a strong dissolving reagent or a combination of dissolving reagents. And the use of potent lysing reagents or combinations of lysing reagents allows for comprehensive nucleic acid amplification or genome analysis, regardless of the type of cell (including those with cell walls and other types of microorganisms). Can be.
  • the method may include removing the lysing reagent and / or contaminants from the gel capsule.
  • the dissolution reagent immersion portion includes means for removing the dissolution reagent and / or contaminants from the gel capsule.
  • the target molecule is part of a cell surface marker or nucleic acid and the goal is to detect its presence itself, the goal may be achieved even if the lysis operation is partial or undissolved. There is.
  • the genomic DNA when attempting to amplify the full length of genomic DNA, the genomic DNA usually has only one molecule in the cell, so that the cell or cell-like structure is completely dissolved and bound from the DNA. It is necessary to sufficiently remove proteins. As a result, even when a sample consisting of hundreds or more kinds of microorganisms such as intestinal microorganisms is targeted, all of them can be uniformly dissolved and whole genome amplification can be performed from all of them. It also makes it possible to prepare the library and finally obtain whole genome sequence information.
  • the present disclosure may include contacting the polynucleotide with an amplification reagent to amplify the polynucleotide within a gel capsule.
  • the device may include an amplification reagent immersion portion for immersing the gel capsule in the amplification reagent.
  • the amplification reagent immersion portion may be provided with means for adjusting the temperature of the gel capsule, if necessary, after immersion in the amplification reagent.
  • Reactions involving heat treatment can lead to redissolution of gels (eg, agarose gels), which can disrupt the individualized shape and invalidate single cell isolation. is there.
  • an enzymatic reaction of about 60 degrees or less is desirable to maintain the gel droplet shape.
  • the constant temperature chain substitution amplification reaction is preferable in that it can be carried out within this temperature range and the entire genomic DNA can be amplified.
  • the enzyme used include phi29 polymerase, Bst polymerase, Aac polymerase, and recombinase polymerase.
  • a PCR primer for specifically detecting the organism and an oligonucleotide for detection such as Taqman probe.
  • the primer / probe can be prepared according to the oligonucleotide sequence design by general PCR, qPCR, or the like.
  • non-patent documents Li X, Zhang W, Wang C, Cui L, Yang CJ. Agarose droplet microfluidics for high-high lily parallell and effective Single 28 mulsion;
  • agarose By adding agarose to the PCR reaction solution as in 10.1039 / c0lc00145 g), it is possible to cool the droplets after PCR in the reaction step and gel-encapsulate the droplets again.
  • the gel capsule can be separated by using flow cytometry or the like.
  • the agarose skeleton (non-patent documents: Leng X, Zhang W, Wang C, Cui L, Yang CJ. ):. 2841-2843 doi:... 10.1039 / c0lc00145g) and beads (Novak R, Zeng Y, Shuga J, et al Single-cell multiplex gene detection and sequencing with microfluidically generated agarose emulsions Angew Chem Int Ed Engl 2011 It is also designed to be appropriately crosslinked with 50 (2): 390-395. Doi: 10.10012 / anie.201006089) so that the augmented by-product stays in the gel capsule. This makes it possible to select gel capsules using a fluorescent DNA-binding dye by retaining short-chain by-products in gel capsules.
  • RNA Ribonucleic acid
  • the purpose is to determine the type and expression level of a gene in an absolute (relative) manner. It is possible to quantify whether it is expressed only.
  • Treatment in gel capsules is advantageous for such amplification.
  • the cells or cell-like structures of interest in the present disclosure are not particularly limited, but are, for example, microorganisms (eg, bacteria, fungi, single cell animals), cells of multicellular organisms (eg, somatic cells, germ cells). , Cultured cells, tumor cells, animal cells, plant cells), intracellular organs (mitochonium, nucleus, chloroplast), viruses.
  • microorganisms eg, bacteria, fungi, single cell animals
  • cells of multicellular organisms eg, somatic cells, germ cells.
  • intracellular organs mitochonium, nucleus, chloroplast
  • RNA For cells of organisms with known genome sequences, which genes are expressed in them RNA In the case of analysis of an organism whose genome sequence and / or gene information is unknown, it is necessary to obtain information on the genome itself before RNA analysis. In that case, amplification of the genome sequence at the single cell level by the method of the present disclosure using gel capsules is advantageous.
  • a sample containing two or more cells or cell-like structures can be used.
  • the two or more cells may be derived from a plurality of organisms.
  • the sample include a microbial sample, a tissue sample, a mixed sample of a symbiotic microorganism and a host organism, and a sample containing microorganisms and cells taken from an animal / human sample.
  • the microbial sample include a bacterial flora sample, a sample containing two or less types of cells or cell-like structures, and a sample containing cells or cell structures other than bacteria such as fungi.
  • samples containing microorganisms and cells taken out from human samples include feces, saliva, sputum, surgical lavage fluid, blood, skin / body mucosa wipes and swabs, and can be used directly. , It may be used after performing an operation for separating cells and microorganisms.
  • the microorganisms that can be targeted are not limited to, but are not limited to, eubacteria, Escherichia coli, bacilli, indigo bacteria, cocci, bacilli, spiral bacteria, gram-negative bacteria, gram-positive bacteria, archaea, fungi, etc. Can be mentioned.
  • Bacteria that can be targeted by the present disclosure for example, Negibacteria, Eobacteria, Deinococci, Deinococci, Deinococcales, Thermales, Chloroflexi, Anaerolineae, Anaerolineales, Caldilineae, Chloroflexales, Herpetosiphonales, Thermomicrobia, Thermomicrobiales, Sphaerobacterales, Ktedonobacteria, Ktedonobacterales, Thermogemmatisporales, Glycobacteria, Cyanobacteria , Gloeobacterophyceae, Gloeobacterales, Nostocophyceae, Synechococcophycidae, Synechococcales, Nostocophycidae, Chroococcales, Oscillatoriales, Nostocales, Pseudanabaenales, Spirochaetes, Spirochaetes, Spirochaetales, Fibrobacteres
  • Encapsulation The process of individually encapsulating cells or cell-like structures containing nucleic acids, or substances derived from them, together with necessary drugs, is also simply referred to as encapsulation.
  • the object to be encapsulated may be one amplified by primary amplification or may be a purified product of another reaction.
  • the object of encapsulation may be an amplified nucleic acid, a gel capsule containing the amplified nucleic acid, a cell or a cell-like structure containing the amplified nucleic acid.
  • the object to be reencapsulated has the polynucleotide in a state where the substance binding to the polynucleotide has been removed, or if the polynucleotide is amplified while maintaining the gel state in the gel capsule, gelation is performed in the encapsulation. It does not have to be, and it does not have to contain a gelling material.
  • the diameter of the capsule may be about 1-250 ⁇ m, more preferably about 10-200 ⁇ m, for example, the diameter of the capsule is about 1 ⁇ m, about 5 ⁇ m, about 10 ⁇ m, about 15 ⁇ m, about 20 ⁇ m, about 25 ⁇ m, about 30 ⁇ m, It may be about 40 ⁇ m, about 50 ⁇ m, about 80 ⁇ m, about 100 ⁇ m, about 150 ⁇ m, about 200 ⁇ m, or about 250 ⁇ m.
  • the diameter of the capsule may increase as the number of encapsulations increases.
  • each capsule when encapsulating the capsules for the second and subsequent times, each capsule may be made of different materials, some may overlap, or all may be made of the same material. You may.
  • the agent required for analysis may be the agent required for nucleic acid amplification.
  • amplification of nucleic acid derived from a single cell or cell-like structure can be performed by any method, but simply one cell or cell-like structure from a large number of cells or cell-like structures.
  • a step of encapsulating cells or cell-like structures one by one in a droplet a step of gelling the droplet to generate a gel capsule, and a step of forming the gel capsule 1
  • a substance in which a polynucleotide containing the genomic DNA of the cell or a portion thereof is eluted into the gel capsule and binds to the genomic DNA or the portion thereof.
  • One cell by method comprising the step of retaining the polynucleotide in the gel capsule in a removed state and the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule.
  • an amplification reagent to amplify the polynucleotide in the gel capsule.
  • the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
  • step A) steps of selecting two or more cells or cell-like structures by a single cell or structure unit and amplifying the nucleic acids contained in the cells or cell-like structures.
  • a sample containing two or more cells or cell-like structures is used to enclose one cell or component in a droplet, and b) the droplet is gelled to form a gel capsule.
  • the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
  • the droplet is a single cell or single cell by flowing a suspension of single cell or single cell-like structures into a microchannel and shearing the suspension with oil. It can be made by encapsulating a suspension of similar structures.
  • the gel capsule may be a hydrogel capsule.
  • the material of the gel capsule may include agarose, acrylamide, a photocurable resin (for example, PEG-DA), PEG, gelatin, sodium alginate, matrigel, collagen and the like.
  • Gelation of the droplets can be performed by configuring the droplets to contain the material of the gel capsule and cooling the prepared droplets. Alternatively, gelation can be performed by giving a stimulus such as light to the droplet.
  • the inclusion of the gel capsule material in the droplets can be done, for example, by including the gel capsule material in a suspension of cells or cell-like structures.
  • the gel capsule may be a hydrogel capsule.
  • hydrogel refers to one in which the solvent or dispersion medium held by the network structure of the polymer substance or colloidal particles is water.
  • Reagents for lysis include lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride.
  • a lysing reagent or a combination of lysing reagents that is strong to some extent.
  • Gram-positive bacteria have a cell wall with a thick peptidoglycan layer, so mild ones alone may not be sufficient to lyse cells.
  • the agent required for analysis may be a second amplification reagent.
  • the number of agents required for analysis may be one or more.
  • the first amplification reagent and the second amplification reagent are identical.
  • the first amplification reagent and the second amplification reagent are different.
  • the amplification reagents used may all be the same or all different.
  • the amplification reagents used may be partially identical.
  • the first amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
  • the second amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
  • the reagent for specific sequence amplification may be a reagent that amplifies a cell type-specific sequence or a species-specific sequence, or a reagent that amplifies a sequence stored in a specific species.
  • the reagent for specific sequence amplification may be a reagent that amplifies a marker gene.
  • one cell or cell-like structure and a first label may be encapsulated in the droplet.
  • one cell or cell-like structure and a second label may be encapsulated in step B).
  • one cell or cell-like structure and one or more labels may be encapsulated in a) and / or step B) above.
  • the first sign and the second sign are the same. In another embodiment, the first and second markers are different. In other embodiments, the labels used may all be the same or all may be different. In certain embodiments, the labels used may be only partially identical. In some embodiments, the label may indicate the yield of total nucleic acid. In other embodiments, the label may indicate the presence or absence of a particular sequence. In certain embodiments, the label may indicate the yield of nucleic acid of a particular sequence. In other embodiments, the label may be an agent for detecting a particular molecule. In certain embodiments, agents for detecting a particular molecule include probes, antibodies, intercalators, tags, radioactive substances, fluorescent dye-binding nucleotides, fluorescently labeled proteins and the like.
  • the nucleic acid contained in the cell or cell-like structure in step A) can be genomic DNA or a portion thereof.
  • step C) may include evaluating the label. In some embodiments, step C) may include evaluating a first label and / or a second label. In other embodiments, the yield of total nucleic acid, the presence or absence of a particular sequence, or the yield of a particular sequence of nucleic acid, as indicated by the label, may be assessed.
  • the encapsulated cells or cell-like structures may be selected. In one embodiment, selection may be based on the presence or absence of a particular gene sequence, the yield of a particular gene or the total nucleic acid yield. In some embodiments, it may be selected when there is a specific gene sequence, or it may be selected when there is no specific gene sequence. In some embodiments, it may be selected if the yield of the particular gene is greater than or equal to the baseline yield. In some embodiments, it may be selected if the total nucleic acid yield is greater than or equal to the reference yield.
  • reagents that specifically detect the presence or absence of a particular gene sequence include antibodies, probes, DNA-binding fluorescent dyes, fluorescent dye-binding nucleotides.
  • the yield of a specific gene or the total nucleic acid yield can be measured by absorbance measurement, fluorescence measurement, agarose gel electrophoresis, or microchip electrophoresis.
  • the method may include detecting nucleic acid having a particular sequence in a sample containing amplified nucleic acid derived from each cell.
  • the step of detecting a nucleic acid having a specific sequence may include amplifying and sequencing the nucleic acid having a specific sequence.
  • sorting may be performed by flow cytometry or the like.
  • a fluorescent DNA-binding dye (intercalator) that labels all nucleic acids with the first label is used.
  • a primer set for detecting a specific gene and a fluorescent DNA-binding dye (intercalator) or a fluorescent labeling position probe are used.
  • the fluorescent dyes used for the first and second labels do not have overlapping fluorescence wavelengths, and the fluorescence intensity can be measured by another channel in flow cytometry.
  • nucleic acid By introducing gel capsules containing the first and second labels into flow cytometry and selecting from the fluorescent signals of each gel capsule, a certain amount of nucleic acid is contained inside the gel capsule and a specific gene is included. Can be recovered. When selecting based on the yield of a specific sequence or the yield of total nucleic acid, the selection was performed by first detecting a gel capsule containing an amplified polynucleotide with a fluorescent intercalator, separating it into a plate by flow cytometry, and further separating it. Nucleic acid is reamplified from the polynucleotide of each gel capsule to prepare an amplified polynucleotide library.
  • the yield of the amplified nucleic acid is measured by absorptiometry, fluorescence measurement, agarose gel electrophoresis, and microchip electrophoresis, and the total amount of nucleic acid is measured.
  • a part of this amplified nucleic acid is used to perform specific gene amplification, gene sequencing or molecular weight determination, and the sequence information and yield of the amplified gene are evaluated, and each sample in the polynucleotide library is evaluated. Evaluate the presence or absence of a particular gene. With reference to the results of either or both of these, a sample is selected and transferred to another plate or the like.
  • the cell type-specific sequence, the species-specific sequence, or the sequence stored in the specific species may be amplified.
  • the marker gene may be amplified in the analysis of step C).
  • the present disclosure is, for example, a system for analyzing cells or cell-like structures, in which [X] two or more cells or cell-like structures or substances derived thereto, or capsules containing them are encapsulated. Drugs and / or encapsulated drug vaults for storing them, desired drugs used for [Y] analysis and / or analytical drug vaults for storing them, and [Z] means for encapsulation. And [W], as needed, provide a system comprising means for performing an analysis with the desired agent.
  • Examples of Y include reagents (polymerizer, primer set, etc.) for amplifying and decoding a specific gene
  • W includes, for example, reagents (fluorescent-labeled probe and intercalator) for detecting the amplified gene.
  • reagents fluorescent-labeled probe and intercalator
  • [Z] includes microfluidic devices, gelling agents, structures for encapsulation (cooling reaction tank, storage, etc.)
  • Examples include reagents (fluorescent-labeled probes and intercalators) for detecting amplified genes and measuring devices (flow cytometry).
  • kits The present disclosure is, for example, a kit for providing a capsule for analyzing a cell or a cell-like structure, which comprises [X] two or more cells or a cell-like structure or a substance derived thereto, or a substance thereof.
  • a kit comprising a drug for encapsulating the capsule and a desired drug for use in the [Y] analysis.
  • Y include reagents for amplifying and decoding a specific gene (polymerase, primer set, etc.) and reagents for detecting the amplified gene (fluorescent-labeled probe and intercalator).
  • a gel capsule containing an amplified and retained 1-cell genome-derived polynucleotide is prepared by the method described in the above section (Method for amplifying polynucleotide in cells). Suspended in a PCR solution containing a primer set corresponding to the gene region targeting the gel capsule containing the amplified polynucleotide and a PCR amplification detection indicator such as a fluorescent DNA intercalator or Taqman probe, and introduced the suspension into a microfluidic device. To do. Shear the suspension with oil to make droplets containing a single gel capsule.
  • PCR After collecting the droplets in an external container, perform a PCR operation.
  • the heat treatment of PCR disintegrates the gel capsule, the inner polynucleotide associates with the primer / enzyme in the PCR solution, and the polynucleotide is amplified in the droplet if the target gene is present.
  • the presence or absence of polynucleotide amplification can be confirmed and detected by the fluorescent color derived from the PCR amplification detection indicator.
  • the gel capsule containing the amplified polynucleotide is also labeled with a fluorescent DNA intercalator, and by using a PCR amplification detection indicator of another fluorescent color in the above means, the gel capsule can be obtained from a pattern of two or more fluorescent colors.
  • the droplet or the re-gelled droplet is selectively collected or eliminated, and the droplet containing the amplified polynucleotide containing the target gene is selectively contained in the containing solution.
  • Example 1 Encapsulation of cells and cell-like structures
  • a 1.5 cm coral branch obtained from Ishikawara in the sea area around Sesoko, Motobu-cho, Kunigami-gun, Okinawa was collected and filtered with a 0.22 ⁇ m diameter filter (DURAPORE membrane filter, GVWP04700, MERCK) to 5 mL of seawater. Collected in a 25 mL tube (2362-025, IWAKI) containing. After sufficiently crushing the coral branches using a scalpel (replacement blade scalpel holder (61-3813-28), replacement blade No. 10 (1-8545-11), AS ONE)), leave it on ice for 3 minutes. Then, large particles such as bone fragments were precipitated.
  • DURAPORE membrane filter GVWP04700, MERCK
  • the supernatant was collected in a 1.5 mL tube (122-10, SSIbio) and centrifuged at 8,000 xg for 5 min (himac CF15RX, Koki Holdings). The supernatant was removed leaving the pellets, and 800 ⁇ L of seawater filtered through a 0.22 ⁇ m diameter filter (DURAPORE membrane filter, GVWP04700, MERCK) was added to suspend the pellets again. Next, a 250 xg centrifuge was performed for 5 min and the supernatant was collected in a new 1.5 mL tube.
  • DURAPORE membrane filter GVWP04700, MERCK
  • the concentration of bacterial cells in the prepared suspension was measured (microscope: CKX41, OLYMPUS, bacterial calculator A161,-5679-01, AS ONE), and ultra-low melting point agarose (A5030) was adjusted to a final concentration of 1.5%.
  • -10G, SIGMA-ALDRICH was added to prepare a mixed suspension of cells and cell-like structures used for gel capsule preparation (final bacterial cell concentration: 4.5 ⁇ 10 3 cells / ⁇ L).
  • a gel capsule having a diameter of 35 ⁇ m was prepared using the prepared mixed suspension of cells and cell-like structures. Subsequently, the gel capsule was immersed in a lytic reagent as a lysis reagent, a portion other than the object to be collected such as a cell wall of a cell was lysed inside the gel capsule, and genomic DNA was eluted into the gel capsule.
  • the gel capsule was immersed in Buffer D2 (QIAGEN), which is an aqueous solution containing potassium hydroxide, which is one of the lytic reagents, to dissolve the residual components and denature the genomic DNA.
  • Buffer D2 QIAGEN
  • the lytic test solution used in this example is lysozyme, achromopeptidase, proteinase K, sodium dodecyl sulfate, and Buffer D2.
  • Potassium hydroxide is also used in a normal DNA amplification reaction step, but since it also has a lytic effect, it is used as one of the lytic reagents in this example.
  • the eluted genomic DNA is not discharged from the gel capsule by the lytic reagent and is retained in the gel capsule.
  • the lytic reagent permeated into the gel capsule is also included in the contaminants.
  • Buffer D2 is added to proceed with the reaction, but a sufficient cleaning effect can be obtained by performing lysis operations with a plurality of reagents step by step and performing centrifugal cleaning in each step. Further, the cells may be lysed with each lytic reagent and then centrifuged.
  • the target genomic DNA can be collected by lysing the cells with a plurality of types of lytic reagents, and the lytic reagent and the poly of the lysed cells can be collected by centrifugation after immersion in the lytic reagent. Contaminants such as components other than nucleotides can be removed, and genomic DNA can be purified without inhibiting the subsequent genomic DNA amplification reaction.
  • the amplification reagent was added to the tube containing the gel capsule holding the denatured genomic DNA in the potassium hydroxide solution (Buffer D2), and the gel capsule was immersed in the amplification reagent.
  • the MDA (Multiple Replication Replication) method using phi29 DNA polymerase, which is a strand-substitution type DNA synthase was used.
  • the whole genome amplification reaction was carried out for 3 hours by immersing in the whole genome amplification reaction reagent REPLI-g Single Cell Kit (QIAGEN).
  • the amplification reagent (REPLI-g Single Cell Kit) contains a component that neutralizes the potassium hydroxide solution (Buffer D2).
  • the gel capsule after whole genome amplification is purified by DPBS (Dulvecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) three times and washed, and then washed with DAPI (Dojin), which is a fluorescent DNA intercalator. Staining was performed using science). The stained gel capsules were washed again with DPBS (Fig. 1).
  • DPBS Dens Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific
  • Example 2-1 Droplet reencapsulation and gene amplification
  • the droplets produced in Example 1 were re-encapsulated.
  • the reagent for PCR was used as the reagent to be inserted at the time of re-encapsulation.
  • PCR reaction solution was prepared.
  • a primer designed for target gene detection and a Taqman probe prepared by Integrated DNA Technologies
  • the primers and probes used in this example were designed to specifically detect a part of the 16S rRNA gene (V3-V4 region) of a microorganism belonging to the genus Endozoicomonas, which is a microorganism coexisting in coral tissue (manufactured by Integrated). DNA Technologies) and, in the presence of the target sequence, exhibit green fluorescence from the FAM after the PCR reaction.
  • a DNA staining reagent SYBR Green, EvaGreen (registered trademark), etc.
  • a gel capsule was added to the prepared PCR reaction solution to prepare a PCR reaction solution in which the gel capsule was suspended.
  • microdroplets having a diameter of 50 ⁇ m (this diameter can be 40 to 60 ⁇ m, etc.) were prepared from a PCR reaction solution in which gel capsules were suspended, and microdroplets were prepared.
  • a gel capsule was encapsulated in the droplet.
  • the gel capsule concentration in the microdroplets at this time is preferably about 0.5 cell / drop. All the prepared droplets were collected in a PCR tube.
  • the recovered material was subjected to a PCR reaction using a thermal cycler.
  • the PCR reaction conditions were as follows: initial heat denaturation at 95 ° C. for 3 minutes, heat denaturation at 95 ° C. for 5 seconds, annealing and extension reaction at 60 ° C. for 30 seconds for 28 cycles, and storage at 4 ° C. after completion of the reaction.
  • the gel capsule encapsulated inside was thermally disintegrated, and the polynucleotide held in the capsule reacted with the primer / enzyme in the PCR solution.
  • the polynucleotide is amplified in the droplet, increasing the intensity of probe-derived fluorescence or DNA intercalator-derived fluorescence.
  • the droplets were observed under a fluorescence microscope to confirm the fluorescence in the droplets.
  • Example 2-2 Detection / selection without reencapsulation and gene amplification
  • the gel capsule prepared in Example 1 is immersed in a recombinase polymerase amplification reaction (RPA) reagent.
  • RPA recombinase polymerase amplification reaction
  • the RPA reagent for example, TwisAmp (registered trademark) series (TwistDx) can be used.
  • Isothermal nucleic acid amplification is performed inside the gel capsule under the conditions of 37 ° C to 42 ° C using a primer / fluorescent probe designed according to the target sequence to be detected.
  • the gel capsule in which the amplified nucleic acid containing the target organism or the target gene is present is specificized using the fluorescent substance accumulated inside the gel capsule as an index. Can be detected.
  • Example 3 Secondary amplification
  • the secondary amplification step was further performed on the samples contained in the individual capsules prepared in Example 2. The procedure is shown below.
  • Example 2 Of those prepared in Example 2, two types of fluorescent droplets were fractionated under a microscope using a micropipette and individually collected in a PCR tube. After breaking the droplets by heating the collected individual droplets at 65 ° C. using a thermal cycler (S1000 thermal cycler, Bio-Rad), secondary amplification by the MDA method in the wells of each tube. Was done. This makes it possible to obtain a 1-cell amplified genomic library containing the sequence of the target gene.
  • S1000 thermal cycler Bio-Rad
  • a library was prepared using the Nextera XT DNA sample prep kit (Illumina, FC-131-1096) for eight samples containing genomic information derived from coral coexisting microorganisms, and Miseq (Illumina, SY-410-).
  • a 2 ⁇ 75 bp paired end read (3.99 Gb) was obtained by whole genome sequencing using 1003).
  • Assembly of sequence data using SPAdes (Bankevich A et al., J Comput Biol. 2012 May; 19 (5): 455-77. This was performed to prepare a Contig.
  • CheckM Parks et al., Genome Res. 2015.
  • Example 3-2 Analysis without secondary amplification
  • the following method is available as a procedure for skipping the secondary amplification step described in Example 3 and proceeding to the analysis.
  • droplets showing two types of fluorescence were collected under a microscope using a micropipette and individually collected in a PCR tube.
  • a thermal cycler S1000 thermal cycler, Bio-Rad
  • the Nextera XT DNA genome prep kit in the wells of each tube.
  • a library can be prepared using (Illumina, FC-131-1096) and a whole genome sequence can be performed using Miseq (Illumina, SY-410-1003).
  • DNA sequence library preparation reagent other than the Nextera XT DNA sample prep kit.
  • an adapter sequence is inserted by a transposase reaction as in the above kit, or a reaction such as fragmentation / ligation is performed to add the adapter sequence, and then a PCR reaction is sufficient to perform sequence analysis. Acquire the amount of DNA. After that, the analysis can be performed in the same manner as described in Example 3.
  • Example 4 When it is desired to selectively acquire data of cells having specific characteristics from various cells) A gel containing a polynucleotide when the purpose is to obtain genomic data of one or more specific microorganisms of interest to those skilled in the art among animal symbiotic microorganisms such as gut flora and marine / soil microorganisms. By confirming the presence or absence of the gene fragment of the target microorganism in advance for the capsule, it is possible to reduce unnecessary gene sequence data acquisition and the cost involved.
  • measurement targets include comparative analysis of microorganisms of the same strain (for example, analysis of subspecies in a group of microorganisms related to diseases, etc.) and microorganisms having a specific gene (for example, secondary metabolites and enzymes produced by microorganisms). It is assumed that the purpose is to search for or analyze bacteria, archaea, fungi, and other eukaryotic cells individually from among various species.
  • Example 5 When a large amount of host DNA or the like is mixed, the analysis sample is feces, saliva, sputum or skin, oral cavity, nasal cavity, ears, genital lavage fluid, surgical cleaning solution, tissue extract or blood, and the microorganisms contained in the sample are analyzed. Contains cells, intracellular small organs, and nucleic acids from many host animals in the sample. Some of these can also be encapsulated inside the gel capsule to perform polynucleotide amplification.
  • the intestinal flora and host animals present in the gastrointestinal tract have a symbiotic relationship in which the host provides an anaerobic environment for colonization of the gastrointestinal tract, while the intestinal flora affects the health of the host. It is known to have.
  • the major effects on the health status of the host include the production of nutrients, the defense against infectious diseases, and the development of the immune system.
  • inflammatory bowel disease is a disease caused by an abnormality of intestinal environmental factors such as intestinal bacteria in addition to a genetic predisposition.
  • Example 6 Example of performing encapsulation for the third time or more
  • Analysis samples include feces, saliva, sputum and skin, oral cavity, nasal cavity, ears, genital lavage fluid, surgical cleaning fluid, tissue extract and blood, or crushed fluid of plants, insects, and animals containing symbiotic microorganisms.
  • each gel capsule is encapsulated with host-derived cells, organelles, nucleic acids, and nucleic acids derived from symbiotic microorganisms. All of these can be encapsulated inside the gel capsule and polynucleotide amplification can be performed.
  • the target may be a combination of two or more microorganisms or host-derived substances.
  • This disclosure is useful in industries that use single cell analysis.

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Abstract

The present disclosure provides an analysis of single cells with high precision. More particularly, the present disclosure provides: a method that is for analyzing cells or cell-like structures and that comprises a step for encapsulating two or more cells or cell-like structures or substances derived therefrom, or capsules containing these elements, together with a desired chemical agent separately for respective cells or cell-like structures or substances derived therefrom, and a step for analyzing the cells or cell-like structures using the desired chemical agent; and a technique related to the method.

Description

ゲルカプセル方式による1細胞ゲノムライブラリーからの配列スクリーニング法Sequence screening method from 1-cell genomic library by gel capsule method
 本開示は、カプセル化法を用いた単一細胞または細胞様構造物の個別の分析の応用に関する。 The present disclosure relates to the application of individual analysis of single cell or cell-like structures using the encapsulation method.
 多様な細胞を含有する様々な生物学的サンプルにおいて、シングルセル毎の解析を行うことが近年少しずつ可能になっている。しかしながら、このような解析には、依然として、細胞の効果的な分離、単一細胞の溶解、全ゲノムの均一増幅、単一細胞増幅ゲノム(SAG)の品質評価、配列決定ライブラリー調製、および配列決定分析を含めたサンプル調製プロセスにおけるいくつかの技術的課題が存在している。そのため、品質とスループットを最大化するために、大規模並列解析を可能にする新規な技術に対する大きな需要がある。 In recent years, it has become possible to analyze each single cell in various biological samples containing various cells. However, such analyzes still include effective cell isolation, single cell lysis, whole-genome uniform amplification, single-cell amplified genome (SAG) quality assessment, sequencing library preparation, and sequencing. There are several technical challenges in the sample preparation process, including definitive analysis. Therefore, there is a great demand for new technologies that enable large-scale parallel analysis in order to maximize quality and throughput.
 環境微生物のうち培養が可能なものはわずか1%に限られており、地球上の微生物の多様性の大部分は未知である。ゲノム配列情報は、生物を理解するための基本情報であり、ゲノム解読により系統、進化、疾患、生物地球化学的循環に関連する微生物の多様性と機能の理解が可能となる。このため、難培養性の機能理解には全ゲノム解析が必要不可欠とされている。この難培養微生物ゲノム解析の方法としては、試料から種々の微生物ゲノムを一括抽出し配列解読するメタゲノム解析が主流であった。 Only 1% of environmental microorganisms can be cultivated, and most of the diversity of microorganisms on the earth is unknown. Genome sequence information is basic information for understanding living organisms, and genome decoding enables understanding of the diversity and function of microorganisms related to lineage, evolution, diseases, and biogeochemical cycle. For this reason, whole-genome analysis is indispensable for understanding the function of refractory culture. As a method for this difficult-to-culture microbial genome analysis, metagenomic analysis, in which various microbial genomes are collectively extracted from a sample and sequenced, has been the mainstream.
 しかし、メタゲノム解析法では、様々な微生物ゲノム配列の情報が混在するデータが得られることになる。このため、その中から興味のある微生物のゲノム情報を取り出して個別に再構築することは容易ではなく、大規模なデータ取得と計算処理が必要とされている。 However, with the metagenomic analysis method, data in which information on various microbial genome sequences is mixed can be obtained. Therefore, it is not easy to extract the genomic information of the microorganism of interest from the information and reconstruct it individually, and large-scale data acquisition and calculation processing are required.
 一方、1細胞解析では細胞一つ一つを始めに分けた後、処理を開始する。単一の細胞に由来する遺伝情報のみを直接的に取得するため、例えばゲノム情報を再構成するための計算難易度はメタゲノム解析に比べて圧倒的に易しい。しかし、細胞内の極小量のゲノム配列を漏れなく解読するためには、高精度な細胞操作と核酸反応が求められる。 On the other hand, in 1-cell analysis, each cell is first divided and then processing is started. Since only the genetic information derived from a single cell is directly acquired, for example, the calculation difficulty for reconstructing the genomic information is overwhelmingly easier than the metagenomic analysis. However, highly accurate cell manipulation and nucleic acid reaction are required to decipher the extremely small amount of intracellular genome sequence without omission.
 1細胞ゲノム解析のステップは、(1)微生物1細胞の分取、(2)微生物の溶解、(3)全ゲノム増幅、(4)増幅ゲノムのシークエンス解析、に大きく分けることができる。しかし、従来の分子生物学実験で用いられてきた数十マイクロリットルの反応系は、1細胞という極小の試料を上記の流れにしたがい精密に扱う上で不適切であった。具体的には、小さく不定形の多様な微生物をフローサイトメトリーなどで分取する際には、生物以外の粒子と微生物を分けて認識し回収することが困難であった。分取した微生物試料の扱いにおいても、実験環境や試料、実験者からの核酸のコンタミネーションが大容量の反応系においては高頻度に生じ、正常な増幅産物が得られる歩留まりは低く、解読した配列の大部分が無関係のコンタミネーション由来になるケースが非常に大きかった。 The steps of 1-cell genome analysis can be roughly divided into (1) separation of 1 cell of microorganism, (2) lysis of microorganism, (3) whole genome amplification, and (4) sequence analysis of amplified genome. However, the reaction system of several tens of microliters used in the conventional molecular biology experiment is inappropriate for precisely handling a very small sample of one cell according to the above flow. Specifically, when a variety of small and amorphous microorganisms were separated by flow cytometry or the like, it was difficult to separately recognize and collect non-living particles and microorganisms. Even when handling the separated microbial samples, contamination of nucleic acids from the experimental environment, samples, and experimenters occurs frequently in a large-capacity reaction system, and the yield at which a normal amplification product can be obtained is low. In very large cases, most of them were derived from unrelated contamination.
 さらに、正しい配列のみを情報処理により抽出しても、30%程度のゲノム解読率に留まっていた。オープンな実験環境ではエアロゾルなどからのコンタミネーションを完全に防ぐことは難しいため、1細胞ゲノム解析実験専用の清浄実験環境が必要とされている。また細胞一つ一つからシークエンスを実行する必要性に対して、反応系のスループット性が極めて低いことも課題であり、高価な分注ロボットなどが実験操作に使用されてきた。 Furthermore, even if only the correct sequence was extracted by information processing, the genome decoding rate remained at about 30%. Since it is difficult to completely prevent contamination from aerosols and the like in an open experimental environment, a clean experimental environment dedicated to single-cell genome analysis experiments is required. Another problem is that the throughput of the reaction system is extremely low with respect to the need to execute the sequence from each cell, and expensive dispensing robots and the like have been used for experimental operations.
 本発明者らは、鋭意研究した結果、一旦シングルセル(単位生物単位)にした後に、再度ゲルカプセル化の手法を適用することで、個別の単一生物単位(例えば、細胞)を集合として保持したままでさらに核酸増幅や検出などを行うことができることを見出し、本開示を完成するに至った。
 本開示の実施形態の例として、以下のものが挙げられる。
(項目1)
(A)2つ以上の細胞または細胞様構造物を、第一の薬剤とともに、細胞または細胞様構造物ごとにカプセル化する工程と、
(B)第一の薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(C)該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに第二の薬剤とともにカプセル化する工程と、
(D)該第二の薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(E)必要に応じて、該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに第Xの薬剤とともにカプセル化する工程であって、Xは3以上の整数である、工程と、
(F)必要に応じて、該第Xの薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(G)(E)および(F)の工程を必要に応じて繰り返す工程と、
(H)該カプセルに対して少なくとも1つの分析を行う工程と
を包含する、細胞または細胞様構造物を分析する方法。
(項目1A)
(A)2つ以上の細胞または細胞様構造物を、第一の薬剤とともに、細胞または細胞様構造物ごとにカプセル化する工程と、
(B)第一の薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(C)該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに第二の薬剤とともにカプセル化する工程と、
(D)該第二の薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(E)必要に応じて、該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに第Xの薬剤とともにカプセル化する工程であって、Xは3以上の整数である、工程と、
(F)必要に応じて、該第Xの薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(G)(E)および(F)の工程を必要に応じて繰り返す工程と、
(H)該カプセルに対して第1~第2および必要に応じて第3~第Xの薬剤に基づく少なくとも1つの分析を行う工程と
を包含する、細胞または細胞様構造物を分析する方法。
(項目2)
(C’)2つ以上の細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、細胞または細胞様構造物あるいはそれに由来する物質ごとに所望の薬剤とともにカプセル化する工程と、
(D’)該所望の薬剤を用いて該細胞または細胞様構造物の分析を行う工程と
を包含する、細胞または細胞様構造物を分析する方法。
(項目3)
(E)~(G)は存在せず、前記第一の薬剤および第二の薬剤は、核酸増幅に必要な薬剤である、上記項目のいずれか一項に記載の方法。
(項目4)
前記所望の薬剤は、核酸増幅に必要な薬剤である、上記項目のいずれか一項に記載の方法。
(項目5)
A)2つ以上の細胞または細胞様構造物を、単一の細胞または構造物単位ごとに選別し、該細胞または細胞様構造物に含まれる核酸を増幅する工程と、
B)該増幅された核酸を含む細胞または細胞様構造物を、分析に必要な薬剤とともに、個別にカプセル化する工程と、
C)該個別にカプセル化された該細胞または細胞様構造物について分析する工程と
を包含する、細胞または細胞様構造物を分析する方法。
(項目6)
前記C)の分析は、核酸増幅によるものであり、前記B)の分析に必要な薬剤は、核酸増幅に必要な薬剤を含む、上記項目のいずれか一項に記載の方法。
(項目7)
 前記A)が、
a)2つ以上の細胞または細胞様構造物を含む試料を用い、液滴中に1つの細胞または成分を封入することと、
b)該液滴をゲル化してゲルカプセルを生成することと、
c)該ゲルカプセルを1種以上の溶解用試薬に浸漬して該細胞または成分を溶解することであって、該細胞または成分中のポリヌクレオチドが該ゲルカプセル内に溶出し該ポリヌクレオチドに結合する物質が除去された状態で前記ゲルカプセル内に保持される、ことと、
d)該ポリヌクレオチドを第1の増幅用試薬に接触させて該ポリヌクレオチドをゲルカプセル内で増幅すること
を包含する、上記項目のいずれか一項に記載の方法。
(項目8)
 前記単一細胞または単一細胞様構造物の懸濁液をマイクロ流路中に流動させ、オイルで前記懸濁液をせん断することにより該単一細胞または単一細胞様構造物を封入した前記液滴が作製されることを特徴とする、上記項目のいずれか一項に記載の方法。
(項目9)
 前記ゲルカプセルがアガロース、アクリルアミド、PEG、ゼラチン、アルギン酸ナトリウム、マトリゲル、コラーゲン又は光硬化性樹脂から形成されることを特徴とする、上記項目のいずれか一項に記載の方法。
(項目10)
 前記溶解用試薬がリゾチーム、ラビアーゼ、ヤタラーゼ、アクロモペプチダーゼ、プロテアーゼ、ヌクレアーゼ、ザイモリアーゼ、キチナーゼ、リソスタフィン、ムタノライシン、ドデシル硫酸ナトリウム、ラウリル硫酸ナトリウム、水酸化カリウム、水酸化ナトリウム、フェノール、クロロホルム、グアニジン塩酸塩、尿素、2-メルカプトエタノール、ジチオトレイトール、TCEP-HCl、コール酸ナトリウム、デオキシコール酸ナトリウム、Triton X-100、Triton X-114、NP-40、Brij-35、Brij-58、Tween 20、Tween 80、オクチルグルコシド、オクチルチオグルコシド、CHAPS、CHAPSO、ドデシル-β-D-マルトシド、Nonidet P-40、およびZwittergent 3-12からなる群から少なくとも1種選択されることを特徴とする、上記項目のいずれか一項に記載の方法。
(項目11)
 前記ゲルカプセルがヒドロゲルカプセルであることを特徴とする、上記項目のいずれか一項に記載の方法。
(項目12)
 前記分析に必要な薬剤が第2の増幅用試薬である、上記項目のいずれか一項に記載の方法。
(項目13)
 前記第1の増幅試薬と前記第2の増幅用試薬とが同一である、上記項目のいずれか一項に記載の方法。(項目14)
 前記第1の増幅試薬と前記第2の増幅用試薬とが異なる、上記項目のいずれか一項に記載の方法。
(項目15)
 前記第1の増幅試薬がゲノムDNAまたはその部分の増幅用試薬または特定配列増幅用試薬である、上記項目のいずれか一項に記載の方法。
(項目16)
 前記第2の増幅試薬がゲノムDNAまたはその部分の増幅用試薬または特定配列増幅用試薬である、上記項目のいずれか一項に記載の方法。
(項目17)
 前記a)において、前記液滴中に前記1つの細胞または細胞様構造物、および第1の標識を封入することを特徴とする、上記項目のいずれか一項に記載の方法。
(項目18)
 前記B)において、前記1つの細胞または細胞様構造物および第2の標識をカプセル化することを特徴とする、上記項目のいずれか一項に記載の方法。
(項目19)
 前記第1の標識と前記第2の標識とが同一である、上記項目のいずれか一項に記載の方法。
(項目20)
 前記第1の標識と前記第2の標識とが異なる、上記項目のいずれか一項に記載の方法。
(項目21)
 前記第1の標識と前記第2の標識とが核酸の量を示すことを特徴とする、上記項目のいずれか一項に記載の方法。
(項目22)
 前記C)が、前記第1の標識および/または前記第2の標識を評価することを含む、上記項目のいずれか一項に記載の方法。
(項目23)
 前記A)における前記細胞または細胞様構造物に含まれる核酸が、ゲノムDNAである、上記項目のいずれか一項に記載の方法。
(項目24)
 前記C)の分析において、種特異的な配列または特定種に保存されている配列の増幅を行うことを特徴とする、上記項目のいずれか一項に記載の方法。
(項目25)
細胞または細胞様構造物を分析するためのシステムであって、
[X]2つ以上の細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、カプセル化するための薬剤および/またはそれらを格納するカプセル化薬剤格納部と、
[Y]分析に使用する所望の薬剤および/またはそれらを格納する分析薬剤格納部と、
[Z]カプセル化のための手段と
[W]必要に応じて、該所望の薬剤を用いて分析を行うための手段と
を備える、システム。
(項目25A)
上記項目のいずれか一項または複数に記載の特徴をさらに含む、項目25に記載のシステム。
(項目26)
細胞または細胞様構造物を分析するためカプセルを提供するためのキットであって、
[X]2つ以上の細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、カプセル化するための薬剤と、
[Y]分析に使用する所望の薬剤と、
を備える、キット。
(項目26A)
上記項目のいずれか一項または複数に記載の特徴をさらに含む、項目26に記載のキット。
(項目27)
細胞または細胞様構造物を分析する方法であって、
(A)2つ以上の細胞または細胞様構造物を、核酸増幅のための薬剤とともに、細胞または細胞様構造物ごとにカプセル化する工程と、
(B)第一の核酸増幅反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(C)該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに核酸増幅のための薬剤とともにカプセル化する工程と、
(D)第二の核酸増幅反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(E)必要に応じて、D)で得られた、該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに第Xの薬剤とともにカプセル化する工程であって、Xは3以上の整数である、工程と、
(F)必要に応じて、該第Xの薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(G)(E)および(F)の工程を必要に応じて繰り返す工程と、
(H)該カプセルに含まれる核酸の配列決定を行う工程と
を包含する、方法。
 
As a result of diligent research, the present inventors held individual single biological units (for example, cells) as a set by once converting them into single cells (unit biological units) and then applying the gel encapsulation method again. We have found that nucleic acid amplification and detection can be performed as it is, and have completed the present disclosure.
Examples of embodiments of the present disclosure include:
(Item 1)
(A) A step of encapsulating two or more cells or cell-like structures together with a first agent for each cell or cell-like structure.
(B) A step of performing a reaction based on the first drug on the cell or cell-like structure in a capsule, and
(C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each cell or cell-like structure or a substance derived thereto together with a second agent.
(D) A step of carrying out a reaction based on the second drug on the cell or cell-like structure in a capsule, and
(E) If necessary, the cell or cell-like structure or a substance derived thereto, or a capsule containing them, is encapsulated for each cell or cell-like structure or a substance derived thereto together with a drug X. In the process, X is an integer of 3 or more.
(F) If necessary, a step of carrying out a reaction based on the Xth drug on the cell or cell-like structure in a capsule, and
A step of repeating the steps (G), (E) and (F) as necessary, and
(H) A method for analyzing a cell or cell-like structure, which comprises the step of performing at least one analysis on the capsule.
(Item 1A)
(A) A step of encapsulating two or more cells or cell-like structures together with a first agent for each cell or cell-like structure.
(B) A step of performing a reaction based on the first drug on the cell or cell-like structure in a capsule, and
(C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each cell or cell-like structure or a substance derived thereto together with a second agent.
(D) A step of carrying out a reaction based on the second drug on the cell or cell-like structure in a capsule, and
(E) If necessary, the cell or cell-like structure or a substance derived thereto, or a capsule containing them, is encapsulated for each cell or cell-like structure or a substance derived thereto together with a drug X. In the process, X is an integer of 3 or more.
(F) If necessary, a step of carrying out a reaction based on the Xth drug on the cell or cell-like structure in a capsule, and
A step of repeating the steps (G), (E) and (F) as necessary, and
(H) A method for analyzing a cell or cell-like structure, which comprises a step of performing at least one analysis based on the first to second and optionally third to X agents on the capsule.
(Item 2)
(C') A step of encapsulating two or more cells or cell-like structures or substances derived thereto, or capsules containing them, for each cell or cell-like structure or substances derived thereto together with a desired drug.
(D') A method for analyzing a cell or cell-like structure, comprising the step of analyzing the cell or cell-like structure with the desired agent.
(Item 3)
The method according to any one of the above items, wherein (E) to (G) do not exist, and the first drug and the second drug are drugs required for nucleic acid amplification.
(Item 4)
The method according to any one of the above items, wherein the desired drug is a drug required for nucleic acid amplification.
(Item 5)
A) A step of selecting two or more cells or cell-like structures for each single cell or structure unit and amplifying nucleic acids contained in the cells or cell-like structures.
B) A step of individually encapsulating the cell or cell-like structure containing the amplified nucleic acid together with the drug required for analysis.
C) A method of analyzing a cell or cell-like structure, comprising the step of analyzing the individually encapsulated cell or cell-like structure.
(Item 6)
The method according to any one of the above items, wherein the analysis of C) is based on nucleic acid amplification, and the drug required for the analysis of B) includes a drug required for nucleic acid amplification.
(Item 7)
A) above
a) Encapsulating one cell or component in a droplet using a sample containing two or more cells or cell-like structures.
b) Gelling the droplets to form gel capsules
c) By immersing the gel capsule in one or more solubilizing reagents to lyse the cells or components, the polynucleotide in the cells or components elutes into the gel capsule and binds to the polynucleotide. It is retained in the gel capsule with the substance to be removed.
d) The method according to any one of the above items, comprising contacting the polynucleotide with a first amplification reagent to amplify the polynucleotide in a gel capsule.
(Item 8)
The single cell or single cell-like structure is encapsulated by flowing the suspension of the single cell or single cell-like structure into a microchannel and shearing the suspension with oil. The method according to any one of the above items, wherein droplets are produced.
(Item 9)
The method according to any one of the above items, wherein the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, matrigel, collagen or a photocurable resin.
(Item 10)
The solubilizing reagents are lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride. , Urea, 2-mercaptoethanol, dithiotreitol, TCEP-HCl, sodium dodecyl, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, The above item, which is characterized in that at least one is selected from the group consisting of Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-hydrochloride, Noidet P-40, and Zwittergent 3-12. The method according to any one of the above.
(Item 11)
The method according to any one of the above items, wherein the gel capsule is a hydrogel capsule.
(Item 12)
The method according to any one of the above items, wherein the agent required for the analysis is the second amplification reagent.
(Item 13)
The method according to any one of the above items, wherein the first amplification reagent and the second amplification reagent are the same. (Item 14)
The method according to any one of the above items, wherein the first amplification reagent and the second amplification reagent are different.
(Item 15)
The method according to any one of the above items, wherein the first amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
(Item 16)
The method according to any one of the above items, wherein the second amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
(Item 17)
The method according to any one of the above items, wherein in the a), the one cell or cell-like structure and the first label are encapsulated in the droplet.
(Item 18)
The method according to any one of the above items, wherein in B), the one cell or cell-like structure and the second label are encapsulated.
(Item 19)
The method according to any one of the above items, wherein the first label and the second label are the same.
(Item 20)
The method according to any one of the above items, wherein the first label and the second label are different.
(Item 21)
The method according to any one of the above items, wherein the first label and the second label indicate the amount of nucleic acid.
(Item 22)
The method according to any one of the above items, wherein C) comprises evaluating the first label and / or the second label.
(Item 23)
The method according to any one of the above items, wherein the nucleic acid contained in the cell or cell-like structure in A) is genomic DNA.
(Item 24)
The method according to any one of the above items, which comprises amplifying a species-specific sequence or a sequence stored in a specific species in the analysis of C).
(Item 25)
A system for analyzing cells or cell-like structures
[X] A drug for encapsulating two or more cells or cell-like structures or a substance derived from them, or a capsule containing them, and / or an encapsulating drug storage unit for storing them.
[Y] A desired drug used for analysis and / or an analytical drug storage unit for storing them,
A system comprising [Z] means for encapsulation and [W] means for performing analysis with the desired agent, if desired.
(Item 25A)
25. The system of item 25, further comprising the features of any one or more of the above items.
(Item 26)
A kit for providing capsules for analyzing cells or cell-like structures.
[X] An agent for encapsulating two or more cells or cell-like structures or substances derived from them, or capsules containing them.
[Y] With the desired drug used for analysis
A kit that includes.
(Item 26A)
26. The kit of item 26, further comprising the features of any one or more of the above items.
(Item 27)
A method of analyzing cells or cell-like structures
(A) A step of encapsulating two or more cells or cell-like structures together with a drug for nucleic acid amplification for each cell or cell-like structure.
(B) A step of performing the first nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and
(C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each of the cell or cell-like structure or a substance derived thereto together with a drug for nucleic acid amplification.
(D) A step of performing a second nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and
(E) If necessary, the cell or cell-like structure or a substance derived from the cell or cell-like structure obtained in D), or a capsule containing them, is prepared for each cell or cell-like structure or a substance derived from the cell or cell-like structure. The step of encapsulating with the drug of X, where X is an integer of 3 or more.
(F) If necessary, a step of carrying out a reaction based on the Xth drug on the cell or cell-like structure in a capsule, and
A step of repeating the steps (G), (E) and (F) as necessary, and
(H) A method comprising the step of sequencing the nucleic acid contained in the capsule.
 本開示において、上記1又は複数の特徴は、明示された組み合わせに加え、さらに組み合わせて提供されうることが意図される。本開示のなおさらなる実施形態及び利点は、必要に応じて以下の詳細な説明を読んで理解すれば、当業者に認識される。 In the present disclosure, it is intended that the above one or more features may be provided in a further combination in addition to the specified combinations. Further embodiments and advantages of the present disclosure will be appreciated by those skilled in the art upon reading and understanding the following detailed description as necessary.
 本開示により、個別の単一生物単位(例えば、「シングルセル」)を集合として保持したままで核酸増幅や検出、選別などを行うことができ、レアな細菌のデータを獲得したい場合、ホストDNAなどが多量に混在する場合、特定の細菌だけのデータが求められ、多様性の評価が不要な場合などに応用可能である。 According to the present disclosure, nucleic acid amplification, detection, selection, etc. can be performed while holding individual single biological units (for example, "single cell") as a set, and when it is desired to acquire rare bacterial data, host DNA When a large amount of such substances are mixed, data on only specific bacteria is required, and it can be applied when evaluation of diversity is unnecessary.
 本開示を用いると、希少な微生物種をもれなく捉えられる確度が高くなる。本開示はまた、事前に微生物種を特定し、全ゲノムシーケンスなどの分析に移行するサンプルを選抜することができる。本開示はまた、特異的な特定遺伝子の有無を事前に調べることで、ある種の微生物を選択的に選抜あるいは排除して、効果的なゲノム解析またはその他の分析を実施することができる。 Using this disclosure, the probability of catching all rare microbial species is high. The present disclosure can also identify microbial species in advance and select samples for transition to analysis such as whole genome sequences. The present disclosure can also selectively select or eliminate certain microorganisms to perform effective genomic or other analyses by preliminarily examining the presence or absence of specific specific genes.
図1は、単一の細胞または細胞様構造物を液滴に封入し、1次増幅するまでの段階を示す図である。1次増幅後にDAPIなどの蛍光性DNAインターカレーターを用いて、ゲルカプセル内の核酸を染色する。FIG. 1 is a diagram showing a step of encapsulating a single cell or cell-like structure in a droplet and performing primary amplification. After the primary amplification, the nucleic acid in the gel capsule is stained using a fluorescent DNA intercalator such as DAPI. 図2は、ゲルカプセルを液滴に再封入する段階を示す図である。ゲルカプセルを液滴に再封入する際に、PCR用プライマーおよび蛍光標識したプローブを添加したPCR反応液などもともに再封入を行う。PCR反応液は、例示であり、これ以外にLAMP反応、SmartAmp法、RPA(リコンビナーゼポリメラーゼ)増幅法も可能である。FIG. 2 is a diagram showing a step of reencapsulating a gel capsule in a droplet. When the gel capsule is re-encapsulated in the droplet, the PCR primer and the PCR reaction solution to which the fluorescently labeled probe is added are also re-encapsulated. The PCR reaction solution is an example, and other than this, a LAMP reaction, a SmartAmp method, and an RPA (recombinase polymerase) amplification method are also possible. 図3は、PCR増幅(あるいは他の手法)および遺伝子検出を示す図である。再封入時にTaqmanプライマーなどの標識を共に封入することにより、特定の遺伝子が増幅された場合に検出することができる。遺伝子検出は、DNA結合性蛍光インターカレーターと特異的遺伝子検出用プライマーの併用でも可能である。FIG. 3 is a diagram showing PCR amplification (or other technique) and gene detection. By encapsulating a label such as Taqman primer together at the time of re-encapsulation, it is possible to detect when a specific gene is amplified. Gene detection can also be performed by using a DNA-binding fluorescent intercalator and a primer for specific gene detection in combination. 図4は、二次増幅の段階を示す図である。二次増幅に供する液滴は、1次増幅後の染色、遺伝子検出の結果に基づき選別することができる。選別は、フローサイトメトリー、蛍光顕微鏡での検鏡、マイクロ流体デバイスでも可能である。FIG. 4 is a diagram showing the stages of secondary amplification. The droplets to be subjected to the secondary amplification can be selected based on the results of staining and gene detection after the primary amplification. Sorting is also possible with flow cytometry, fluorescence microscopy, and microfluidic devices. 図5は、PCRによる確認およびライブラリー調製の段階を示す図である。FIG. 5 is a diagram showing the steps of confirmation by PCR and library preparation. 図6は、ゲルカプセル方式による1細胞ゲノムライブラリーからの配列スクリーニング法の全体的なフローを示す模式図である。FIG. 6 is a schematic diagram showing the overall flow of the sequence screening method from the 1-cell genome library by the gel capsule method. 図7は、遺伝子検出時のゲルカプセルを示す図である。図7AはDAPIによるゲルカプセルの染色を示し、図7BはFAMによるゲルカプセルの染色を示す。蛍光を示さない液滴、MDAによって増幅されたDNAに結合したDAPI由来の蛍光(青色)のみを示す液滴、DAPI由来の蛍光およびプローブ由来の蛍光(FAM:緑色)の2種類の蛍光を示す液滴、の3種類が確認できる。FIG. 7 is a diagram showing a gel capsule at the time of gene detection. FIG. 7A shows staining of gel capsules with DAPI and FIG. 7B shows staining of gel capsules with FAM. Two types of fluorescence are shown: droplets that do not show fluorescence, droplets that show only DAPI-derived fluorescence (blue) bound to DNA amplified by MDA, and DAPI-derived fluorescence and probe-derived fluorescence (FAM: green). Three types of droplets can be confirmed.
 以下、本開示を最良の形態を示しながら説明する。本明細書の全体にわたり、単数形の表現は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。従って、単数形の冠詞(例えば、英語の場合は「a」、「an」、「the」など)は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。また、本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。したがって、他に定義されない限り、本明細書中で使用される全ての専門用語及び科学技術用語は、本開示の属する分野の当業者によって一般的に理解されるのと同じ意味を有する。矛盾する場合、本明細書(定義を含めて)が優先する。 Hereinafter, the present disclosure will be described while showing the best form. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise stated. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the concept of their plural, unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Thus, unless otherwise defined, all terminology and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, this specification (including definitions) takes precedence.
 本開示は、カプセル化により、その中で個別に分離して生成した集合に対して反応を行った集合に対して、さらにカプセル化技術を用いて別又は同じ反応を当該集合に対して行うことで、効率よいスクリーニングを可能とする技術に関する。具体的には、遺伝子配列解析に応用され、多様な微生物から並列調製された増幅ポリヌクレオチドから、遺伝子配列を参照して微生物特異的に検出し、選抜することができる。 In the present disclosure, an encapsulation technique is used to perform another or the same reaction on a set that has been reacted with respect to a set that is individually separated and generated. And regarding the technology that enables efficient screening. Specifically, it can be applied to gene sequence analysis and can be specifically detected and selected from amplified polynucleotides prepared in parallel from various microorganisms by referring to the gene sequence.
 (定義等)
 以下に本明細書において特に使用される用語の定義及び/又は基本的技術内容を適宜説明する。
(Definition, etc.)
The definitions and / or basic technical contents of terms particularly used in the present specification will be described below as appropriate.
 本明細書において、「細胞」とは、遺伝情報を有する分子を内包する粒子であって、(単独で可能かどうかにかかわらず)複製されることが可能である任意の粒子を指す。本明細書における「細胞」としては、単細胞生物の細胞、細菌、多細胞生物由来の細胞、真菌などが包含される。 As used herein, the term "cell" refers to a particle that contains a molecule that carries the genetic information and is any particle that can be replicated (whether or not it is possible alone). The term "cell" as used herein includes cells of unicellular organisms, bacteria, cells derived from multicellular organisms, fungi and the like.
 本明細書において、「細胞様構造物」とは、遺伝情報を有する分子を内包する任意の粒子を指す。本明細書における「細胞様構造物」としては、細胞内小器官、例えば、ミトコンドリア、細胞核、および葉緑体、ならびにウイルスなどが包含される。 In the present specification, the "cell-like structure" refers to any particle containing a molecule having genetic information. As used herein, "cell-like structures" include organelles such as mitochondria, cell nuclei, and chloroplasts, and viruses.
 本明細書において、「ゲル」とは、コロイド溶液(ゾル)において、高分子物質またはコロイド粒子がその相互作用により全体として網目構造をつくり、溶媒あるいは分散媒である液相を多量に含んだまま流動性を失った状態のことをいう。本明細書において、「ゲル化」とは、溶液を「ゲル」の状態に変化させることをいう。 In the present specification, the term "gel" refers to a colloidal solution (sol) in which a polymer substance or colloidal particles interact with each other to form a network structure as a whole and contain a large amount of a liquid phase as a solvent or a dispersion medium. A state in which fluidity is lost. As used herein, "gelling" means changing a solution into a "gel" state.
 本明細書において、「カプセル」とは、その中に細胞または細胞様構造物を保持することが可能なカプセル状のものをいい、ゲルでできている場合は、ゲルカプセルといいゲル状の微粒子状構造体である。本明細書において「カプセル化」とは、細胞または細胞様構造物などをカプセルの中に封入する処理をいう。本明細書において「ゲルカプセル化」とは、細胞または細胞様構造物などをゲルであるカプセルの中に封入する処理をいう。 As used herein, the term "capsule" refers to a capsule-like substance capable of holding cells or cell-like structures therein, and when it is made of gel, it is referred to as a gel-like fine particle. It is a shaped structure. As used herein, the term "encapsulation" refers to the process of encapsulating cells or cell-like structures in a capsule. As used herein, the term "gel encapsulation" refers to a process of encapsulating cells or cell-like structures in a gel capsule.
 本明細書において、「ゲルカプセル」とは、その中に細胞または細胞様構造物を保持することが可能なゲル状の微粒子状構造体を指す。 In the present specification, the "gel capsule" refers to a gel-like fine particle structure capable of holding a cell or a cell-like structure therein.
 本明細書において、「遺伝子分析」とは生体サンプル中の核酸(DNA、RNA等)の状態を調べることをいう。1つの実施形態では、遺伝子分析は、核酸増幅反応を利用するものを挙げることができる。これらを含め、遺伝子分析の例としては、配列決定、遺伝子型判定・多型分析(SNP分析、コピー数多型、制限酵素断片長多型、リピート数多型)、発現解析、蛍光消光プローブ(Quenching Probe:Q-Probe)、SYBR green法、融解曲線分析、リアルタイムPCR、定量RT-PCR、デジタルPCRなどを挙げることができる。 In the present specification, "gene analysis" means examining the state of nucleic acids (DNA, RNA, etc.) in a biological sample. In one embodiment, the gene analysis can include those that utilize a nucleic acid amplification reaction. Examples of gene analysis including these include sequencing, genotyping / polymorphism analysis (SNP analysis, copy number polymorphism, restriction enzyme fragment length polymorphism, repeat number polymorphism), expression analysis, fluorescence quenching probe ( Quenching Probe: Q-Probe), SYBR green method, melting curve analysis, real-time PCR, quantitative RT-PCR, digital PCR and the like can be mentioned.
 本明細書において、「シングルセルレベル」とは、1つの細胞または細胞様構造物に含まれる遺伝情報またはその他の生体分子の情報に対して、他の細胞または細胞様構造物に含まれる遺伝情報またはその他の生体分子の情報と区別した状態で処理を行うことをいう。例えば、「単一生物単位レベル」または「シングルセルレベル」でのポリヌクレオチドを増幅する場合、それぞれある単一生物単位、またはある細胞もしくは細胞様構造物中のポリヌクレオチドと、他の単一生物単位、または他の細胞もしくは細胞様構造物中のポリヌクレオチドが区別可能な状態でそれぞれの増幅が行われる。 As used herein, the term "single cell level" refers to genetic information contained in one cell or cell-like structure or information on other biomolecules, as opposed to genetic information contained in another cell or cell-like structure. Or, it means that the processing is performed in a state of being distinguished from the information of other biomolecules. For example, when amplifying a polynucleotide at the "single biological unit level" or "single cell level", the polynucleotide in one single biological unit or cell or cell-like structure, and another single organism, respectively. Each amplification takes place with the polynucleotides in the unit, or other cell or cell-like structure, distinguishable.
 本明細書において、「シングルセル解析」とは、1つの細胞または細胞様構造物に含まれる遺伝情報またはその他の生体分子の情報を、他の細胞または細胞様構造物に含まれる遺伝情報またはその他の生体分子の情報と区別した状態で解析することを指す。 As used herein, "single cell analysis" refers to genetic information or other biomolecule information contained in one cell or cell-like structure, and genetic information or other information contained in another cell or cell-like structure. It refers to analysis in a state that is distinguished from the information on biomolecules.
 本明細書において、「遺伝情報」とは、1つの細胞または細胞様構造物に含まれる遺伝子その他情報をコードする核酸の情報を指し、特定の遺伝子配列の有無、特定の遺伝子の収量または全核酸収量を含む。 As used herein, the term "genetic information" refers to information on a nucleic acid encoding a gene or other information contained in one cell or cell-like structure, and refers to the presence or absence of a specific gene sequence, the yield of a specific gene, or the total nucleic acid. Including yield.
 本明細書において、「核酸情報」とは、1つの細胞または細胞様構造物に含まれる核酸の情報を指し、特定の遺伝子配列の有無、特定の遺伝子の収量または全核酸収量を含む。 In the present specification, "nucleic acid information" refers to information on nucleic acids contained in one cell or cell-like structure, and includes the presence or absence of a specific gene sequence, the yield of a specific gene, or the total nucleic acid yield.
 本明細書において、「同一性」とは、2つの核酸分子間の配列類似性を指す。同一性は、比較のためにアライメントしうる各配列中の位置を比較することによって決定することができる。 In the present specification, "identity" refers to sequence similarity between two nucleic acid molecules. Identity can be determined by comparing positions in each sequence that can be aligned for comparison.
 (好ましい実施形態)
 以下に好ましい実施形態の説明を記載するが、この実施形態は本開示の例示であり、本発明の範囲はそのような好ましい実施形態に限定されないことが理解されるべきである。当業者はまた、以下のような好ましい実施例を参考にして、本開示の範囲内にある改変、変更などを容易に行うことができることが理解されるべきである。これらの実施形態について、当業者は適宜、1または複数の任意の実施形態を組み合わせ得る。
(Preferable embodiment)
Although a description of preferred embodiments will be described below, it should be understood that this embodiment is an example of the present disclosure and the scope of the invention is not limited to such preferred embodiments. It should be understood that those skilled in the art can also easily make modifications, changes, etc. within the scope of the present disclosure with reference to the following preferred embodiments. For these embodiments, one of ordinary skill in the art may optionally combine one or more arbitrary embodiments.
 (カプセル化技術の応用)
 本開示の1つの局面において、本開示は、(A)2つ以上の細胞または細胞様構造物を、第一の薬剤とともに、細胞または細胞様構造物ごとにカプセル化する工程と、(B)第一の薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、(C)該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに第二の薬剤とともにカプセル化する工程と、(D)該第二の薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、(E)必要に応じて、該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに第Xの薬剤とともにカプセル化する工程であって、Xは3以上の整数である、工程と、(F)必要に応じて、該第Xの薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、(G)(E)および(F)の工程を必要に応じて繰り返す工程と、(H)該カプセルに対して分析、例えば、第1~第Xの薬剤に基づく少なくとも1つの分析を行う工程とを包含する、細胞または細胞様構造物を分析する方法を提供する。ここで、Xは3以上の整数であり、任意の整数が選択される。(E)~(G)は任意工程であり、存在しない場合は、2回のカプセル化がなされる実施形態となる。2つ以上の細胞または細胞様構造物を、第一の薬剤とともにカプセル化し、第一の薬剤に基づく反応を細胞または細胞様構造物に対して行うだけでは、多量のサンプルを分析するのは非常に困難である場合もあるが、第一の薬剤に基づく反応を行った細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、第二の薬剤とともにカプセル化し、第二の薬剤を用いて細胞または細胞様構造物の分析を行うことにより、カプセルをチューブまたはプレートなどの容器内で分析することが不要となり、多量のサンプルを迅速かつ精確に分析することができ、また、第一の薬剤および第二の薬剤の反応に異なる種類の薬剤を用いて反応させる場合、第一の薬剤を用いて一次スクリーニングし、これを第二および/または第三~第Xの薬剤を用いてさらに(第二次またはさらに高次の)スクリーニングすることができる。
(Application of encapsulation technology)
In one aspect of the disclosure, the disclosure comprises (A) encapsulating two or more cells or cell-like structures, together with a first agent, for each cell or cell-like structure, and (B). A step of performing a reaction based on the first drug on the cell or cell-like structure in a capsule, and (C) the cell or cell-like structure or a substance derived from the substance, or a capsule containing them, the cell. Alternatively, a step of encapsulating each cell-like structure or a substance derived thereto together with a second drug, and (D) a step of performing a reaction based on the second drug on the cell or cell-like structure in the capsule. And (E), if necessary, the cells or cell-like structures or substances derived from them, or capsules containing them, are encapsulated together with the drug X for each of the cells or cell-like structures or substances derived from them. In the step of making, X is an integer of 3 or more, and (F), if necessary, a reaction based on the Xth drug is carried out on the cell or cell-like structure in a capsule. A step, a step of repeating steps (G), (E) and (F) as needed, and (H) an analysis on the capsule, eg, at least one analysis based on the first to X agents. Provided is a method for analyzing a cell or a cell-like structure, including the steps to be performed. Here, X is an integer of 3 or more, and an arbitrary integer is selected. (E) to (G) are optional steps, and if they do not exist, the embodiment is encapsulated twice. It is very difficult to analyze a large number of samples simply by encapsulating two or more cells or cell-like structures with a first drug and performing a reaction based on the first drug on the cells or cell-like structures. Although it may be difficult, the cells or cell-like structures that have undergone a reaction based on the first drug, or substances derived from the cells, or capsules containing them, are encapsulated together with the second drug, and the second drug is used. By analyzing cells or cell-like structures using, it is not necessary to analyze capsules in a container such as a tube or plate, and a large number of samples can be analyzed quickly and accurately. When the reaction of one drug and the second drug is reacted with different types of drugs, the first drug is used for primary screening, which is then used with the second and / or third to X drugs. Further (secondary or higher) screening can be performed.
 別の局面では、本開示は、一旦単一の細胞または細胞様構造物ごとに調製または処理がなされている場合において適用される局面を提供する。ここにおいて、本開示は、(C’)2つ以上の細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、細胞または細胞様構造物あるいはそれに由来する物質ごとに所望の薬剤とともにカプセル化する工程と、(D’)該所望の薬剤を用いて該細胞または細胞様構造物の分析を行う工程とを包含する、細胞または細胞様構造物を分析する方法を提供する。 In another aspect, the disclosure provides an aspect that applies once a single cell or cell-like structure has been prepared or processed. Here, in the present disclosure, (C') two or more cells or cell-like structures or substances derived thereto, or capsules containing them, are desired agents for each cell or cell-like structure or substances derived thereto. Provided is a method for analyzing a cell or cell-like structure, which comprises a step of encapsulating with and (D') analyzing the cell or cell-like structure using the desired agent.
 本開示の方法を用いることで、多量のサンプルの中から、目的の遺伝子を含む、および/または含まない細胞または細胞様構造物を選択して、分析に供することが可能となる。例えば、レアな細菌のデータのみを獲得したい場合、ホストDNAなどが多量に混在し、目的の細胞または細胞様構造物の割合が低い場合、特定の細菌だけのデータが求められ、多様性の評価が不要な場合、本開示は有用である。 By using the method of the present disclosure, it is possible to select a cell or cell-like structure containing and / or not containing the gene of interest from a large amount of samples and subject it to analysis. For example, if you want to obtain only data on rare bacteria, if a large amount of host DNA is mixed and the proportion of target cells or cell-like structures is low, data on only specific bacteria is required, and evaluation of diversity is required. This disclosure is useful when is not required.
 1つの実施形態において、カプセルをさらにカプセル化する場合、それぞれのカプセルは別の材料で形成されてもよく、同一の材料で形成されてもよい。特定の実施形態において、工程(A)のカプセルはゲルカプセルであってもよい。1つの実施形態において薬剤は、核酸増幅に必要な薬剤であってもよい。あるいは薬剤は、核酸プローブであってもよいし、抗体等のタンパク質やその他抗原を特定する薬剤でもよい。特定の実施形態において、複数の(第一~第Xの)薬剤は、それぞれ独立して同一であってもよく、異なってもよい。例えば、核酸増幅試薬の他、特定の核酸の分解薬剤、修飾剤、標識、各種酵素等を含んでいてもよい。 In one embodiment, when the capsules are further encapsulated, each capsule may be made of a different material or the same material. In certain embodiments, the capsule of step (A) may be a gel capsule. In one embodiment, the agent may be an agent required for nucleic acid amplification. Alternatively, the drug may be a nucleic acid probe, or a drug that identifies a protein such as an antibody or other antigen. In a particular embodiment, the plurality of (first to X) agents may be independently the same or different. For example, in addition to the nucleic acid amplification reagent, it may contain a specific nucleic acid degrading agent, a modifier, a label, various enzymes, and the like.
 工程(H)における分析は、カプセルに対して分析を行うものであり、それぞれのカプセル化ごとに都度分析してもよく、あるいはしなくてもよい。途中で行う場合は、スクリーニングのような機能を果たすことができ、最終的に行う場合は、評価も兼ねた分析を行うこともできるがこれらに限定されない。1つの実施形態において、工程(H)の分析は、核酸増幅をともなってもよく、ともなわなくともよい。一部の実施形態において、工程(H)の分析は、含まれるおよび/または増幅された核酸の配列決定、全核酸の収量、特定配列の有無、特定配列の核酸の収量を分析してもよい。他の実施形態において、工程(D)の分析は、タンパク質、糖鎖、脂質等を対象とした分析でもよい。配列決定には次世代シーケンサ(NGS)等を用いることができ、全ゲノムシーケンスを取得してもよい。 The analysis in the step (H) is to analyze the capsules, and it may or may not be analyzed for each encapsulation. If it is performed in the middle, it can perform a function such as screening, and if it is finally performed, an analysis that also serves as an evaluation can be performed, but the present invention is not limited to these. In one embodiment, the analysis of step (H) may or may not include nucleic acid amplification. In some embodiments, the analysis of step (H) may analyze the sequencing of the nucleic acids contained and / or amplified, the yield of total nucleic acids, the presence or absence of specific sequences, the yield of nucleic acids of specific sequences. .. In another embodiment, the analysis of step (D) may be an analysis targeting proteins, sugar chains, lipids and the like. A next-generation sequencer (NGS) or the like can be used for sequencing, and a whole genome sequence may be obtained.
 1つの実施形態において、所望の薬剤は、核酸増幅に必要な薬剤であってもよい。他の実施形態において、所望の薬剤は、特定分子を検出するための薬剤であってもよい。特定の実施形態において、特定分子を検出するための薬剤として、プローブ、抗体、インターカレーター、タグ、放射性物質、蛍光色素結合ヌクレオチド、蛍光標識タンパク質などが挙げられる。 In one embodiment, the desired drug may be a drug required for nucleic acid amplification. In other embodiments, the desired agent may be an agent for detecting a particular molecule. In certain embodiments, agents for detecting a particular molecule include probes, antibodies, intercalators, tags, radioactive substances, fluorescent dye-binding nucleotides, fluorescently labeled proteins and the like.
 一つの具体的な実施形態では、本開示は、細胞または細胞様構造物を分析する方法であって、
(A)2つ以上の細胞または細胞様構造物を、核酸増幅のための薬剤とともに、細胞または細胞様構造物ごとにカプセル化する工程と、
(B)第一の核酸増幅反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(C)該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに核酸増幅のための薬剤とともにカプセル化する工程と、
(D)第二の核酸増幅反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(E)必要に応じて、D)で得られた、該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに第Xの薬剤とともにカプセル化する工程であって、Xは3以上の整数である、工程と、
(F)必要に応じて、該第Xの薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
(G)(E)および(F)の工程を必要に応じて繰り返す工程と、
(H)該カプセルに含まれる核酸の配列決定を行う工程と
を包含する、方法を提供する。(E)で用いられる薬剤は上記したものと同様に種々の薬剤が使用され得る。
In one specific embodiment, the disclosure is a method of analyzing a cell or cell-like structure.
(A) A step of encapsulating two or more cells or cell-like structures together with a drug for nucleic acid amplification for each cell or cell-like structure.
(B) A step of performing the first nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and
(C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each of the cell or cell-like structure or a substance derived thereto together with a drug for nucleic acid amplification.
(D) A step of performing a second nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and
(E) If necessary, the cell or cell-like structure or a substance derived from the cell or cell-like structure obtained in D), or a capsule containing them, is prepared for each cell or cell-like structure or a substance derived from the cell or cell-like structure. The step of encapsulating with the drug of X, where X is an integer of 3 or more.
(F) If necessary, a step of carrying out a reaction based on the Xth drug on the cell or cell-like structure in a capsule, and
A step of repeating the steps (G), (E) and (F) as necessary, and
(H) Provided is a method including the step of sequencing the nucleic acid contained in the capsule. As the drug used in (E), various drugs may be used in the same manner as those described above.
 本開示の具体的な実施形態において、本開示は、A)2つ以上の細胞または細胞様構造物を、単一の細胞または構造物単位ごとに選別し、該細胞または細胞様構造物に含まれる核酸を増幅する工程と、B)該増幅された核酸を含む細胞または細胞様構造物を、分析に必要な薬剤とともに、個別にカプセル化する工程と、C)該個別にカプセル化された該細胞または細胞様構造物について分析する工程とを包含する方法を提供する。増幅、カプセル化、分析などの詳細は、以下更に説明する。 In a specific embodiment of the disclosure, the disclosure A) selects two or more cells or cell-like structures by a single cell or structure unit and includes the cells or cell-like structures. The steps of amplifying the amplified nucleic acid, B) individually encapsulating the cell or cell-like structure containing the amplified nucleic acid together with the drug required for analysis, and C) the individually encapsulated said. Provided are methods that include the steps of analyzing cells or cell-like structures. Details such as amplification, encapsulation, and analysis will be described further below.
(1次増幅)
 前記工程A)は1次増幅ともいい、下記に記載の方法にて行うことができる。
(Primary amplification)
The step A) is also referred to as primary amplification and can be performed by the method described below.
 (細胞中のポリヌクレオチド増幅方法)
 一つの局面において、本開示は、細胞中のポリヌクレオチドを増幅する方法を提供する。この増幅方法は、2つ以上の細胞または細胞様構造物(例えば、ウイルス、小器官(Mt,Nuc)等を含む)を含む試料を用い、該細胞または細胞様構造物を1細胞または構造物単位ずつ液滴中に封入する工程と、該液滴をゲル化してゲルカプセルを生成する工程と、該ゲルカプセルを1種以上の溶解用試薬に浸漬して前記細胞または細胞様構造物を溶解する工程であって、該細胞中のポリヌクレオチドが該ゲルカプセル内に溶出し該ポリヌクレオチドに結合する物質が除去された状態で前記ゲルカプセル内に保持される、工程と、該ポリヌクレオチドを増幅用試薬に接触させて該ポリヌクレオチドをゲルカプセル内で増幅する工程とを含む。本開示の増幅方法は、いわゆるシングルセルレベルでのゲノムまたはそれに類似する遺伝子集合物を個別に増幅し得るものである。本開示の増幅方法は、個別のゲノム増幅を、非常に簡便な手法で実現するものであり、そのため、100個単位、1000個単位、1万個単位、10万個単位あるいはそれ以上の単位の細胞について一時期にゲノム情報を取得することができ、それゆえライブラリーとすることもできるものである。本開示の一実施形態において、当該ポリヌクレオチドを増幅用試薬に接触させて当該ポリヌクレオチドをゲルカプセル内で増幅する工程は、当該ポリヌクレオチドをゲルカプセル内でゲル状態を保ちながら増幅することもできる。
(Method for amplifying polynucleotides in cells)
In one aspect, the present disclosure provides a method of amplifying a polynucleotide in a cell. This amplification method uses a sample containing two or more cells or cell-like structures (including, for example, viruses, organs (Mt, Nuc), etc.) and uses the cells or cell-like structures as one cell or structure. A step of encapsulating the droplets unit by unit, a step of gelling the droplets to form gel capsules, and a step of immersing the gel capsules in one or more lysis reagents to lyse the cells or cell-like structures. The step of amplifying the polynucleotide and the step of eluting the polynucleotide in the cell into the gel capsule and retaining the substance in the gel capsule in a state where the substance binding to the polynucleotide is removed. It comprises contacting with a reagent for amplification and amplifying the polynucleotide in a gel capsule. The amplification method of the present disclosure can individually amplify a genome at the so-called single cell level or a gene assembly similar thereto. The amplification method of the present disclosure realizes individual genome amplification by a very simple method, and therefore, in units of 100 units, 1000 units, 10,000 units, 100,000 units, or more. Genomic information about cells can be obtained at one time, and therefore can be used as a library. In one embodiment of the present disclosure, the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
 本開示の方法における液滴中への封入工程は、下記(液滴生成)の項または他の項に詳述されている任意の実施形態を採用することができる。 As the encapsulation step in the droplet in the method of the present disclosure, any embodiment detailed in the following (droplet generation) section or other sections can be adopted.
 1つの実施形態では、本開示の増幅方法において対象としうる細胞または細胞様構造物は、2つ以上の任意の数字であり、例えば、10個以上、50個以上、100個以上、500個以上、1000個以上、5000個以上、1万個以上、5万個以上、10万個以上、50万個以上、100万個以上、500万個以上、1000万個以上であり得る。本開示の増幅方法は、従来のシングルセル反応系、例えば、0.2mL、1.5mLマイクロチューブ反応系を用いるよりも多数の細胞を対象とし得る。 In one embodiment, the cells or cell-like structures that can be targeted in the amplification methods of the present disclosure are any number of two or more, eg, 10 or more, 50 or more, 100 or more, 500 or more. , 1000 or more, 5000 or more, 10,000 or more, 50,000 or more, 100,000 or more, 500,000 or more, 1 million or more, 5 million or more, 10 million or more. The amplification method of the present disclosure can target a larger number of cells than using conventional single cell reaction systems, such as 0.2 mL, 1.5 mL microtube reaction systems.
 本開示の増幅方法において対象とし得る細胞または細胞様構造物は、(細胞および細胞様構造物)の項に説明されている任意のものを採用することができる。1つの好ましい実施形態では、細胞が対象とされ得る。別の実施形態では、細胞様構造物が対象とされ、その中でも、ウイルス、あるいはミトコンドリア、核等の細胞小器官等を対象とすることができる。 As the cell or cell-like structure that can be targeted in the amplification method of the present disclosure, any cell or cell-like structure described in the section (cells and cell-like structure) can be adopted. In one preferred embodiment, cells can be targeted. In another embodiment, cell-like structures are targeted, among which viruses or organelles such as mitochondria and nuclei can be targeted.
 本開示の増幅方法において、提供される細胞または細胞様構造物を含む試料は、どのような形で提供されてもよい。試料に含まれる媒体は、(細胞および細胞様構造物)の項から選択した任意の細胞または細胞様構造物に対して適切な媒体(バッファー、塩、栄養素や他の成分等を含む)を選択することができる。このような成分としては、液滴生成に適した成分であればどのような成分を使用してもよい。ゲル化する際にも適切な成分であることが好ましい。そのような成分としては、PBS、Tris-HCl、TE、HEPESなどの緩衝液のほか、滅菌水、海水、人工海水、各種液体培地等を挙げることができるがこれらに限定されない。液滴を生成するためには、界面活性剤を含まない水またはバッファーなどの媒体が好ましい場合がある。 In the amplification method of the present disclosure, the sample containing the provided cells or cell-like structures may be provided in any form. For the medium contained in the sample, select an appropriate medium (including buffers, salts, nutrients, other components, etc.) for any cell or cell-like structure selected from the section (cells and cell-like structures). can do. As such a component, any component may be used as long as it is a component suitable for droplet generation. It is preferable that the component is also suitable for gelation. Examples of such components include, but are not limited to, buffer solutions such as PBS, Tris-HCl, TE, and HEPES, as well as sterile water, seawater, artificial seawater, and various liquid media. In order to generate droplets, a detergent-free medium such as water or buffer may be preferred.
 本明細書において、細胞または細胞様構造物を1細胞または構造物単位ずつの液滴中への封入は、(液滴作製)の項に記載される任意の実施形態を採用することができる。代表的には、マイクロ流路を用い、細胞または細胞様構造物の懸濁液をマイクロ流路中に流動させ、懸濁液をせん断することにより、1つずつの細胞または細胞様構造物を封入した液滴を作製することができ、(液滴作製)での説明の他、実施例において例示される代表例を参考に、当業者は、適宜成分やパラメータを調製して実施することができる。 In the present specification, any embodiment described in the section (Drop production) can be adopted for encapsulation of cells or cell-like structures in droplets of cells or structure units one by one. Typically, a microchannel is used to flow a suspension of cells or cell-like structures into the microchannel and shear the suspension to create individual cells or cell-like structures. Encapsulated droplets can be produced, and in addition to the explanation in (Droplet preparation), those skilled in the art can appropriately prepare and carry out components and parameters with reference to the representative examples exemplified in the examples. it can.
 本開示の増幅方法において、液滴をゲル化してゲルカプセルを生成する工程は、下記(ゲル化)の項に記載される任意の実施形態を採用することができる。 In the amplification method of the present disclosure, any embodiment described in the following (gelation) section can be adopted as the step of gelling the droplet to form a gel capsule.
 1つの実施形態では、ゲル化は、液滴あるいは液滴の材料(例えば、細胞または細胞様構造物を含む試料)にゲルカプセルの材料が含まれるように構成した作製した液滴を冷却することによって行うことができるし、あるいは、光等の刺激を与えることでゲル化させることもできる。 In one embodiment, gelation cools a droplet made so that the material of the droplet or droplet (eg, a sample containing a cell or cell-like structure) contains the material of a gel capsule. Alternatively, it can be gelled by giving a stimulus such as light.
 ゲルカプセルの材料としては、下記(ゲル化)の項に記載される任意の材料を用いることができる。 As the material of the gel capsule, any material described in the following (gelation) section can be used.
 本開示において、細胞または細胞様構造物を溶解する工程は、ゲルカプセルを1種以上の溶解用試薬に浸漬して実現することができ、下記(溶解)の項目に記載される任意の実施形態を採用することができる。 In the present disclosure, the step of lysing a cell or cell-like structure can be realized by immersing a gel capsule in one or more lysing reagents, and any embodiment described in the following (dissolution) item. Can be adopted.
 ここで、細胞または細胞様構造物を溶解する工程では、細胞中のポリヌクレオチドが該ゲルカプセル内に溶出しそのポリヌクレオチドに結合する物質が除去された状態でこのゲルカプセル内に保持されるように処理されることが重要である。本開示の一実施形態において、当該ポリヌクレオチドを増幅用試薬に接触させて当該ポリヌクレオチドをゲルカプセル内で増幅する工程は、当該ポリヌクレオチドをゲルカプセル内でゲル状態を保ちながら増幅することもできる。 Here, in the step of lysing a cell or a cell-like structure, the polynucleotide in the cell is eluted into the gel capsule and retained in the gel capsule in a state where the substance binding to the polynucleotide is removed. It is important to be processed in. In one embodiment of the present disclosure, the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
 このように、ポリヌクレオチドに結合する物質が除去された状態を維持するためあるいはポリヌクレオチドをゲルカプセル内でゲル状態を保ちながら増幅するためには、多種類の溶解剤を段階的あるいは同時に加えることで、細胞または細胞様構造物の細胞壁・細胞膜構造を確実に破壊し、細胞内に含まれるタンパク質、ポリヌクレオチドに結合する物質までを変性させることが必要である。溶解は細胞外層の破壊から段階的に試薬を加えて達成される。さらに、溶解操作後にゲルカプセル内に残存する溶解物および前記溶解試薬は、後段のポリヌクレオチド増幅を阻害するため、適切な洗浄液を用いてゲルカプセル内を通液させ、前記阻害物質をゲルカプセル外に放出することが望ましい場合がある。これらの操作をゲルカプセル内で完遂するために、ポリヌクレオチドをゲルカプセル内に保持しながら、各種薬液と細胞溶解物の浸透・放出を達成するヒドロゲル構造を取ることが望ましい場合がある。ゲルカプセルを用いることにより、遺伝物質を保持したまま、残留試薬を希薄化することができる。このステップは繰り返すことも可能である。阻害が出ないレベルにまで試薬を希薄化することで、下流の操作、例えば、増幅反応をスムーズに行うことができる。 In this way, in order to maintain the state in which the substance binding to the polynucleotide is removed or to amplify the polynucleotide while maintaining the gel state in the gel capsule, various kinds of solubilizers are added stepwise or simultaneously. Therefore, it is necessary to surely destroy the cell wall / cell membrane structure of cells or cell-like structures, and to denature proteins and substances that bind to polynucleotides contained in cells. Lysis is achieved by adding reagents step by step from the destruction of the extracellular layer. Further, in order to inhibit the polynucleotide amplification in the subsequent stage, the lysate remaining in the gel capsule and the lysis reagent after the dissolution operation are passed through the gel capsule using an appropriate washing solution, and the inhibitor is transferred to the outside of the gel capsule. It may be desirable to release to. In order to complete these operations in the gel capsule, it may be desirable to have a hydrogel structure that achieves penetration and release of various drug solutions and cell lysates while retaining the polynucleotide in the gel capsule. By using a gel capsule, the residual reagent can be diluted while retaining the genetic material. This step can be repeated. By diluting the reagent to a level that does not cause inhibition, downstream operations, such as amplification reactions, can be performed smoothly.
 本開示において、ポリヌクレオチドをゲルカプセル内で増幅する工程は、ポリヌクレオチドを増幅用試薬に接触させることにより実現することができ、下記(増幅)に規定される任意の実施形態を採用することができる。 In the present disclosure, the step of amplifying a polynucleotide in a gel capsule can be realized by contacting the polynucleotide with an amplification reagent, and any embodiment defined in the following (amplification) can be adopted. it can.
 (液滴生成)
 本開示は、2つ以上の細胞または細胞様構造物を含む試料を用い、細胞または細胞様構造物を1細胞または構造物単位ずつ液滴中に封入することを包含し得る。また、本開示において、装置は、細胞または細胞様構造物を1細胞または構造物単位ずつ液滴中に封入する液滴作製部を備え得る。
(Drop generation)
The present disclosure may include encapsulating cells or cell-like structures in droplets, one cell or structural unit at a time, using a sample containing two or more cells or cell-like structures. Further, in the present disclosure, the device may include a droplet making section in which cells or cell-like structures are encapsulated in droplets one by one or in units of structures.
 液滴作製は、例えば、マイクロ流路を用いて行うことができる。液滴作製部は、マイクロ流路を備え得る。細胞または細胞様構造物の懸濁液をマイクロ流路中に流動させ、懸濁液をせん断することにより、1つずつの細胞または細胞様構造物を封入した液滴を作製し得る。せん断は、一定間隔で行い得る。懸濁液のせん断は、オイルを用いて行うことができる。オイルとしては、例えば、鉱物油(例えば、ライトミネラルオイル)、植物油、シリコーンオイル、フッ素化オイル、を用い得る。懸濁液の濃度、流路中の流速、せん断の間隔を調整し、当業者は、液滴あたり1つ以上の細胞または細胞様構造物が封入されないように液滴作製を行うことが可能である。 Droplet production can be performed using, for example, a microchannel. The droplet making section may include a microchannel. By flowing a suspension of cells or cell-like structures into a microchannel and shearing the suspension, droplets encapsulating each cell or cell-like structure can be made. Shearing can be done at regular intervals. Shearing of the suspension can be done with oil. As the oil, for example, mineral oil (for example, light mineral oil), vegetable oil, silicone oil, fluorinated oil, etc. can be used. By adjusting the concentration of the suspension, the flow velocity in the flow path, and the shear interval, those skilled in the art can prepare droplets so that one or more cells or cell-like structures are not enclosed in each droplet. is there.
 液滴の直径は、約1~250μm、より好ましくは約10~200μmであってよく、例えば、液滴の直径は、約1μm、約5μm、約10μm、約15μm、約20μm、約25μm、約30μm、約40μm、約50μm、約80μm、約100μm、約150μm、約200μm、または約250μmであってよい。 The diameter of the droplet may be from about 1 to 250 μm, more preferably from about 10 to 200 μm, for example, the diameter of the droplet is about 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about. It may be 30 μm, about 40 μm, about 50 μm, about 80 μm, about 100 μm, about 150 μm, about 200 μm, or about 250 μm.
 (ゲル化)
 本開示は、液滴をゲル化してゲルカプセルを生成する工程を包含し得る。また、本開示において、装置は、液滴をゲル化してゲルカプセルを生成するゲルカプセル生成部を備え得る。液滴のゲル化は、液滴にゲルカプセルの材料が含まれるように構成し、作製した液滴を冷却することによって行うことができる。あるいは、液滴に対して光等の刺激を与えることによってゲル化を行うこともできる。液滴にゲルカプセルの材料が含まれるようにするには、例えば、細胞または細胞様構造物の懸濁液にゲルカプセルの材料を含めておくことによって行うことができる。
(Gelification)
The present disclosure may include the step of gelling a droplet to form a gel capsule. Further, in the present disclosure, the device may include a gel capsule generating unit that gels droplets to form gel capsules. Gelation of the droplets can be performed by configuring the droplets to contain the material of the gel capsule and cooling the prepared droplets. Alternatively, gelation can be performed by giving a stimulus such as light to the droplet. The inclusion of the gel capsule material in the droplets can be done, for example, by including the gel capsule material in a suspension of cells or cell-like structures.
 ゲルカプセルの直径は、約1~250μm、より好ましくは約10~200μmであってよく、例えば、約1μm、約5μm、約10μm、約15μm、約20μm、約25μm、約30μm、約40μm、約50μm、約80μm、約100μm、約150μm、約200μm、または約250μmであってよい。ゲルカプセルの直径は、作製する液滴と同じであってもよいが、ゲル化に際して直径が変化してもよい。 The diameter of the gel capsule may be about 1-250 μm, more preferably about 10-200 μm, eg, about 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 40 μm, about. It may be 50 μm, about 80 μm, about 100 μm, about 150 μm, about 200 μm, or about 250 μm. The diameter of the gel capsule may be the same as that of the droplet to be produced, but the diameter may change during gelation.
 ゲルカプセルの材料は、アガロース、アクリルアミド、光硬化性樹脂(例えば、PEG-DA)、PEG、ゼラチン、アルギン酸ナトリウム、マトリゲル、コラーゲンなどを含み得る。 The material of the gel capsule may include agarose, acrylamide, a photocurable resin (for example, PEG-DA), PEG, gelatin, sodium alginate, matrigel, collagen and the like.
 ゲルカプセルは、ヒドロゲルカプセルであってよい。本明細書において、「ヒドロゲル」とは、高分子物質またはコロイド粒子の網目構造によって保持されている溶媒あるいは分散媒が水であるものを指す。 The gel capsule may be a hydrogel capsule. As used herein, the term "hydrogel" refers to one in which the solvent or dispersion medium held by the network structure of the polymer substance or colloidal particles is water.
 大量の細胞からまとめてDNAを取り出す際などは、フェノール・クロロホルム抽出とエタノール沈殿によってDNAを精製し得る。しかしながら、単一細胞からの遺伝物質の取得・分析を企図する場合、1細胞毎の遺伝物質の量は非常に微量であり、ロスなく個別に核酸のみの状態に変換する必要がある。単一細胞に対しては、一般的なバルクスケールでの手順で核酸精製を試みても、全く核酸が取り出せないか、あるいは、夾雑物由来の核酸しか抽出できない結果になる。1細胞実験ではコンタミや標的遺伝物質のロスは大きな問題となるが、単一の細胞または細胞様構造物を封入したゲルカプセルを用いることによって、精製した遺伝物質(例えば、DNA)をゲルカプセル中に保持することができ、また、外部からの分子の夾雑の可能性を排除することができる。また、操作面でも非常に簡単な操作で、大量の1細胞を並列処理することができる。ゲル化した液滴を含む試験管を遠心し、上清を除去し、洗浄液に置換するというステップを行うことができる。あるいは、ゲル化した液滴をフィルターでろ過し、上清を除去したのち、洗浄液を通液させ、最後にゲルカプセルを回収するというステップでも行うことができる。ゲルカプセルを用いることにより、遺伝物質を保持したまま、残留試薬を希薄化することができる。このステップは繰り返すことも可能である。阻害が出ないレベルにまで試薬を希薄化することで、下流の操作、例えば、増幅反応をスムーズに行うことができる。 When extracting DNA from a large number of cells at once, DNA can be purified by phenol / chloroform extraction and ethanol precipitation. However, when attempting to obtain and analyze a genetic substance from a single cell, the amount of the genetic substance per cell is very small, and it is necessary to individually convert the genetic substance into a nucleic acid-only state without loss. For a single cell, even if nucleic acid purification is attempted by a general bulk scale procedure, the result is that no nucleic acid can be extracted or only nucleic acid derived from impurities can be extracted. Loss of contamination and target genetic material is a major problem in single-cell experiments, but by using a gel capsule containing a single cell or cell-like structure, purified genetic material (eg, DNA) can be placed in the gel capsule. It can be retained in the cell, and the possibility of contamination of molecules from the outside can be eliminated. In addition, a large amount of one cell can be processed in parallel with a very simple operation. The steps of centrifuging the test tube containing the gelled droplets, removing the supernatant and replacing it with a cleaning solution can be performed. Alternatively, the gelled droplets can be filtered through a filter to remove the supernatant, the cleaning solution is passed through the mixture, and finally the gel capsules are collected. By using a gel capsule, the residual reagent can be diluted while retaining the genetic material. This step can be repeated. By diluting the reagent to a level that does not cause inhibition, downstream operations, such as amplification reactions, can be performed smoothly.
 本開示の1つの局面において、ゲルカプセルまたはその材料を含む組成物が提供され得る。上述または後述の点から、かかる組成物は、細胞中の核酸をシングルセルレベルで増幅するために有用であり得る。また、かかる組成物は、ゲノムライブラリーを作製するために有用であり得る。さらなる実施形態において、ゲルカプセルまたはその材料と、シングルセル状態の細胞とを含む組成物が提供され得る。上述または後述の点から、かかる組成物は、細胞中の核酸をシングルセルレベルで増幅するために有用であり得る。また、かかる組成物は、ゲノムライブラリーを作製するために有用であり得る。かかる組成物は、シングルセルレベルで細胞中の核酸を配列決定するために有用であり得る。 In one aspect of the present disclosure, a composition comprising a gel capsule or a material thereof may be provided. From the points described above or below, such compositions may be useful for amplifying nucleic acids in cells at the single cell level. Also, such compositions can be useful for making genomic libraries. In a further embodiment, a composition comprising a gel capsule or a material thereof and cells in a single cell state can be provided. From the points described above or below, such compositions may be useful for amplifying nucleic acids in cells at the single cell level. Also, such compositions can be useful for making genomic libraries. Such compositions can be useful for sequencing intracellular nucleic acids at the single cell level.
 (溶解)
 本開示は、ゲルカプセルを1種以上の溶解用試薬に浸漬して前記細胞または細胞様構造物を溶解することを包含し得る。また、本開示において、装置は、ゲルカプセルを溶解用試薬に浸漬する溶解用試薬浸漬部を備え得る。溶解の際、細胞中のポリヌクレオチドがゲルカプセル内に溶出しポリヌクレオチドに結合する物質が除去された状態でゲルカプセル内に保持され得る。溶解用試薬には、例えば、酵素、界面活性剤、その他変性剤、還元剤およびpH調節剤があり、それらの組み合わせもまた用いることができる。本開示の1つの局面では、細胞中の核酸をシングルセルレベルで増幅するための、溶解用試薬を含む組成物が提供され得る。本開示の一実施形態において、当該ポリヌクレオチドを増幅用試薬に接触させて当該ポリヌクレオチドをゲルカプセル内で増幅する工程は、当該ポリヌクレオチドをゲルカプセル内でゲル状態を保ちながら増幅することもできる。
(Dissolution)
The present disclosure may include immersing a gel capsule in one or more lysis reagents to lyse the cell or cell-like structure. Further, in the present disclosure, the device may include a dissolution reagent immersion portion for immersing the gel capsule in the dissolution reagent. Upon lysis, the polynucleotide in the cell may be eluted in the gel capsule and retained in the gel capsule with the substance binding to the polynucleotide removed. Dissolving reagents include, for example, enzymes, surfactants, other denaturing agents, reducing agents and pH regulators, and combinations thereof can also be used. In one aspect of the disclosure, a composition comprising a lysing reagent for amplifying nucleic acids in cells at the single cell level may be provided. In one embodiment of the present disclosure, the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
 溶解用試薬は、リゾチーム、ラビアーゼ、ヤタラーゼ、アクロモペプチダーゼ、プロテアーゼ、ヌクレアーゼ、ザイモリアーゼ、キチナーゼ、リソスタフィン、ムタノライシン、ドデシル硫酸ナトリウム、ラウリル硫酸ナトリウム、水酸化カリウム、水酸化ナトリウム、フェノール、クロロホルム、グアニジン塩酸塩、尿素、2-メルカプトエタノール、ジチオトレイトール、TCEP-HCl、コール酸ナトリウム、デオキシコール酸ナトリウム、Triton X-100、Triton X-114、NP-40、Brij-35、Brij-58、Tween 20、Tween 80、オクチルグルコシド、オクチルチオグルコシド、CHAPS、CHAPSO、ドデシル-β-D-マルトシド、Nonidet P-40、Zwittergent 3-12からなる群から選択される少なくとも1つを含み得る。溶解用試薬は、リゾチーム、アクロモペプチダーゼ、プロテアーゼ、ドデシル硫酸ナトリウム及び水酸化カリウムからなる群から少なくとも1種選択される場合がある。 Reagents for lysis include lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride. , Urea, 2-mercaptoethanol, dithiotreitol, TCEP-HCl, sodium dodecyl, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, It may include at least one selected from the group consisting of Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Noidet P-40, Zwittergent 3-12. The lysis reagent may be selected from at least one group consisting of lysozyme, achromopeptidase, protease, sodium dodecyl sulfate and potassium hydroxide.
 細胞または細胞様構造物における一部の配列の有無を検出することのみを目的とする場合には、積極的な細胞または細胞様構造物の溶解を行う必要は必ずしもなく、物理的な刺激または熱的な刺激による、細胞または細胞様構造物からの核酸の漏出に基づいて検出を行うことも可能である。しかしながら、ゲノム全域などの大量の情報を単一細胞から得るためには、積極的に細胞または細胞様構造物を破壊し、その中の遺伝物質を完全な状態で細胞から単離することが好ましい。また、ゲルカプセルを用いている場合、熱的・機械的な刺激は、ゲルカプセルの崩壊につながるおそれもあり、溶解試薬を用いることが好ましい場合があり得る。 If the only purpose is to detect the presence or absence of some sequences in a cell or cell-like structure, it is not always necessary to actively lyse the cell or cell-like structure, and physical stimulation or heat. It is also possible to perform detection based on the leakage of nucleic acid from cells or cell-like structures by stimulus. However, in order to obtain a large amount of information such as the entire genome from a single cell, it is preferable to actively destroy the cell or cell-like structure and isolate the genetic substance in the cell from the cell in a complete state. .. Further, when a gel capsule is used, thermal / mechanical irritation may lead to disintegration of the gel capsule, and it may be preferable to use a dissolving reagent.
 多様な微生物について、細胞ごとに核酸の増幅または分析を行う場合、溶解試薬または溶解試薬の組合せとして、ある程度強力なものを用いることが望ましい。例えば、グラム陽性菌は厚いペプチドグリカン層を有する細胞壁を有するため、緩和なもののみでは細胞が十分に溶解できない可能性がある。 When amplifying or analyzing nucleic acids for each cell of various microorganisms, it is desirable to use a lytic reagent or a combination of lysing reagents that is strong to some extent. For example, Gram-positive bacteria have a cell wall with a thick peptidoglycan layer, so mild ones alone may not be sufficient to lyse cells.
 強力な溶解試薬は、DNA増幅等の反応を阻害する可能性があり、下流の反応の前に十分に除去されることが好ましい。ゲルカプセルを用いている場合には、ゲルカプセルによって解析または増幅の対象となる遺伝物質が保持されるため、遺伝物質が少量である単一細胞解析においても溶解試薬の除去を行うことができ、そのため、強力な溶解試薬または溶解試薬の組合せを用いることが可能である。そして、強力な溶解試薬または溶解試薬の組合せを用いることは、多様な細胞(細胞壁を有するものやその他の種類の微生物を含む)の種類を問わず、網羅的な核酸の増幅またはゲノム解析を可能にし得る。方法は、ゲルカプセルから溶解用試薬および/または夾雑物質を除去する工程を含み得る。また、溶解用試薬浸漬部は、ゲルカプセルから溶解用試薬および/または夾雑物質を除去する手段を備える。 A strong lysing reagent may inhibit a reaction such as DNA amplification, and is preferably sufficiently removed before the downstream reaction. When a gel capsule is used, the gel capsule retains the genetic substance to be analyzed or amplified, so that the lytic reagent can be removed even in a single cell analysis in which the amount of the genetic substance is small. Therefore, it is possible to use a strong dissolving reagent or a combination of dissolving reagents. And the use of potent lysing reagents or combinations of lysing reagents allows for comprehensive nucleic acid amplification or genome analysis, regardless of the type of cell (including those with cell walls and other types of microorganisms). Can be. The method may include removing the lysing reagent and / or contaminants from the gel capsule. In addition, the dissolution reagent immersion portion includes means for removing the dissolution reagent and / or contaminants from the gel capsule.
 標的分子が細胞表面マーカーや核酸の一部であり、その存在自体を検知することが目的である場合には、溶解操作が部分的、あるいは溶解操作がなされなくても、目的を達成できる可能性がある。他方で、ゲノムDNAの全長の増幅を企図する場合、ゲノムDNAは、細胞中に1分子しか存在しないことが通常であるため、完全に細胞または細胞様構造物の溶解が進み、またDNAから結合タンパク質類を十分に除去する必要がある。これにより、腸内微生物のような数百種以上の微生物からなる検体を対象とした際にも、そのすべてを均質に溶解し、その全てから全ゲノム増幅を行うことが可能となる。また、それにより、ライブラリー調製を行い、最終的に全ゲノム配列情報を得ることが可能となる。 If the target molecule is part of a cell surface marker or nucleic acid and the goal is to detect its presence itself, the goal may be achieved even if the lysis operation is partial or undissolved. There is. On the other hand, when attempting to amplify the full length of genomic DNA, the genomic DNA usually has only one molecule in the cell, so that the cell or cell-like structure is completely dissolved and bound from the DNA. It is necessary to sufficiently remove proteins. As a result, even when a sample consisting of hundreds or more kinds of microorganisms such as intestinal microorganisms is targeted, all of them can be uniformly dissolved and whole genome amplification can be performed from all of them. It also makes it possible to prepare the library and finally obtain whole genome sequence information.
 (増幅)
 本開示は、ポリヌクレオチドを増幅用試薬に接触させてポリヌクレオチドをゲルカプセル内で増幅することを包含し得る。また、本開示において、装置は、ゲルカプセルを増幅用試薬に浸漬する増幅用試薬浸漬部を備え得る。増幅用試薬浸漬部は、増幅用試薬への浸漬後、必要に応じて、ゲルカプセルの温度を調節するための手段を備え得る。
(amplification)
The present disclosure may include contacting the polynucleotide with an amplification reagent to amplify the polynucleotide within a gel capsule. Further, in the present disclosure, the device may include an amplification reagent immersion portion for immersing the gel capsule in the amplification reagent. The amplification reagent immersion portion may be provided with means for adjusting the temperature of the gel capsule, if necessary, after immersion in the amplification reagent.
 加熱処理(80度以上)を伴う反応はゲル(例えば、アガロースゲル)の再溶解を招く可能性があるため、個別粒子化していた形状を崩壊させ、単一細胞隔離を無効化してしまう場合がある。この場合、約60度以下の酵素反応がゲル液滴形状を保つために望ましい。恒温鎖置換増幅反応(multiple displacement amplification)は、この温度の範囲内で実行可能であり、また、ゲノムDNA全域の増幅が可能である点で好ましい。用いる酵素としては、例えば、phi29ポリメラーゼ、Bstポリメラーゼ、Aacポリメラーゼ、リコンビナーゼポリメラーゼが挙げられる。 Reactions involving heat treatment (80 ° C or higher) can lead to redissolution of gels (eg, agarose gels), which can disrupt the individualized shape and invalidate single cell isolation. is there. In this case, an enzymatic reaction of about 60 degrees or less is desirable to maintain the gel droplet shape. The constant temperature chain substitution amplification reaction is preferable in that it can be carried out within this temperature range and the entire genomic DNA can be amplified. Examples of the enzyme used include phi29 polymerase, Bst polymerase, Aac polymerase, and recombinase polymerase.
 特定の細胞(例えば、特定の微生物)を検出することを目的としたPCRを実施する場合、その微生物に応じた特定プライマーを使用することが一般的である。しかしながら、ゲノム全体の増幅を行う場合には、ランダムプライマーを用いることが好ましい。 When performing PCR for the purpose of detecting a specific cell (for example, a specific microorganism), it is common to use a specific primer corresponding to the microorganism. However, when amplifying the entire genome, it is preferable to use a random primer.
 本開示に記載のない他種の生物を対象とする際には、当該生物を特異的に検出するためのPCRプライマーおよびTaqmanプローブ等検出用のオリゴヌクレオチドを設計することで対応が可能である。プライマー・プローブの作成法は一般的なPCR・qPCR等でのオリゴヌクレオチド配列設計に準じて行うことができる。 When targeting other species of organisms not described in the present disclosure, it is possible to deal with it by designing a PCR primer for specifically detecting the organism and an oligonucleotide for detection such as Taqman probe. The primer / probe can be prepared according to the oligonucleotide sequence design by general PCR, qPCR, or the like.
 また、非特許文献(Leng X, Zhang W, Wang C, Cui L, Yang CJ. Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR. Lab Chip. 2010;10(21):2841-2843. doi:10.1039/c0lc00145g)のようにPCR反応液中にアガロースを加えておくことで、反応工程にて、PCR後に冷却を行い、再度ドロップレットをゲルカプセル化することも可能である。この場合、ゲルカプセルとしてフローサイトメトリーなどを使って分取することができる。 In addition, non-patent documents (Leng X, Zhang W, Wang C, Cui L, Yang CJ. Agarose droplet microfluidics for high-high lily parallell and effective Single 28 mulsion; By adding agarose to the PCR reaction solution as in 10.1039 / c0lc00145 g), it is possible to cool the droplets after PCR in the reaction step and gel-encapsulate the droplets again. In this case, the gel capsule can be separated by using flow cytometry or the like.
 また、プライマーの修飾として、アガロース骨格(非特許文献:Leng X, Zhang W, Wang C, Cui L, Yang CJ. Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR. Lab Chip. 2010;10(21):2841-2843. doi:10.1039/c0lc00145g)やビーズ(Novak R, Zeng Y, Shuga J, et al. Single-cell multiplex gene detection and sequencing with microfluidically generated agarose emulsions. Angew Chem Int Ed Engl. 2011;50(2):390-395. doi:10.1002/anie.201006089)との架橋が適宜なされた形に設計され、増副産物がゲルカプセル内にとどまるようにすることもできる。これにより短鎖の増副産物もゲルカプセルに留めて蛍光DNA結合性色素を使ったゲルカプセルの選抜が可能である。 In addition, as a modification of the primer, the agarose skeleton (non-patent documents: Leng X, Zhang W, Wang C, Cui L, Yang CJ. ):. 2841-2843 doi:... 10.1039 / c0lc00145g) and beads (Novak R, Zeng Y, Shuga J, et al Single-cell multiplex gene detection and sequencing with microfluidically generated agarose emulsions Angew Chem Int Ed Engl 2011 It is also designed to be appropriately crosslinked with 50 (2): 390-395. Doi: 10.10012 / anie.201006089) so that the augmented by-product stays in the gel capsule. This makes it possible to select gel capsules using a fluorescent DNA-binding dye by retaining short-chain by-products in gel capsules.
 mRNAはゲノムDNAを元に細胞内に数千から数万種存在し、個々の分子量も多い。このためRNAを対象とした発現解析では、遺伝子の種類や発現量を絶対(相対)的に求めることが目的となるため、遺伝子の一部(数十塩基)だけを読み取って、どの遺伝子がどれだけ発現しているかを定量することが可能である。ゲノムDNAを対象とする場合、原則的にゲノムDNAは1細胞に1分子しか存在しないため、その1分子しか無い配列情報を漏らさず増やすことが、その数百万塩基の全てを解読するためには必要となり得る。ゲルカプセル中での処理は、そのような増幅にとって有利である。また、配列決定のための核酸を、1細胞から一部ずつの断片的な情報ではなく、全体として得られることはシングルセル解析において有利である。 There are thousands to tens of thousands of types of mRNA in cells based on genomic DNA, and each has a large molecular weight. For this reason, in expression analysis for RNA, the purpose is to determine the type and expression level of a gene in an absolute (relative) manner. It is possible to quantify whether it is expressed only. When targeting genomic DNA, in principle, there is only one molecule of genomic DNA in one cell, so increasing the sequence information of only one molecule without leaking it is necessary to decode all of the millions of bases. May be needed. Treatment in gel capsules is advantageous for such amplification. In addition, it is advantageous in single cell analysis that the nucleic acid for sequencing can be obtained as a whole from one cell instead of fragmentary information.
 (細胞および細胞様構造物)
 本開示において対象とする細胞または細胞様構造物は、特段限定されるものではないが、例えば、微生物(例えば、細菌、真菌、単細胞動物)、多細胞生物の細胞(例えば、体細胞、生殖細胞、培養細胞、腫瘍細胞、動物細胞、植物細胞)、細胞内器官(ミトコンドリア、核、葉緑体)、ウイルスが挙げられる。
(Cells and cell-like structures)
The cells or cell-like structures of interest in the present disclosure are not particularly limited, but are, for example, microorganisms (eg, bacteria, fungi, single cell animals), cells of multicellular organisms (eg, somatic cells, germ cells). , Cultured cells, tumor cells, animal cells, plant cells), intracellular organs (mitochonium, nucleus, chloroplast), viruses.
 ゲノム配列既知の生物の細胞については、その中でどの遺伝子が発現しているかRNA
 を計測するという意図が発生し得るが、ゲノム配列および/または遺伝子の情報が未知の生物の解析の場合には、RNA解析の前に、ゲノム自体の情報を得る必要がある。その場合には、ゲルカプセルを用いる本開示の方法によるシングルセルレベルでのゲノム配列の増幅は有利である。
For cells of organisms with known genome sequences, which genes are expressed in them RNA
In the case of analysis of an organism whose genome sequence and / or gene information is unknown, it is necessary to obtain information on the genome itself before RNA analysis. In that case, amplification of the genome sequence at the single cell level by the method of the present disclosure using gel capsules is advantageous.
 本開示においては、2つ以上の細胞または細胞様構造物を含む試料を用いることができる。2つ以上の細胞は、複数の生物に由来するものであってもよい。試料として、例えば、微生物試料、組織試料、共生微生物および宿主生物の混合試料、動物・ヒト検体より取り出した微生物および細胞を含む試料が挙げられる。微生物試料としては、細菌叢試料の他、2種以下の細胞または細胞様構造物を含有する試料、真菌等の細菌以外の細胞または細胞用構造物が含有される試料が挙げられる。ヒト検体より取り出した微生物および細胞を含む試料としては、糞便、唾液、喀痰、手術洗浄液、血液、皮膚・身体粘膜の拭い液やスワブなどが挙げられ、直接的に利用することも可能であるが、細胞や微生物を分離する操作を行ってから使用してもよい。 In the present disclosure, a sample containing two or more cells or cell-like structures can be used. The two or more cells may be derived from a plurality of organisms. Examples of the sample include a microbial sample, a tissue sample, a mixed sample of a symbiotic microorganism and a host organism, and a sample containing microorganisms and cells taken from an animal / human sample. Examples of the microbial sample include a bacterial flora sample, a sample containing two or less types of cells or cell-like structures, and a sample containing cells or cell structures other than bacteria such as fungi. Examples of samples containing microorganisms and cells taken out from human samples include feces, saliva, sputum, surgical lavage fluid, blood, skin / body mucosa wipes and swabs, and can be used directly. , It may be used after performing an operation for separating cells and microorganisms.
 本開示において、対象とし得る微生物は、限定されるものではないが、真正細菌、大腸菌、枯草菌、藍色細菌、球菌、桿菌、ラセン菌、グラム陰性菌、グラム陽性菌、古細菌、真菌などが挙げられる。本開示で対象とし得る細菌は、例えば、Negibacteria、Eobacteria、Deinococci、Deinococci、Deinococcales、Thermales、Chloroflexi、Anaerolineae、Anaerolineales、Caldilineae、Chloroflexales、Herpetosiphonales、Thermomicrobia、Thermomicrobiales、Sphaerobacterales、Ktedonobacteria、Ktedonobacterales、Thermogemmatisporales、Glycobacteria、Cyanobacteria、Gloeobacterophyceae、Gloeobacterales、Nostocophyceae、Synechococcophycidae、Synechococcales、Nostocophycidae、Chroococcales、Oscillatoriales、Nostocales、Pseudanabaenales、Spirochaetes、Spirochaetes、Spirochaetales、Fibrobacteres、Fibrobacteria、Gemmatimonadetes、Gemmatimonadetes、Gemmatimonadales、Chlorobi、Chlorobea、Chlorobiales、Ignavibacteria、Ignavibacteriales、Bacteroidetes、Bacteroidia、Bacteroidales、Flavobacteriia、Flavobacteriales、Sphingobacteriia、Sphingobacteriales、Cytophagia、Cytophagales、Planctomycetes、Planctomycea、Planctomycetales、Phycisphaerae、Phycisphaerales、Chlamydiae、Chlamydiae、Chlamydiales、Verrucomicrobia、Verrucomicrobiae、Verrucomicrobiales、Opitutae、Opitutales、Puniceicoccales、Spartobacteria、Chthoniobacterales、Lentisphaerae、Lentisphaeria、Lentisphaerales、Victivallales、Proteobacteria、Alphaproteobacteria、Rhodospirillales、Rickettsiales、Rhodobacterales、Sphingomonadales、Caulobacterales、Rhizobiales、Parvularculales、Kordiimonadales、Sneathiellales、Kiloniellales、Betaproteobacteria、Burkholderiales、Hydrogenophilales、Methylophilales、Neisseriales、Nitrosomonadales、Rhodocyclales、Procabacteriales、Gammaproteobacteria、Chromatiales、Acidithiobacillales、Xanthomonadales、Cardiobacteriales、Thiotrichales、Legionellales、Methylococcales、Oceanospirillales、Pseudomonadales、Alteromonadales、Vibrionales、Aeromonadales、Enterobacteriales、Pasteurellales、Deltaproteobacteria、Desulfurellales、Desulfovibrionales、Desulfobacterales、Desulfarculales、Desulfuromonadales、Syntrophobacterales、Bdellovibrionales、Myxococcales、Epsilonproteobacteria、Campylobacterales、Nautiliales、Acidobacteria、Acidobacteria、Acidobacteriales、Holophagae、Holophagales、Acanthopleuribacterales、Aquificae、Aquificae、Aquificales、Deferribacteres、Deferribacteres、Geovibriales、Thermodesulfobacteria、Thermodesulfobacteria、Thermodesulfobacteriales、Nitrospirae、Nitrospira、Nitrospirales、Fusobacteria、Fusobacteriia、Fusobacteriales、Synergistetes、Synergistia、Synergistales、Caldiserica、Caldisericia、Caldisericales、Elusimicrobia、Elusimicrobia、Elusimicrobiales、Armatimonadetes、Armatimonadia、Armatimonadales、Chthonomonadetes、Chthonomonadales、Fimbriimonadia、Fimbriimonadales、Posibacteria、Thermotogae、Thermotogae、Thermotagales、Firmicutes、Bacilli、Bacillales、Lactobacillales、Clostridia、Clostridiales、Halanaerobiales、Thermoanaerobacterales、Natranaerobiales、Negativicutes、Selenomonadales、Erysipelotrichia、Erysipelotrichales、Thermolithobacteria、Thermolithobacterales、Tenericutes、Mollicutes、Mycoplasmatales、Entomoplasmatales、Acholeplasmatales、Anaeroplasmatales、Actinobacteria、Actinobacteria、Actinomycetales、Actinopolysporales、Bifidobacteriales、Catenulisporales、Corynebacteriales、Frankiales、Glycomycetales、Jiangellales、Kineosporiales、Micrococcales、Micromonosporales、Propionibacteriales、Pseudonocardiales、Streptomycetales、Streptosporangiales、Dictyoglomi、Dictyoglomia、Dictyoglomales、Chrysiogenetes、Chrysiogenetes、Chrysiogenales、Haloplasmatalesなどの細菌が挙げられる。また、それらのうちの複数が混在する試料において、細胞毎の網羅的な解析を行うことができる。 In the present disclosure, the microorganisms that can be targeted are not limited to, but are not limited to, eubacteria, Escherichia coli, bacilli, indigo bacteria, cocci, bacilli, spiral bacteria, gram-negative bacteria, gram-positive bacteria, archaea, fungi, etc. Can be mentioned. Bacteria that can be targeted by the present disclosure, for example, Negibacteria, Eobacteria, Deinococci, Deinococci, Deinococcales, Thermales, Chloroflexi, Anaerolineae, Anaerolineales, Caldilineae, Chloroflexales, Herpetosiphonales, Thermomicrobia, Thermomicrobiales, Sphaerobacterales, Ktedonobacteria, Ktedonobacterales, Thermogemmatisporales, Glycobacteria, Cyanobacteria , Gloeobacterophyceae, Gloeobacterales, Nostocophyceae, Synechococcophycidae, Synechococcales, Nostocophycidae, Chroococcales, Oscillatoriales, Nostocales, Pseudanabaenales, Spirochaetes, Spirochaetes, Spirochaetales, Fibrobacteres, Fibrobacteria, Gemmatimonadetes, Gemmatimonadetes, Gemmatimonadales, Chlorobi, Chlorobea, Chlorobiales, Ignavibacteria, Ignavibacteriales, Bacteroidetes, Bacteroidia , Bacteroidales, Flavobacteriia, Flavobacteriales, Sphingobacteriia, Sphingobacteriales, Cytophagia, Cytophagales, Planctomycetes, Planctomycea, Planctomycetales, Phycisphaerae, Phycisphaerales, Chlamydiae, Chlamydiae, Chlamydiales, Verrucomicrobia, Verrucomicrobiae, Verrucomicrobiales, Opitutae, Opitutales, Puniceicoccales, Spartobacteria, Chthoniobacterales, Lentisphaerae, Lentisphaeria, Lentisphaerales, Victivallales, Proteobacteria, Alphaproteobacteria, Rhodospirillales, Rickettsiales, Rhodobacterales, Sphingomonadales, Caulobacterales, Rhizobiales, Parvularculales, Kordiimonadales, Sneathiellales, Kiloniellales, Betaproteobacteria, Burkholderiales, Hydrogenophilales, Methylophilales, Neisseriales, Nitrosomonadales, Rhodocyclales, Procabacteriales, Gammaproteobacteria, Chromatiales, Acidithiobacillales, Xanthomonadales, Cardiobacteriales, Thiotrichales, Legionellales, Methylococcales, Oceanospirillales, Pseudomonadales, Alteromonadales, Vibrionales, Aeromonadales, Enterobacteriales, Pasteurellales, Deltaproteobacteria, Desulfurellales, Desulfovibrionales, Desulfobacterales, Desulfarculales, Desulfuromonadales, Syntrophobacterales, Bdellovibrionales, Myxococcales, Epsilonproteobacteria, Campilobacterales, Nautiliales, Acidobacteria, Acidobacteria, Acidobacteriales, Holofagae, Holofagaales, Aquificaeobacteria, Aquificae, Aquificae, Aquificae, Aquificae, Aquificae, Aquificae, Aquificae es, Geovibriales, Thermodesulfobacteria, Thermodesulfobacteria, Thermodesulfobacteriales, Nitrospirae, Nitrospira, Nitrospirales, Fusobacteria, Fusobacteriia, Fusobacteriales, Synergistetes, Synergistia, Synergistales, Caldiserica, Caldisericia, Caldisericales, Elusimicrobia, Elusimicrobia, Elusimicrobiales, Armatimonadetes, Armatimonadia, Armatimonadales, Chthonomonadetes, Chthonomonadales, Fimbriimonadia, Fimbriimonadales, Posibacteria, Thermotogae, Thermotogae, Thermotagales, Firmicutes, Bacilli, Bacillales, Lactobacillales, Clostridia, Clostridiales, Halanaerobiales, Thermoanaerobacterales, Natranaerobiales, Negativicutes, Selenomonadales, Erysipelotrichia, Erysipelotrichales, Thermolithobacteria, Thermolithobacterales, Tenericutes, Mollicutes, Mycoplasmatales, Entomoplasmatales, Acholeplasmatales, Anaeroplasmatales, Actinobacteria, Actinobacteria, Actinomycetales, Actinopolysporales, Bifidobacteriales, Catenulisporales, Corynebacteriales, Frankiales, Glycomycetales, Jiangellales, Kineosporiales, Micrococcales, Micromonosporales, Propionibacteri ares, Pseudonocardiales, Streptomycetales, Streptosporangiales, Dictyoglomia, Dictyoglomia, Dictyoglomales, Chrysiogenetes, Chrysiogenetes, Chrysiogenetes, Chrysiogenetes, etc. In addition, it is possible to perform a comprehensive analysis for each cell in a sample in which a plurality of them coexist.
(カプセル化)
 核酸を含む細胞または細胞様構造物、あるいはそれに由来する物質を、必要な薬剤とともに、個別にカプセル化する工程を単にカプセル化ともいう。カプセル化される対象は、1次増幅により増幅されたものであってもよく、他の反応の精製物であってもよい。カプセル化の対象は、増幅された核酸、増幅された核酸を含むゲルカプセル、増幅された核酸を含む細胞または細胞様構造物であってもよい。再カプセル化の対象が、ポリヌクレオチドに結合する物質が除去された状態でポリヌクレオチドを有する場合、あるいは、ポリヌクレオチドをゲルカプセル内でゲル状態を保ちながら増幅する場合、カプセル化においてゲル化を行わなくともよく、ゲル化材料を含まなくともよい。カプセルの直径は、約1~250μm、より好ましくは約10~200μmであってよく、例えば、カプセルの直径は、約1μm、約5μm、約10μm、約15μm、約20μm、約25μm、約30μm、約40μm、約50μm、約80μm、約100μm、約150μm、約200μm、または約250μmであってよい。カプセルの直径は、カプセル化の回数が増えるごとに大きくなってもよい。
(Encapsulation)
The process of individually encapsulating cells or cell-like structures containing nucleic acids, or substances derived from them, together with necessary drugs, is also simply referred to as encapsulation. The object to be encapsulated may be one amplified by primary amplification or may be a purified product of another reaction. The object of encapsulation may be an amplified nucleic acid, a gel capsule containing the amplified nucleic acid, a cell or a cell-like structure containing the amplified nucleic acid. If the object to be reencapsulated has the polynucleotide in a state where the substance binding to the polynucleotide has been removed, or if the polynucleotide is amplified while maintaining the gel state in the gel capsule, gelation is performed in the encapsulation. It does not have to be, and it does not have to contain a gelling material. The diameter of the capsule may be about 1-250 μm, more preferably about 10-200 μm, for example, the diameter of the capsule is about 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, It may be about 40 μm, about 50 μm, about 80 μm, about 100 μm, about 150 μm, about 200 μm, or about 250 μm. The diameter of the capsule may increase as the number of encapsulations increases.
 1つの実施形態において、カプセルを二回目以降のカプセル化する場合、それぞれのカプセルは別の材料で形成されてもよく、一部材料が重複してもよく、あるいはすべてが同一の材料で形成されてもよい。1つの実施形態において、分析に必要な薬剤は、核酸増幅に必要な薬剤であってもよい。 In one embodiment, when encapsulating the capsules for the second and subsequent times, each capsule may be made of different materials, some may overlap, or all may be made of the same material. You may. In one embodiment, the agent required for analysis may be the agent required for nucleic acid amplification.
 (シングルセル解析)
 本開示において、1つずつの細胞または細胞様構造物由来の核酸の増幅は、任意の方法によって行われ得るが、多数の細胞または細胞様構造物から簡便に1つずつの細胞または細胞様構造物由来の核酸増幅を行うためには、細胞または細胞様構造物を1つずつ液滴中に封入する工程と、当該液滴をゲル化してゲルカプセルを生成する工程と、当該ゲルカプセルを1種以上の溶解用試薬に浸漬して細胞を溶解する工程であって、当該細胞のゲノムDNAまたはその部分を含むポリヌクレオチドが当該ゲルカプセル内に溶出し当該ゲノムDNAまたはその部分に結合する物質が除去された状態で当該ゲルカプセル内に保持される、工程と、当該ポリヌクレオチドを増幅用試薬に接触させて当該ポリヌクレオチドをゲルカプセル内で増幅する工程とを含む、方法によって1つずつの細胞または細胞様構造物由来の増幅核酸を得ることが好ましい場合がある。本開示の一実施形態において、当該ポリヌクレオチドを増幅用試薬に接触させて当該ポリヌクレオチドをゲルカプセル内で増幅する工程は、当該ポリヌクレオチドをゲルカプセル内でゲル状態を保ちながら増幅することもできる。
(Single cell analysis)
In the present disclosure, amplification of nucleic acid derived from a single cell or cell-like structure can be performed by any method, but simply one cell or cell-like structure from a large number of cells or cell-like structures. In order to perform nucleic acid amplification derived from a substance, a step of encapsulating cells or cell-like structures one by one in a droplet, a step of gelling the droplet to generate a gel capsule, and a step of forming the gel capsule 1 In the step of lysing a cell by immersing it in a lysis reagent of seeds or more, a substance in which a polynucleotide containing the genomic DNA of the cell or a portion thereof is eluted into the gel capsule and binds to the genomic DNA or the portion thereof. One cell by method, comprising the step of retaining the polynucleotide in the gel capsule in a removed state and the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule. Alternatively, it may be preferable to obtain an amplified nucleic acid derived from a cell-like structure. In one embodiment of the present disclosure, the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
 1つの実施形態において、工程A)(2つ以上の細胞または細胞様構造物を、単一の細胞または構造物単位ごとに選別し、該細胞または細胞様構造物に含まれる核酸を増幅する工程)は、a)2つ以上の細胞または細胞様構造物を含む試料を用い、液滴中に1つの細胞または成分を封入することと、b)該液滴をゲル化してゲルカプセルを生成することと、c)該ゲルカプセルを1種以上の溶解用試薬に浸漬して該細胞または成分を溶解することであって、該細胞または成分中のポリヌクレオチドが該ゲルカプセル内に溶出し該ポリヌクレオチドに結合する物質が除去された状態で前記ゲルカプセル内に保持される、ことと、d)該ポリヌクレオチドを第1の増幅用試薬に接触させて該ポリヌクレオチドをゲルカプセル内で増幅することを包含してもよい。本開示の一実施形態において、当該ポリヌクレオチドを増幅用試薬に接触させて当該ポリヌクレオチドをゲルカプセル内で増幅する工程は、当該ポリヌクレオチドをゲルカプセル内でゲル状態を保ちながら増幅することもできる。 In one embodiment, step A) (steps of selecting two or more cells or cell-like structures by a single cell or structure unit and amplifying the nucleic acids contained in the cells or cell-like structures. In), a) a sample containing two or more cells or cell-like structures is used to enclose one cell or component in a droplet, and b) the droplet is gelled to form a gel capsule. C) Immersing the gel capsule in one or more lysis reagents to lyse the cells or components, in which the polynucleotides in the cells or components are eluted into the gel capsule and the poly The substance that binds to the nucleotide is retained in the gel capsule in a removed state, and d) the polynucleotide is brought into contact with the first amplification reagent to amplify the polynucleotide in the gel capsule. May be included. In one embodiment of the present disclosure, the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
 1つの実施形態において、液滴は、単一細胞または単一細胞様構造物の懸濁液をマイクロ流路中に流動させ、オイルで懸濁液をせん断することにより単一細胞または単一細胞様構造物の懸濁液を封入することで作製され得る。一部の実施形態において、ゲルカプセルはヒドロゲルカプセルであってもよい。 In one embodiment, the droplet is a single cell or single cell by flowing a suspension of single cell or single cell-like structures into a microchannel and shearing the suspension with oil. It can be made by encapsulating a suspension of similar structures. In some embodiments, the gel capsule may be a hydrogel capsule.
 ゲルカプセルの材料は、アガロース、アクリルアミド、光硬化性樹脂(例えば、PEG-DA)、PEG、ゼラチン、アルギン酸ナトリウム、マトリゲル、コラーゲンなどを含み得る。液滴のゲル化は、液滴にゲルカプセルの材料が含まれるように構成し、作製した液滴を冷却することによって行うことができる。あるいは、液滴に対して光等の刺激を与えることによってゲル化を行うこともできる。液滴にゲルカプセルの材料が含まれるようにするには、例えば、細胞または細胞様構造物の懸濁液にゲルカプセルの材料を含めておくことによって行うことができる。 The material of the gel capsule may include agarose, acrylamide, a photocurable resin (for example, PEG-DA), PEG, gelatin, sodium alginate, matrigel, collagen and the like. Gelation of the droplets can be performed by configuring the droplets to contain the material of the gel capsule and cooling the prepared droplets. Alternatively, gelation can be performed by giving a stimulus such as light to the droplet. The inclusion of the gel capsule material in the droplets can be done, for example, by including the gel capsule material in a suspension of cells or cell-like structures.
 ゲルカプセルは、ヒドロゲルカプセルであってよい。本明細書において、「ヒドロゲル」とは、高分子物質またはコロイド粒子の網目構造によって保持されている溶媒あるいは分散媒が水であるものを指す。 The gel capsule may be a hydrogel capsule. As used herein, the term "hydrogel" refers to one in which the solvent or dispersion medium held by the network structure of the polymer substance or colloidal particles is water.
 溶解用試薬は、リゾチーム、ラビアーゼ、ヤタラーゼ、アクロモペプチダーゼ、プロテアーゼ、ヌクレアーゼ、ザイモリアーゼ、キチナーゼ、リソスタフィン、ムタノライシン、ドデシル硫酸ナトリウム、ラウリル硫酸ナトリウム、水酸化カリウム、水酸化ナトリウム、フェノール、クロロホルム、グアニジン塩酸塩、尿素、2-メルカプトエタノール、ジチオトレイトール、TCEP-HCl、コール酸ナトリウム、デオキシコール酸ナトリウム、Triton X-100、Triton X-114、NP-40、Brij-35、Brij-58、Tween 20、Tween 80、オクチルグルコシド、オクチルチオグルコシド、CHAPS、CHAPSO、ドデシル-β-D-マルトシド、Nonidet P-40、およびZwittergent 3-12からなる群から少なくとも1種選択され得る。 Reagents for lysis include lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride. , Urea, 2-mercaptoethanol, dithiotreitol, TCEP-HCl, sodium dodecyl, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, At least one can be selected from the group consisting of Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Noidet P-40, and Zwittergent 3-12.
 多様な微生物について、細胞ごとに核酸の増幅または分析を行う場合、溶解試薬または溶解試薬の組合せとして、ある程度強力なものを用いることが望ましい。例えば、グラム陽性菌は厚いペプチドグリカン層を有する細胞壁を有するため、緩和なもののみでは細胞が十分に溶解できない可能性がある。 When amplifying or analyzing nucleic acids for each cell of various microorganisms, it is desirable to use a lysing reagent or a combination of lysing reagents that is strong to some extent. For example, Gram-positive bacteria have a cell wall with a thick peptidoglycan layer, so mild ones alone may not be sufficient to lyse cells.
 1つの実施形態において、分析に必要な薬剤が第2の増幅用試薬であってもよい。特定の実施形態において、分析に必要な薬剤は、1つまたそれより多くてもよい。一部の実施形態において、第1の増幅試薬と第2の増幅用試薬とが同一である。他の実施形態において、第1の増幅試薬と第2の増幅用試薬とが異なる。特定の実施形態において、使用する増幅試薬は全て同一でもよく、全て異なってもよい。他の実施形態において、使用する増幅試薬は、一部のみ同一であってもよい。 In one embodiment, the agent required for analysis may be a second amplification reagent. In certain embodiments, the number of agents required for analysis may be one or more. In some embodiments, the first amplification reagent and the second amplification reagent are identical. In other embodiments, the first amplification reagent and the second amplification reagent are different. In a particular embodiment, the amplification reagents used may all be the same or all different. In other embodiments, the amplification reagents used may be partially identical.
 1つの実施形態において、第1の増幅試薬と第2の増幅用試薬を使用する場合、第1の増幅試薬はゲノムDNAまたはその部分の増幅用試薬または特定配列増幅用試薬である。別の実施形態において、第2の増幅試薬はゲノムDNAまたはその部分の増幅用試薬または特定配列増幅用試薬である。特定の実施形態において、特定配列増幅用試薬は、細胞種特異的な配列もしくは種特異的な配列を増幅する試薬または特定種に保存されている配列を増幅する試薬であってもよい。一部の実施形態において、特定配列増幅用試薬はマーカー遺伝子を増幅する試薬であってもよい。 In one embodiment, when the first amplification reagent and the second amplification reagent are used, the first amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence. In another embodiment, the second amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence. In certain embodiments, the reagent for specific sequence amplification may be a reagent that amplifies a cell type-specific sequence or a species-specific sequence, or a reagent that amplifies a sequence stored in a specific species. In some embodiments, the reagent for specific sequence amplification may be a reagent that amplifies a marker gene.
 1つの実施形態において、前記a)において、液滴中に1つの細胞または細胞様構造物、および第1の標識を封入してもよい。別の実施形態において、工程B)において、1つの細胞または細胞様構造物および第2の標識を封入してもよい。他の実施形態において、前記a)および/または工程B)において、1つの細胞または細胞様構造物および1つまたはそれより多くの標識を封入してもよい。 In one embodiment, in a) above, one cell or cell-like structure and a first label may be encapsulated in the droplet. In another embodiment, one cell or cell-like structure and a second label may be encapsulated in step B). In other embodiments, one cell or cell-like structure and one or more labels may be encapsulated in a) and / or step B) above.
 1つの実施形態において、第1の標識と第2の標識とが同一である。別の実施形態において、第1の標識と第2の標識とが異なる。他の実施形態において、使用する標識が全て同一でもよく、全て異なってもよい。特定の実施形態において、使用する標識は、一部のみ同一であってもよい。一部の実施形態において、標識は全核酸の収量を示してもよい。他の実施形態において、標識は特定配列の有無を示してもよい。特定の実施形態において、標識は、特定配列の核酸の収量を示してもよい。他の実施形態において、標識は、特定分子を検出するための薬剤であってもよい。特定の実施形態において、特定分子を検出するための薬剤として、プローブ、抗体、インターカレーター、タグ、放射性物質、蛍光色素結合ヌクレオチド、蛍光標識タンパク質などが挙げられる。 In one embodiment, the first sign and the second sign are the same. In another embodiment, the first and second markers are different. In other embodiments, the labels used may all be the same or all may be different. In certain embodiments, the labels used may be only partially identical. In some embodiments, the label may indicate the yield of total nucleic acid. In other embodiments, the label may indicate the presence or absence of a particular sequence. In certain embodiments, the label may indicate the yield of nucleic acid of a particular sequence. In other embodiments, the label may be an agent for detecting a particular molecule. In certain embodiments, agents for detecting a particular molecule include probes, antibodies, intercalators, tags, radioactive substances, fluorescent dye-binding nucleotides, fluorescently labeled proteins and the like.
 1つの実施形態において、工程A)における細胞または細胞様構造物に含まれる核酸は、ゲノムDNAまたはその部分であり得る。 In one embodiment, the nucleic acid contained in the cell or cell-like structure in step A) can be genomic DNA or a portion thereof.
 1つの実施形態において、工程C)は、標識を評価することを含み得る。一部の実施形態において、工程C)は、第1の標識および/または第2の標識を評価することを含み得る。他の実施形態において、標識により示される、全核酸の収量、特定配列の有無または特定配列の核酸の収量を評価してもよい。 In one embodiment, step C) may include evaluating the label. In some embodiments, step C) may include evaluating a first label and / or a second label. In other embodiments, the yield of total nucleic acid, the presence or absence of a particular sequence, or the yield of a particular sequence of nucleic acid, as indicated by the label, may be assessed.
 1つの実施形態において、工程C)の分析において、カプセル化された該細胞または細胞様構造物の選択を行ってもよい。1つの実施形態において、選択は、特定の遺伝子配列の有無、特定の遺伝子の収量または全核酸収量に基づいて行ってもよい。一部の実施形態において、特定の遺伝子配列がある場合に選択してもよく、特定の遺伝子配列が無い場合に選択してもよい。一部の実施形態において、特定の遺伝子の収量が基準となる収量より多い場合選択してもよく、低い場合に選択してもよい。一部の実施形態において、全核酸収量が、基準となる収量より多い場合に選択してもよく、低い場合に選択してもよい。 In one embodiment, in the analysis of step C), the encapsulated cells or cell-like structures may be selected. In one embodiment, selection may be based on the presence or absence of a particular gene sequence, the yield of a particular gene or the total nucleic acid yield. In some embodiments, it may be selected when there is a specific gene sequence, or it may be selected when there is no specific gene sequence. In some embodiments, it may be selected if the yield of the particular gene is greater than or equal to the baseline yield. In some embodiments, it may be selected if the total nucleic acid yield is greater than or equal to the reference yield.
 特定の実施形態において、特定の遺伝子配列の有無を、特定の遺伝子配列を特異的に検出する試薬、アガロースゲル電気泳動、マイクロチップ電気泳動、PCR、qPCR、遺伝子配列決定(サンガーシーケンシング、NGS)からなる群から選択される手段により検出する。一部の実施形態において、特定の遺伝子配列を特異的に検出する試薬として、抗体、プローブ、DNA結合性蛍光色素、蛍光色素結合ヌクレオチドが挙げられる。 In certain embodiments, reagents that specifically detect the presence or absence of a particular gene sequence, agarose gel electrophoresis, microchip electrophoresis, PCR, qPCR, gene sequencing (Sanger sequencing, NGS). Detected by means selected from the group consisting of. In some embodiments, reagents that specifically detect a particular gene sequence include antibodies, probes, DNA-binding fluorescent dyes, fluorescent dye-binding nucleotides.
 特定の実施形態において、特定の遺伝子の収量または全核酸収量を吸光度測定、蛍光光度測定、アガロースゲル電気泳動、マイクロチップ電気泳動により測定することができる。方法は、1つずつの細胞由来の増幅核酸を含む試料において、特定の配列を有する核酸を検出する工程を含み得る。特定の配列を有する核酸を検出する工程は、特定の配列を有する核酸を増幅および配列解読することを含み得る。 In a specific embodiment, the yield of a specific gene or the total nucleic acid yield can be measured by absorbance measurement, fluorescence measurement, agarose gel electrophoresis, or microchip electrophoresis. The method may include detecting nucleic acid having a particular sequence in a sample containing amplified nucleic acid derived from each cell. The step of detecting a nucleic acid having a specific sequence may include amplifying and sequencing the nucleic acid having a specific sequence.
 1つの実施形態において、選別は、フローサイトメトリーなどによって行ってもよい。この場合の方法としては、第一の標識で全核酸を標識する蛍光性DNA結合色素(インターカレーター)を用いる。第二の標識で、特定遺伝子を検出するプライマーセットおよび蛍光性DNA結合色素(インターカレーター)または蛍光標識位プローブを用いる。ここで、第一と第二の標識に用いる蛍光色素は、蛍光波長が重ならず、フローサイトメトリーにおいて別チャンネルで蛍光強度を計測できるものとする。第一・第二の標識を含むゲルカプセルをフローサイトメトリーに導入し、各ゲルカプセルの蛍光シグナルから選抜を行うことで、ゲルカプセル内部で一定量の核酸を含み、且つ特定遺伝子を内包するものを回収することができる。特定配列の収量または全核酸の収量に基づいて選択する場合、選別は、はじめに増幅ポリヌクレオチドを含むゲルカプセルを蛍光インターカレーターで検知してフローサイトメトリーにてプレートに分取し、さらに分取した各ゲルカプセルのポリヌクレオチドから核酸の再増幅を行い、増幅ポリヌクレオチドライブラリーを調製する。ここで、本増幅核酸の収量を吸光度測定、蛍光光度測定、アガロースゲル電気泳動、マイクロチップ電気泳動により測定し、全核酸量を測定する。あるいは、本増幅核酸の一部を用いて、特定の遺伝子増幅を行い、遺伝子配列決定または分子量決定を行って、増幅した遺伝子の配列情報や収量を評価し、ポリヌクレオチドライブラリー中の各サンプルの特定の遺伝子の有無を評価する。これらのいずれか又は双方の結果を参考に、サンプルを選抜し別のプレート等へ移送する。 In one embodiment, sorting may be performed by flow cytometry or the like. As a method in this case, a fluorescent DNA-binding dye (intercalator) that labels all nucleic acids with the first label is used. In the second labeling, a primer set for detecting a specific gene and a fluorescent DNA-binding dye (intercalator) or a fluorescent labeling position probe are used. Here, it is assumed that the fluorescent dyes used for the first and second labels do not have overlapping fluorescence wavelengths, and the fluorescence intensity can be measured by another channel in flow cytometry. By introducing gel capsules containing the first and second labels into flow cytometry and selecting from the fluorescent signals of each gel capsule, a certain amount of nucleic acid is contained inside the gel capsule and a specific gene is included. Can be recovered. When selecting based on the yield of a specific sequence or the yield of total nucleic acid, the selection was performed by first detecting a gel capsule containing an amplified polynucleotide with a fluorescent intercalator, separating it into a plate by flow cytometry, and further separating it. Nucleic acid is reamplified from the polynucleotide of each gel capsule to prepare an amplified polynucleotide library. Here, the yield of the amplified nucleic acid is measured by absorptiometry, fluorescence measurement, agarose gel electrophoresis, and microchip electrophoresis, and the total amount of nucleic acid is measured. Alternatively, a part of this amplified nucleic acid is used to perform specific gene amplification, gene sequencing or molecular weight determination, and the sequence information and yield of the amplified gene are evaluated, and each sample in the polynucleotide library is evaluated. Evaluate the presence or absence of a particular gene. With reference to the results of either or both of these, a sample is selected and transferred to another plate or the like.
 1つの実施形態において、工程C)の分析において、細胞種特異的な配列もしくは種特異的な配列または特定種に保存されている配列の増幅を行ってもよい。一部の実施形態において、工程C)の分析においてマーカー遺伝子の増幅を行ってもよい。 In one embodiment, in the analysis of step C), the cell type-specific sequence, the species-specific sequence, or the sequence stored in the specific species may be amplified. In some embodiments, the marker gene may be amplified in the analysis of step C).
 (3回以上のカプセル化を行う場合の例)
上記の方法で選抜操作を行った後に、残存したゲルカプセルに対し、別の特定配列を有する核酸を検出する工程として、再度カプセル化から実施することも可能である。
(Example of encapsulation 3 times or more)
After performing the selection operation by the above method, it is also possible to carry out from encapsulation again as a step of detecting a nucleic acid having another specific sequence in the remaining gel capsule.
 (システム)
 本開示は、例えば、細胞または細胞様構造物を分析するためのシステムであって、[X]2つ以上の細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、カプセル化するための薬剤および/またはそれらを格納するカプセル化薬剤格納部と、[Y]分析に使用する所望の薬剤および/またはそれらを格納する分析薬剤格納部と、[Z]カプセル化のための手段と[W]必要に応じて、該所望の薬剤を用いて分析を行うための手段とを備える、システムを提供する。Yとしては、例えば、特定の遺伝子を増幅・解読するための試薬(ポリメラーゼ、プライマーセットほか)が挙げられ、Wとしては、例えば、増幅した遺伝子を検出するための試薬(蛍光標識プローブやインターカレーター)・計測する機器(フローサイトメトリー)などが挙げられ、[Z]としてマイクロ流体デバイスやゲル化剤、カプセル化するための構造物(冷却反応槽や貯留部など)が挙げられ、[W]として、増幅した遺伝子を検出するための試薬(蛍光標識プローブやインターカレーター)・計測する機器(フローサイトメトリー)が挙げられる。
(system)
The present disclosure is, for example, a system for analyzing cells or cell-like structures, in which [X] two or more cells or cell-like structures or substances derived thereto, or capsules containing them are encapsulated. Drugs and / or encapsulated drug vaults for storing them, desired drugs used for [Y] analysis and / or analytical drug vaults for storing them, and [Z] means for encapsulation. And [W], as needed, provide a system comprising means for performing an analysis with the desired agent. Examples of Y include reagents (polymerizer, primer set, etc.) for amplifying and decoding a specific gene, and W includes, for example, reagents (fluorescent-labeled probe and intercalator) for detecting the amplified gene. ) ・ Measuring equipment (flow cytometry), etc., and [Z] includes microfluidic devices, gelling agents, structures for encapsulation (cooling reaction tank, storage, etc.), [W] Examples include reagents (fluorescent-labeled probes and intercalators) for detecting amplified genes and measuring devices (flow cytometry).
 (キット)
 本開示は、例えば、細胞または細胞様構造物を分析するためカプセルを提供するためのキットであって、[X]2つ以上の細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、カプセル化するための薬剤と、[Y]分析に使用する所望の薬剤と、を備える、キットを提供する。Yとしては、例えば、特定の遺伝子を増幅・解読するための試薬(ポリメラーゼ、プライマーセットほか)、増幅した遺伝子を検出するための試薬(蛍光標識プローブやインターカレーター)が挙げられる。
(kit)
The present disclosure is, for example, a kit for providing a capsule for analyzing a cell or a cell-like structure, which comprises [X] two or more cells or a cell-like structure or a substance derived thereto, or a substance thereof. Provided is a kit comprising a drug for encapsulating the capsule and a desired drug for use in the [Y] analysis. Examples of Y include reagents for amplifying and decoding a specific gene (polymerase, primer set, etc.) and reagents for detecting the amplified gene (fluorescent-labeled probe and intercalator).
 (微生物での例)
 本手法は特に希少な対象微生物を特異的に検出回収する目的で利用される。増幅保持された1細胞ゲノム由来ポリヌクレオチドを含むゲルカプセルを上述の(細胞中のポリヌクレオチド増幅方法)の項に記載の方法で調製する。増幅ポリヌクレオチドを含むゲルカプセルを標的とする遺伝子領域に対応したプライマーセット、および蛍光DNAインターカレーターまたはTaqmanプローブ等のPCR増幅検出指示薬を含むPCR溶液に懸濁し、懸濁液をマイクロ流体デバイスに導入する。オイルで前記懸濁液をせん断し、単一のゲルカプセルを含む液滴を作製する。液滴を外部容器に回収後、PCR操作を行う。PCRの熱処理によりゲルカプセルは崩壊し、内部のポリヌクレオチドとPCR溶液中のプライマー・酵素が会合し、標的遺伝子が存在する場合には液滴内でポリヌクレオチドが増幅される。ポリヌクレオチド増幅の有無は、前記PCR増幅検出指示薬由来の蛍光色で確認・検出が可能である。また、増幅ポリヌクレオチドを含むゲルカプセルについても蛍光DNAインターカレーターで標識をしておき、前記手段では別蛍光色のPCR増幅検出指示薬を用いることで、2色以上の蛍光色のパターンからゲルカプセルの封入有無と標的遺伝子配列の有無を同時検出することも可能である。前記蛍光色を参照し、液滴あるいは再度ゲル化させた液滴を選択的に回収あるいは排除して、標的遺伝子を含む増幅ポリヌクレオチドを含む液滴を収容溶液に選抜収容する。
(Example with microorganisms)
This method is used for the purpose of specifically detecting and recovering a particularly rare target microorganism. A gel capsule containing an amplified and retained 1-cell genome-derived polynucleotide is prepared by the method described in the above section (Method for amplifying polynucleotide in cells). Suspended in a PCR solution containing a primer set corresponding to the gene region targeting the gel capsule containing the amplified polynucleotide and a PCR amplification detection indicator such as a fluorescent DNA intercalator or Taqman probe, and introduced the suspension into a microfluidic device. To do. Shear the suspension with oil to make droplets containing a single gel capsule. After collecting the droplets in an external container, perform a PCR operation. The heat treatment of PCR disintegrates the gel capsule, the inner polynucleotide associates with the primer / enzyme in the PCR solution, and the polynucleotide is amplified in the droplet if the target gene is present. The presence or absence of polynucleotide amplification can be confirmed and detected by the fluorescent color derived from the PCR amplification detection indicator. In addition, the gel capsule containing the amplified polynucleotide is also labeled with a fluorescent DNA intercalator, and by using a PCR amplification detection indicator of another fluorescent color in the above means, the gel capsule can be obtained from a pattern of two or more fluorescent colors. It is also possible to simultaneously detect the presence or absence of inclusion and the presence or absence of the target gene sequence. With reference to the fluorescent color, the droplet or the re-gelled droplet is selectively collected or eliminated, and the droplet containing the amplified polynucleotide containing the target gene is selectively contained in the containing solution.
 本明細書において引用された、科学文献、特許、特許出願などの参考文献は、その全体が、各々具体的に記載されたのと同じ程度に本明細書において参考として援用される。 References such as scientific literature, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety to the same extent as they are specifically described.
 以上、本開示を、理解の容易のために好ましい実施形態を示して説明してきた。以下に、実施例に基づいて本開示を説明するが、上述の説明および以下の実施例は、例示の目的のみに提供され、本開示を限定する目的で提供したものではない。したがって、本開示の範囲は、本明細書に具体的に記載された実施形態にも実施例にも限定されず、特許請求の範囲によってのみ限定される。 The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for purposes of illustration only and not for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in the present specification, and is limited only by the scope of claims.
 以下、本開示の実施例を記載する。
 試薬類は具体的には実施例中に記載した製品を使用したが、他メーカー(Sigma-Aldrich、和光純薬、ナカライ、R&D Systems、USCN Life Science INC等)の同等品でも代用可能である。
Hereinafter, examples of the present disclosure will be described.
Specifically, the reagents described in the examples were used, but equivalent products of other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nacalai Tesque, R & D Systems, USCN Life Science INC, etc.) can be substituted.
 (実施例1:細胞および細胞様構造物の封入)
 本実施例では図1に示すように、一次増幅までの段階を行った。
(Example 1: Encapsulation of cells and cell-like structures)
In this example, as shown in FIG. 1, the steps up to the primary amplification were performed.
 (材料および方法)
 1.5 cmのサンゴ枝(沖縄県国頭郡本部町瀬底周辺海域の石川原から入手)を採取し、0.22 μm径のフィルター(DURAPOREメンブレンフィルター, GVWP04700, MERCK)でろ過した海水5 mLを含んだ25 mLチューブ(2362-025, IWAKI)に回収した。メス(替刃メスホルダー(61-3813-28), 替刃No.10(1-8545-11)を使用, アズワン))を用いてサンゴ枝を十分に破砕した後、氷上で3分間静置して骨片等の大きな粒子を沈殿させた。上清を1.5 mLチューブ(1212-10, SSIbio)に回収し、8,000 xgで5 min遠心分離(himac CF15RX, 工機ホールディングス)した。ペレットを残して上清を除き、0.22 μm径のフィルター(DURAPOREメンブレンフィルター, GVWP04700, MERCK)でろ過した海水800 μLを加えてペレットを再度懸濁した。次に、250 xgで5 min遠心分離を行い、上清を新しい1.5 mLチューブに回収した。8,000 xgでの遠心分離、上清の除去および250 xgでの遠心と上清の回収操作を再度繰り返した後、8,000 xgでの遠心分離と上清の除去をさらに3回行った。得られたペレットに対し、0.22 μm径のフィルター(DURAPOREメンブレンフィルター, GVWP04700, MERCK)でろ過した海水100 μLを加えて懸濁し、サンゴ組織内微生物、サンゴ細胞およびサンゴミトコンドリアの混合懸濁液を取得した。調製した懸濁液中の細菌細胞濃度を測定し(顕微鏡:CKX41, OLYMPUS、バクテリア計算盤A161,2-5679-01, アズワン)、終濃度1.5%になるように超低融点アガロース(A5030-10G, SIGMA-ALDRICH)を加えることで、ゲルカプセル作製に用いる細胞および細胞様構造物の混合懸濁液を調製した(細菌細胞終濃度:4.5×10cells/μL)。
(Materials and methods)
A 1.5 cm coral branch (obtained from Ishikawara in the sea area around Sesoko, Motobu-cho, Kunigami-gun, Okinawa) was collected and filtered with a 0.22 μm diameter filter (DURAPORE membrane filter, GVWP04700, MERCK) to 5 mL of seawater. Collected in a 25 mL tube (2362-025, IWAKI) containing. After sufficiently crushing the coral branches using a scalpel (replacement blade scalpel holder (61-3813-28), replacement blade No. 10 (1-8545-11), AS ONE)), leave it on ice for 3 minutes. Then, large particles such as bone fragments were precipitated. The supernatant was collected in a 1.5 mL tube (122-10, SSIbio) and centrifuged at 8,000 xg for 5 min (himac CF15RX, Koki Holdings). The supernatant was removed leaving the pellets, and 800 μL of seawater filtered through a 0.22 μm diameter filter (DURAPORE membrane filter, GVWP04700, MERCK) was added to suspend the pellets again. Next, a 250 xg centrifuge was performed for 5 min and the supernatant was collected in a new 1.5 mL tube. Centrifugation at 8,000 xg, removal of the supernatant, and centrifugation at 250 xg and recovery of the supernatant were repeated again, followed by centrifugation at 8,000 xg and removal of the supernatant three more times. .. To the obtained pellets, 100 μL of seawater filtered through a 0.22 μm diameter filter (DURAPORE membrane filter, GVWP04700, MERCK) was added and suspended, and a mixed suspension of microorganisms in coral tissue, coral cells and coral mitochondria was suspended. Was acquired. The concentration of bacterial cells in the prepared suspension was measured (microscope: CKX41, OLYMPUS, bacterial calculator A161,-5679-01, AS ONE), and ultra-low melting point agarose (A5030) was adjusted to a final concentration of 1.5%. -10G, SIGMA-ALDRICH) was added to prepare a mixed suspension of cells and cell-like structures used for gel capsule preparation (final bacterial cell concentration: 4.5 × 10 3 cells / μL).
 調製した細胞および細胞様構造物の混合懸濁液を用いて、直径35 μmのゲルカプセルを作製した。続いて、溶解用試薬としての溶菌試薬にゲルカプセルを浸漬し、ゲルカプセル内部で細胞の細胞壁等の収集目的物以外の部分を溶解し、ゲルカプセル内にゲノムDNAを溶出させた。 A gel capsule having a diameter of 35 μm was prepared using the prepared mixed suspension of cells and cell-like structures. Subsequently, the gel capsule was immersed in a lytic reagent as a lysis reagent, a portion other than the object to be collected such as a cell wall of a cell was lysed inside the gel capsule, and genomic DNA was eluted into the gel capsule.
 具体的には、溶菌試薬の1種である水酸化カリウムを含む水溶液であるBuffer D2(QIAGEN社)にゲルカプセルを浸漬し、残存成分の溶解とゲノムDNAの変性を行った。本実施例で使用する溶菌試液は、上述のとおり、リゾチーム、アクロモペプチダーゼ、プロテアーゼK、ドデシル硫酸ナトリウム及びBuffer D2である。なお、水酸化カリウムは通常のDNA増幅反応工程でも使用するが、溶菌の効果も兼ねていることから、本実施例では溶菌試薬の一つとしている。ゲルカプセルの溶菌試薬への浸漬は短時間であるため、溶出させたゲノムDNAが溶菌試薬によりゲルカプセル外に流出されることはなく、ゲルカプセル内に保持される。本実施例では、ゲルカプセルに浸透した溶菌試薬も夾雑物質に含まれるものとする。 Specifically, the gel capsule was immersed in Buffer D2 (QIAGEN), which is an aqueous solution containing potassium hydroxide, which is one of the lytic reagents, to dissolve the residual components and denature the genomic DNA. As described above, the lytic test solution used in this example is lysozyme, achromopeptidase, proteinase K, sodium dodecyl sulfate, and Buffer D2. Potassium hydroxide is also used in a normal DNA amplification reaction step, but since it also has a lytic effect, it is used as one of the lytic reagents in this example. Since the gel capsule is immersed in the lytic reagent for a short time, the eluted genomic DNA is not discharged from the gel capsule by the lytic reagent and is retained in the gel capsule. In this example, the lytic reagent permeated into the gel capsule is also included in the contaminants.
 本実施例は、Buffer D2を入れて反応を進行しているが、複数の試薬での溶菌操作を段階的に行い、各工程で遠心洗浄を行うことで十分な洗浄効果を得ることができる。また、各溶菌試薬により細胞を溶解した後に遠心洗浄を行ってもよい。 In this example, Buffer D2 is added to proceed with the reaction, but a sufficient cleaning effect can be obtained by performing lysis operations with a plurality of reagents step by step and performing centrifugal cleaning in each step. Further, the cells may be lysed with each lytic reagent and then centrifuged.
 このように、複数種類の溶菌試薬により細胞の溶解を行うことで、目的のゲノムDNAを採取することができ、溶菌試薬への浸漬後に遠心洗浄を行うことで、溶菌試薬や溶解した細胞のポリヌクレオチド以外の成分等の夾雑物質を除去し、続くゲノムDNA増幅反応を阻害することのなくゲノムDNAを精製することができる。 In this way, the target genomic DNA can be collected by lysing the cells with a plurality of types of lytic reagents, and the lytic reagent and the poly of the lysed cells can be collected by centrifugation after immersion in the lytic reagent. Contaminants such as components other than nucleotides can be removed, and genomic DNA can be purified without inhibiting the subsequent genomic DNA amplification reaction.
 水酸化カリウム溶液(Buffer D2)中で変性したゲノムDNAを保持するゲルカプセルを含むチューブに増幅用試薬を加え、ゲルカプセルを増幅用試薬に浸漬した。具体的には、鎖置換型DNA合成酵素であるphi29DNAポリメラーゼを用いたMDA(Multiple Displacement Amplification)法を使用した。ここでは、全ゲノム増幅反応試薬REPLI-g Single Cell Kit(QIAGEN社)に浸漬し、3時間の全ゲノム増幅反応を行った。増幅用試薬(REPLI-g Single Cell Kit)には水酸化カリウム溶液(Buffer D2)を中和する成分が含まれている。全ゲノム増幅後のゲルカプセルを、DPBSし(Dulbecco’s Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific)を用いて3回遠心分離して洗浄し、蛍光性DNAインターカレーターであるDAPI(同仁科学)を用いて染色を行った。染色後のゲルカプセルはDPBSを用いて再度洗浄した(図1)。 The amplification reagent was added to the tube containing the gel capsule holding the denatured genomic DNA in the potassium hydroxide solution (Buffer D2), and the gel capsule was immersed in the amplification reagent. Specifically, the MDA (Multiple Replication Replication) method using phi29 DNA polymerase, which is a strand-substitution type DNA synthase, was used. Here, the whole genome amplification reaction was carried out for 3 hours by immersing in the whole genome amplification reaction reagent REPLI-g Single Cell Kit (QIAGEN). The amplification reagent (REPLI-g Single Cell Kit) contains a component that neutralizes the potassium hydroxide solution (Buffer D2). The gel capsule after whole genome amplification is purified by DPBS (Dulvecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) three times and washed, and then washed with DAPI (Dojin), which is a fluorescent DNA intercalator. Staining was performed using science). The stained gel capsules were washed again with DPBS (Fig. 1).
 (実施例2-1:液滴の再封入および遺伝子増幅)
 本実施例では実施例1で生産した液滴の再封入を行った。この実施例では、再封入の際に入れる試薬はPCR用の試薬を用いた。
(Example 2-1: Droplet reencapsulation and gene amplification)
In this example, the droplets produced in Example 1 were re-encapsulated. In this example, the reagent for PCR was used as the reagent to be inserted at the time of re-encapsulation.
 (材料および方法)
次にPCR反応液の調製を行った。PCR反応液はPrimeTime Gene Expression Master Mix(Integrated DNA Technologies, 1055770)に標的遺伝子検出用にデザインしたプライマーおよびTaqmanプローブ(作製は、Integrated DNA Technologies)を添加した。本実施例で使用したプライマーおよびプローブは、サンゴ組織内共在微生物であるEndozoicomonas属の微生物の16S rRNA遺伝子(V3-V4領域)の一部を特異的に検出するように設計(作製は、Integrated DNA Technologies)し、標的の配列が存在する場合、PCR反応後にFAM由来の緑色の蛍光を呈する。また、Taqmanプローブ等検出用のオリゴヌクレオチドを用いず、蛍光性DNAインターカレーターをPCR反応液に添加して増幅を検知する設計も可能である。この場合は、前述のゲルカプセルの染色に用いた蛍光色素と波長の異なる蛍光を示すDNA染色試薬(SYBR GreenやEvaGreen(登録商標)等)を用いた。次に、調製したPCR反応液にゲルカプセルを加え、ゲルカプセルを懸濁したPCR反応液を調製した。
(Materials and methods)
Next, a PCR reaction solution was prepared. For the PCR reaction solution, a primer designed for target gene detection and a Taqman probe (prepared by Integrated DNA Technologies) were added to Prime Time Gene Expression Master Mix (Integrated DNA Technologies, 1055770). The primers and probes used in this example were designed to specifically detect a part of the 16S rRNA gene (V3-V4 region) of a microorganism belonging to the genus Endozoicomonas, which is a microorganism coexisting in coral tissue (manufactured by Integrated). DNA Technologies) and, in the presence of the target sequence, exhibit green fluorescence from the FAM after the PCR reaction. It is also possible to design to detect amplification by adding a fluorescent DNA intercalator to the PCR reaction solution without using an oligonucleotide for detection such as Taqman probe. In this case, a DNA staining reagent (SYBR Green, EvaGreen (registered trademark), etc.) exhibiting fluorescence having a wavelength different from that of the fluorescent dye used for staining the gel capsule described above was used. Next, a gel capsule was added to the prepared PCR reaction solution to prepare a PCR reaction solution in which the gel capsule was suspended.
 自作したポリジメチルシロキサン製のマイクロ流路を用いて、ゲルカプセルが懸濁されたPCR反応液から直径50 μm(この直径は、40~60μmなどであり得る)微小液滴の作製を行い、微小液滴内へのゲルカプセルの封入を行った。このときの微小液滴内のゲルカプセル濃度は、0.5cell/droplet程度であることが好ましい。作製した液滴すべてをPCRチューブに回収した。 Using a self-made microchannel made of polydimethylsiloxane, microdroplets having a diameter of 50 μm (this diameter can be 40 to 60 μm, etc.) were prepared from a PCR reaction solution in which gel capsules were suspended, and microdroplets were prepared. A gel capsule was encapsulated in the droplet. The gel capsule concentration in the microdroplets at this time is preferably about 0.5 cell / drop. All the prepared droplets were collected in a PCR tube.
 この回収したものについて、サーマルサイクラーを用いてPCR反応を行った。PCRの反応条件は、初期熱変性を95℃, 3分、熱変性を95℃, 5秒、アニーリングと伸長反応を60℃, 30秒で28サイクル行い、反応終了後は4℃で保存した。 The recovered material was subjected to a PCR reaction using a thermal cycler. The PCR reaction conditions were as follows: initial heat denaturation at 95 ° C. for 3 minutes, heat denaturation at 95 ° C. for 5 seconds, annealing and extension reaction at 60 ° C. for 30 seconds for 28 cycles, and storage at 4 ° C. after completion of the reaction.
 本反応工程において、内部に封入されたゲルカプセルは熱崩壊し、カプセル内に保持されていたポリヌクレオチドとPCR溶液中のプライマー・酵素が反応した。標的遺伝子が存在する場合には液滴内でポリヌクレオチドが増幅され、プローブ由来の蛍光もしくはDNAインターカレーター由来の蛍光の強度が増加する。PCR反応後、蛍光顕微鏡下でドロップレットの観察を行い、液滴内の蛍光を確認した。 In this reaction step, the gel capsule encapsulated inside was thermally disintegrated, and the polynucleotide held in the capsule reacted with the primer / enzyme in the PCR solution. In the presence of the target gene, the polynucleotide is amplified in the droplet, increasing the intensity of probe-derived fluorescence or DNA intercalator-derived fluorescence. After the PCR reaction, the droplets were observed under a fluorescence microscope to confirm the fluorescence in the droplets.
 観察の結果、蛍光を示さない液滴、MDAによって増幅されたDNAに結合したDAPI由来(同仁化学, D523)の蛍光(青色)のみを示す液滴、DAPI由来の蛍光およびプローブ由来の蛍光(FAM:緑色)の2種類の蛍光を示す液滴、の3種類が確認された(図7)。この内、DAPI由来の蛍光を示す液滴中、蛍光プローブ由来の蛍光を示した液滴の割合は0.8% (4/500)であった。 As a result of observation, droplets showing no fluorescence, droplets showing only fluorescence (blue) derived from DAPI (Dojin Kagaku, D523) bound to DNA amplified by MDA, fluorescence derived from DAPI and fluorescence derived from probe (FAM). : Two types of fluorescent droplets (green) were confirmed (Fig. 7). Among the droplets showing fluorescence derived from DAPI, the ratio of the droplets showing fluorescence derived from the fluorescent probe was 0.8% (4/500).
 (実施例2-2:再封入および遺伝子増幅を伴わない検出・選抜)
 実施例2のように液滴の再封入および遺伝子増幅を行わずに、実施例1で調製したゲルカプセルの状態で標的とする生物・遺伝子を検出・選抜する方法として次のような方法がある。
(Example 2-2: Detection / selection without reencapsulation and gene amplification)
There are the following methods for detecting and selecting the target organism / gene in the state of the gel capsule prepared in Example 1 without reencapsulating the droplet and amplifying the gene as in Example 2. ..
 実施例1で調製したゲルカプセルをリコンビナーゼポリメラーゼ増幅反応(RPA)試薬に浸漬する。本RPA試薬としては、例えばTwisAmp(登録商標)シリーズ(TwistDx社)を用いることができる。検出する標的配列に合わせて設計したプライマー・蛍光プローブを用い、ゲルカプセル内部で37℃から42℃の条件で等温核酸増幅を行う。この際、ゲルカプセルの形状を崩壊させること無く核酸増幅をすることができるため、ゲルカプセル内部に蓄積される蛍光物質を指標に、目的生物あるいは目的遺伝子を含む増幅核酸が存在するゲルカプセルを特異的に検出することができる。このため、実施例2のようにドロップレットへの再封入の手間なく、標的の単一細胞または細胞様構造物の個別の分析へとサンプルを選抜することができる。
 (実施例3:二次増幅)
 次に、本実施例では、実施例2で調整した、個々のカプセルに含まれる試料について、さらに二次増幅のステップを行った。
 以下にその手順を示す。
 (材料および方法)
The gel capsule prepared in Example 1 is immersed in a recombinase polymerase amplification reaction (RPA) reagent. As the RPA reagent, for example, TwisAmp (registered trademark) series (TwistDx) can be used. Isothermal nucleic acid amplification is performed inside the gel capsule under the conditions of 37 ° C to 42 ° C using a primer / fluorescent probe designed according to the target sequence to be detected. At this time, since nucleic acid amplification can be performed without destroying the shape of the gel capsule, the gel capsule in which the amplified nucleic acid containing the target organism or the target gene is present is specificized using the fluorescent substance accumulated inside the gel capsule as an index. Can be detected. Therefore, the sample can be selected for individual analysis of the target single cell or cell-like structure without the hassle of reencapsulation in the droplet as in Example 2.
(Example 3: Secondary amplification)
Next, in this example, the secondary amplification step was further performed on the samples contained in the individual capsules prepared in Example 2.
The procedure is shown below.
(Materials and methods)
 実施例2で調製したものの内、2種類の蛍光を示すドロップレットを顕微鏡下でマイクロピペットを用いて分取し、PCRチューブに個別に回収した。回収した個々の液滴に対してサーマルサイクラーを用いて65℃で加熱を行うこと(S1000 サーマルサイクラー, Bio-Rad)によりドロップレットを壊した後、各チューブのウェル内でMDA法による二次増幅を行った。これにより、標的遺伝子の配列を含んだ1細胞増幅ゲノムライブラリーを獲得することができる。 Of those prepared in Example 2, two types of fluorescent droplets were fractionated under a microscope using a micropipette and individually collected in a PCR tube. After breaking the droplets by heating the collected individual droplets at 65 ° C. using a thermal cycler (S1000 thermal cycler, Bio-Rad), secondary amplification by the MDA method in the wells of each tube. Was done. This makes it possible to obtain a 1-cell amplified genomic library containing the sequence of the target gene.
 調製された1細胞増幅ゲノムライブラリー(12個)を対象として、ドロップレット内でのPCRで使用したものとは異なるプライマーセットを用いて16S rRNA遺伝子を対象としたPCRを行い、PCR産物をサンガー法でシークエンスした。8サンプルにおいてPCR産物が取得され、配列解析の結果サンゴ共在微生物由来のゲノム情報を含むことが確認された。一方で、PCR後にDAPI由来の蛍光のみを示す液滴を分取した場合では、全ての液滴(n=12)が宿主由来のミトコンドリアの配列を含んでいることが明らかとなった。
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
The prepared 1-cell amplified genomic library (12 cells) was subjected to PCR targeting the 16S rRNA gene using a primer set different from that used in the PCR in the droplet, and the PCR product was sangered. Sequenced by law. PCR products were obtained in 8 samples, and as a result of sequence analysis, it was confirmed that they contained genomic information derived from coral coexisting microorganisms. On the other hand, when droplets showing only DAPI-derived fluorescence were collected after PCR, it was revealed that all the droplets (n = 12) contained the host-derived mitochondrial sequence.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
 サンゴ共在微生物由来のゲノム情報を含む8個のサンプルについて、Nextera XT DNA sample prep kit(Illumina社, FC-131-1096)を用いてライブラリー調製を行い、Miseq(Illumina社, SY-410-1003)を用いた全ゲノムシークエンスによって2×75bpのペアエンドリード(3.99Gb)を取得した。SPAdes(Bankevich A et al., J Comput Biol. 2012 May;19(5):455-77. http://doi.org/10.1089/cmb.2012.0021)を用いてシークエンスデータのアセンブリを行い、Contigを作製した。また、ゲノム解読率(コンプリート率)およびコンタミネーション度の評価にはCheckM(Parks et al., Genome Res. 2015. 25: 1043-1055, doi:10.1101/gr.186072.114)を用いてデフォルト条件で解析を行った。この結果、8個のサンプル中5個でゲノム解読率(コンプリート率)が50%を超えた。またコンタミネーション度は1個のサンプルを除いて5%以下となり、8個中4個のサンプルが国際基準Minimum information about a single amplified genome(MISAG)の基準におけるMedium quality draftに分類されることが明らかとなった。(表2参照)。
Figure JPOXMLDOC01-appb-T000003
A library was prepared using the Nextera XT DNA sample prep kit (Illumina, FC-131-1096) for eight samples containing genomic information derived from coral coexisting microorganisms, and Miseq (Illumina, SY-410-). A 2 × 75 bp paired end read (3.99 Gb) was obtained by whole genome sequencing using 1003). Assembly of sequence data using SPAdes (Bankevich A et al., J Comput Biol. 2012 May; 19 (5): 455-77. This was performed to prepare a Contig. In addition, CheckM (Parks et al., Genome Res. 2015. 25: 1043-155, doi: 10.1101 / gr.186072.114) was used to evaluate the genome decoding rate (complete rate) and the degree of contamination. The analysis was performed under the default conditions. As a result, the genome decoding rate (complete rate) exceeded 50% in 5 out of 8 samples. In addition, the degree of contamination is 5% or less except for one sample, and it is clear that 4 out of 8 samples are classified into the Medium quality genome according to the international standard Minimum information about single amplified genome (MISAG). It became. (See Table 2).
Figure JPOXMLDOC01-appb-T000003
 (実施例3-2:二次増幅を伴わない解析)
 実施例3に記載の二次増幅の工程をスキップして解析に進行する手順として、下記の方法がある。
 実施例2で調製したものの内、2種類の蛍光を示すドロップレットを顕微鏡下でマイクロピペットを用いて分取し、PCRチューブに個別に回収した。回収した個々の液滴に対してサーマルサイクラーを用いて65℃で加熱を行うこと(S1000 サーマルサイクラー,Bio-Rad)によりドロップレットを壊した後、各チューブのウェル内でNextera XT DNA sample prep kit(Illumina社、FC-131-1096)を用いてライブラリー調製を行い、Miseq(Illumina社、SY-410-1003)を用いた全ゲノムシークエンスを行うことができる。この際には、Nextera XT DNA sample prep kit以外の一般的に入手可能なDNAシークエンスライブラリ調製試薬を使うことも可能である。ドロップレットより回収したDNAに対し、上記キットのようにトランスポゼース反応によりアダプター配列を挿入する、または断片化・ライゲーションなどの反応を行い、アダプター配列を付加したのち、PCR反応によってシークエンス解析の実行に足るDNA量を獲得する。以降は、実施例3に記載の内容と同様に解析することができる。
(Example 3-2: Analysis without secondary amplification)
The following method is available as a procedure for skipping the secondary amplification step described in Example 3 and proceeding to the analysis.
Of the two types of fluorescent droplets prepared in Example 2, droplets showing two types of fluorescence were collected under a microscope using a micropipette and individually collected in a PCR tube. After breaking the droplets by heating the collected individual droplets at 65 ° C. using a thermal cycler (S1000 thermal cycler, Bio-Rad), the Nextera XT DNA genome prep kit in the wells of each tube. A library can be prepared using (Illumina, FC-131-1096) and a whole genome sequence can be performed using Miseq (Illumina, SY-410-1003). In this case, it is also possible to use a generally available DNA sequence library preparation reagent other than the Nextera XT DNA sample prep kit. For the DNA collected from the droplet, an adapter sequence is inserted by a transposase reaction as in the above kit, or a reaction such as fragmentation / ligation is performed to add the adapter sequence, and then a PCR reaction is sufficient to perform sequence analysis. Acquire the amount of DNA. After that, the analysis can be performed in the same manner as described in Example 3.
(実施例4:多様な細胞の中から特定の特徴を有する細胞のデータを選択的に獲得したい場合)
 腸内細菌などの動物共生微生物や海洋・土壌微生物の中で、当業者が注目する特定の1種以上の微生物のゲノムデータを獲得することが目的であった場合には、ポリヌクレオチドを含むゲルカプセルに対し、事前に標的とする微生物の遺伝子断片の有無を事前確認しておくことで、不要な遺伝子配列データ取得とそれにかかるコストを削減することができる。測定対象例としては、同一系統微生物の比較解析(例えば、疾患等に関連する微生物系統群における亜種の解析)や特定の遺伝子(例えば、微生物の生産する二次代謝産物や酵素)を有する微生物の探索、あるいは多系統の生物種を含む中から細菌・アーキア・真菌・その他真核細胞等を個別に選択して解析することなどを目的とした場合が想定される。
(Example 4: When it is desired to selectively acquire data of cells having specific characteristics from various cells)
A gel containing a polynucleotide when the purpose is to obtain genomic data of one or more specific microorganisms of interest to those skilled in the art among animal symbiotic microorganisms such as gut flora and marine / soil microorganisms. By confirming the presence or absence of the gene fragment of the target microorganism in advance for the capsule, it is possible to reduce unnecessary gene sequence data acquisition and the cost involved. Examples of measurement targets include comparative analysis of microorganisms of the same strain (for example, analysis of subspecies in a group of microorganisms related to diseases, etc.) and microorganisms having a specific gene (for example, secondary metabolites and enzymes produced by microorganisms). It is assumed that the purpose is to search for or analyze bacteria, archaea, fungi, and other eukaryotic cells individually from among various species.
 (実施例5:ホストDNAなどが多量に混在する場合)
解析試料が、糞便、唾液、喀痰や皮膚、口腔、鼻腔、耳、生殖器などの拭い液、手術洗浄液、あるいは組織抽出物や血液であり、当該試料中に含まれる微生物を解析対象とした場合には、試料中に多くのホスト動物由来の細胞、細胞内小器官、核酸が含まれる。これらの一部も、ゲルカプセル内部に封入されポリヌクレオチド増幅が実行されうる。このため、増幅ポリヌクレオチドを含むゲルカプセルに対し、事前に標的とする微生物の遺伝子断片あるいはホスト由来の遺伝子断片の有無を事前確認しておくことで、ホスト由来のポリヌクレオチドを含む不要な遺伝子配列データ取得を避け、それにかかるコストを削減することができる。測定例としては、ヒト由来試料からの微生物検出、例えば、血液や喀痰からの病原性微生物の探索や組織内部に共生する微生物解析などが想定される。
 あるいは、ホスト由来のポリヌクレオチドを積極的に解析することで、目的とする微生物や他の細胞の解析と複合的な解析に応用することもできる。
 例えば、消化管に存在する腸内細菌叢と宿主動物は、宿主が消化管という細菌叢定着のための嫌気的環境を提供すると同時に、腸内細菌叢は宿主の健康に影響を及ぼすという共生関係を持つことが知られている。例えば、ホストの健康状態への影響として、大きなものは栄養素の産生と感染症に対する防御・免疫系の発達などが挙げられる。また、他の例を挙げると、炎症性腸疾患は、遺伝的素因に加え、腸内細菌を初めとする腸内環境因子の異常が相まって起こる疾患である。
 このような病態等については、ホスト自体の遺伝子情報等の解析と、腸内微生物叢、つまり細菌、ウイルス、真菌とを含めた統合的解析と、宿主生理機能への作用機序を明らかにすることが必要であり、本開示の技術はこれらに寄与し得る。また、腸内微生物叢と種々の疾患の病態との関わりや代謝産物を中心とした機能解析も、ホスト自体の遺伝子情報等も含めて解析することでより深化した解析を行うことができる。
 また、腸管においては、腸内細菌と生体側は栄養素を競合して取り合う関係である一方、生体のために腸内細菌は栄養素の代謝や分解も併せて行う。腸内細菌に由来する代謝物がどのような働きを示すかについては、ホスト側の遺伝子解析、メタボローム解析や生化学的解析等のデータを取得し、腸内細菌の機能については細菌のゲノム配列情報から機能を類推し、生成する代謝物、メタボローム等種々のデータを取得し、ホスト・腸内細菌双方のデータを複合的に解析することで、両者の関係を知ることができる。
(Example 5: When a large amount of host DNA or the like is mixed)
When the analysis sample is feces, saliva, sputum or skin, oral cavity, nasal cavity, ears, genital lavage fluid, surgical cleaning solution, tissue extract or blood, and the microorganisms contained in the sample are analyzed. Contains cells, intracellular small organs, and nucleic acids from many host animals in the sample. Some of these can also be encapsulated inside the gel capsule to perform polynucleotide amplification. Therefore, by confirming in advance the presence or absence of the gene fragment of the target microorganism or the gene fragment derived from the host in the gel capsule containing the amplified polynucleotide, an unnecessary gene sequence containing the polynucleotide derived from the host is confirmed. Data acquisition can be avoided and the cost involved can be reduced. As a measurement example, detection of microorganisms from a human-derived sample, for example, search for pathogenic microorganisms from blood or sputum, and analysis of microorganisms symbiotic inside tissues are assumed.
Alternatively, by positively analyzing the polynucleotide derived from the host, it can be applied to the analysis combined with the analysis of the target microorganism or other cells.
For example, the intestinal flora and host animals present in the gastrointestinal tract have a symbiotic relationship in which the host provides an anaerobic environment for colonization of the gastrointestinal tract, while the intestinal flora affects the health of the host. It is known to have. For example, the major effects on the health status of the host include the production of nutrients, the defense against infectious diseases, and the development of the immune system. In addition, to give another example, inflammatory bowel disease is a disease caused by an abnormality of intestinal environmental factors such as intestinal bacteria in addition to a genetic predisposition.
Regarding such pathological conditions, analysis of the genetic information of the host itself, integrated analysis including the intestinal microflora, that is, bacteria, viruses, and fungi, and the mechanism of action on the host physiological function will be clarified. It is necessary, and the techniques of the present disclosure may contribute to these. In addition, the relationship between the intestinal microflora and the pathophysiology of various diseases and functional analysis centered on metabolites can be further deepened by analyzing the genetic information of the host itself.
Further, in the intestinal tract, the intestinal bacteria and the living body side compete with each other for nutrients, while the intestinal bacteria also metabolize and decompose nutrients for the living body. Regarding the function of metabolites derived from gut flora, we obtained data such as gene analysis, metabolome analysis and biochemical analysis on the host side, and regarding the function of gut flora, the genome sequence of the bacteria. The relationship between the two can be known by inferring the function from the information, acquiring various data such as metabolomes and metabolomes to be produced, and analyzing the data of both the host and the intestinal bacteria in a complex manner.
 (実施例6:3回目以上のカプセル化を行う例)
 解析試料が、糞便、唾液、喀痰や皮膚、口腔、鼻腔、耳、生殖器などの拭い液、手術洗浄液、あるいは組織抽出物や血液、または共生微生物を含む植物、昆虫、動物の破砕液などであり、当該試料中に含まれる微生物とホスト細胞の双方を解析対象とする場合が想定される。この場合は、各ゲルカプセルにホスト由来の細胞、細胞内小器官、核酸、共生微生物由来の核酸が封入される。これらのすべてが、ゲルカプセル内部に封入されポリヌクレオチド増幅が実行されうる。このため、増幅ポリヌクレオチドを含むゲルカプセルに対し、標的とするホスト由来の遺伝子断片・微生物由来の遺伝子断片を段階的に検出・選抜することで、解析対象とする遺伝子配列データのみを特異的に取得し、それにかかるコストを削減することができる。例えば、3回以上のカプセル化反応を想定し、1回目のカプセル化ではゲルカプセル内部でのポリヌクレオチド増幅を実行し、2回目のカプセル化にてホスト細胞由来の遺伝子断片をゲルカプセル内で増幅・検出したのち、必要に応じて選抜する。その後に、残余ゲルカプセルに対し3回目のカプセル化を実行し、微生物由来の遺伝子断片をゲルカプセル内で増幅・検出したのち、必要に応じて選抜する。2回目以降の順序は、必要に応じて変更しうる。また対象は、2以上の微生物同士あるいはホスト由来物同士などの組み合わせであっても良い。
(Example 6: Example of performing encapsulation for the third time or more)
Analysis samples include feces, saliva, sputum and skin, oral cavity, nasal cavity, ears, genital lavage fluid, surgical cleaning fluid, tissue extract and blood, or crushed fluid of plants, insects, and animals containing symbiotic microorganisms. , It is assumed that both the microorganism and the host cell contained in the sample are to be analyzed. In this case, each gel capsule is encapsulated with host-derived cells, organelles, nucleic acids, and nucleic acids derived from symbiotic microorganisms. All of these can be encapsulated inside the gel capsule and polynucleotide amplification can be performed. Therefore, by stepwise detecting and selecting gene fragments derived from the target host and gene fragments derived from microorganisms in the gel capsule containing the amplified polynucleotide, only the gene sequence data to be analyzed can be specifically detected. You can get it and reduce the cost of it. For example, assuming three or more encapsulation reactions, the first encapsulation executes polynucleotide amplification inside the gel capsule, and the second encapsulation amplifies the gene fragment derived from the host cell in the gel capsule.・ After detection, select as necessary. After that, the residual gel capsule is encapsulated for the third time, and the gene fragment derived from the microorganism is amplified and detected in the gel capsule, and then selected as necessary. The order after the second time can be changed as needed. Further, the target may be a combination of two or more microorganisms or host-derived substances.
 (注記)
 以上のように、本開示の好ましい実施形態を用いて本開示を例示してきたが、本開示は、特許請求の範囲によってのみその範囲が解釈されるべきであることが理解される。本明細書において引用した特許、特許出願及び他の文献は、その内容自体が具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。本願は、日本国特許庁に2019年4月26日に出願された、特願2019-85829に対して優先権主張をするものであり、同出願の内容自体は具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。
(Note)
As described above, the present disclosure has been illustrated using the preferred embodiments of the present disclosure, but it is understood that the scope of the present disclosure should be interpreted only by the scope of claims. The patents, patent applications and other documents cited herein should be incorporated herein by reference in their content as they are specifically described herein. Is understood. This application claims priority to Japanese Patent Application No. 2019-85829 filed with the Japan Patent Office on April 26, 2019, and the content of the application itself is specifically described in the present specification. It is understood that the content should be incorporated as a reference to this specification as it is.
 本開示は、シングルセル解析を用いる産業において有用である。 This disclosure is useful in industries that use single cell analysis.

Claims (27)

  1. (A)2つ以上の細胞または細胞様構造物を、第一の薬剤とともに、細胞または細胞様構造物ごとにカプセル化する工程と、
    (B)第一の薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
    (C)該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに第二の薬剤とともにカプセル化する工程と、
    (D)該第二の薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
    (E)必要に応じて、該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに第Xの薬剤とともにカプセル化する工程であって、Xは3以上の整数である、工程と、
    (F)必要に応じて、該第Xの薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
    (G)(E)および(F)の工程を必要に応じて繰り返す工程と、
    (H)該カプセルに対して少なくとも1つの分析を行う工程と
    を包含する、細胞または細胞様構造物を分析する方法。
    (A) A step of encapsulating two or more cells or cell-like structures together with a first agent for each cell or cell-like structure.
    (B) A step of performing a reaction based on the first drug on the cell or cell-like structure in a capsule, and
    (C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each cell or cell-like structure or a substance derived thereto together with a second agent.
    (D) A step of carrying out a reaction based on the second drug on the cell or cell-like structure in a capsule, and
    (E) If necessary, the cell or cell-like structure or a substance derived thereto, or a capsule containing them, is encapsulated for each cell or cell-like structure or a substance derived thereto together with a drug X. In the process, X is an integer of 3 or more.
    (F) If necessary, a step of carrying out a reaction based on the Xth drug on the cell or cell-like structure in a capsule, and
    A step of repeating the steps (G), (E) and (F) as necessary, and
    (H) A method for analyzing a cell or cell-like structure, which comprises the step of performing at least one analysis on the capsule.
  2. (C’)2つ以上の細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、細胞または細胞様構造物あるいはそれに由来する物質ごとに所望の薬剤とともにカプセル化する工程と、
    (D’)該所望の薬剤を用いて該細胞または細胞様構造物の分析を行う工程と
    を包含する、細胞または細胞様構造物を分析する方法。
    (C') A step of encapsulating two or more cells or cell-like structures or substances derived thereto, or capsules containing them, for each cell or cell-like structure or substances derived thereto together with a desired drug.
    (D') A method for analyzing a cell or cell-like structure, comprising the step of analyzing the cell or cell-like structure with the desired agent.
  3. (E)~(G)は存在せず、前記第一の薬剤および第二の薬剤は、核酸増幅に必要な薬剤である、請求項1に記載の方法。 The method according to claim 1, wherein (E) to (G) do not exist, and the first agent and the second agent are agents required for nucleic acid amplification.
  4. 前記所望の薬剤は、核酸増幅に必要な薬剤である、請求項2に記載の方法。 The method according to claim 2, wherein the desired drug is a drug required for nucleic acid amplification.
  5. A)2つ以上の細胞または細胞様構造物を、単一の細胞または構造物単位ごとに選別し、該細胞または細胞様構造物に含まれる核酸を増幅する工程と、
    B)該増幅された核酸を含む細胞または細胞様構造物を、分析に必要な薬剤とともに、個別にカプセル化する工程と、
    C)該個別にカプセル化された該細胞または細胞様構造物について分析する工程と
    を包含する、細胞または細胞様構造物を分析する方法。
    A) A step of selecting two or more cells or cell-like structures for each single cell or structure unit and amplifying nucleic acids contained in the cells or cell-like structures.
    B) A step of individually encapsulating the cell or cell-like structure containing the amplified nucleic acid together with the drug required for analysis.
    C) A method of analyzing a cell or cell-like structure, comprising the step of analyzing the individually encapsulated cell or cell-like structure.
  6. 前記C)の分析は、核酸増幅によるものであり、前記B)の分析に必要な薬剤は、核酸増幅に必要な薬剤を含む、請求項5に記載の方法。 The method according to claim 5, wherein the analysis of C) is based on nucleic acid amplification, and the drug required for the analysis of B) includes a drug required for nucleic acid amplification.
  7.  前記A)が、
    a)2つ以上の細胞または細胞様構造物を含む試料を用い、液滴中に1つの細胞または成分を封入することと、
    b)該液滴をゲル化してゲルカプセルを生成することと、
    c)該ゲルカプセルを1種以上の溶解用試薬に浸漬して該細胞または成分を溶解することであって、該細胞または成分中のポリヌクレオチドが該ゲルカプセル内に溶出し該ポリヌクレオチドに結合する物質が除去された状態で前記ゲルカプセル内に保持される、ことと、
    d)該ポリヌクレオチドを第1の増幅用試薬に接触させて該ポリヌクレオチドをゲルカプセル内で増幅すること
    を包含する、請求項5または6に記載の方法。
    A) above
    a) Encapsulating one cell or component in a droplet using a sample containing two or more cells or cell-like structures.
    b) Gelling the droplets to form gel capsules
    c) By immersing the gel capsule in one or more solubilizing reagents to lyse the cells or components, the polynucleotide in the cells or components elutes into the gel capsule and binds to the polynucleotide. It is retained in the gel capsule with the substance to be removed.
    d) The method of claim 5 or 6, comprising contacting the polynucleotide with a first amplification reagent to amplify the polynucleotide in a gel capsule.
  8.  前記単一細胞または単一細胞様構造物の懸濁液をマイクロ流路中に流動させ、オイルで前記懸濁液をせん断することにより該単一細胞または単一細胞様構造物を封入した前記液滴が作製されることを特徴とする、請求項7に記載の方法。 The single cell or single cell-like structure is encapsulated by flowing the suspension of the single cell or single cell-like structure into a microchannel and shearing the suspension with oil. The method according to claim 7, wherein droplets are produced.
  9.  前記ゲルカプセルがアガロース、アクリルアミド、PEG、ゼラチン、アルギン酸ナトリウム、マトリゲル、コラーゲン又は光硬化性樹脂から形成されることを特徴とする、請求項5~8のいずれか1項に記載の方法。 The method according to any one of claims 5 to 8, wherein the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, matrigel, collagen or a photocurable resin.
  10.  前記溶解用試薬がリゾチーム、ラビアーゼ、ヤタラーゼ、アクロモペプチダーゼ、プロテアーゼ、ヌクレアーゼ、ザイモリアーゼ、キチナーゼ、リソスタフィン、ムタノライシン、ドデシル硫酸ナトリウム、ラウリル硫酸ナトリウム、水酸化カリウム、水酸化ナトリウム、フェノール、クロロホルム、グアニジン塩酸塩、尿素、2-メルカプトエタノール、ジチオトレイトール、TCEP-HCl、コール酸ナトリウム、デオキシコール酸ナトリウム、Triton X-100、Triton X-114、NP-40、Brij-35、Brij-58、Tween 20、Tween 80、オクチルグルコシド、オクチルチオグルコシド、CHAPS、CHAPSO、ドデシル-β-D-マルトシド、Nonidet P-40、およびZwittergent 3-12からなる群から少なくとも1種選択されることを特徴とする、請求項7~9のいずれか1項に記載の方法。 The solubilizing reagents are lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride. , Urea, 2-mercaptoethanol, dithiotreitol, TCEP-HCl, sodium dodecyl, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, A claim, wherein at least one is selected from the group consisting of Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Noidet P-40, and Zwittergent 3-12. The method according to any one of 7 to 9.
  11.  前記ゲルカプセルがヒドロゲルカプセルであることを特徴とする、請求項7~10のいずれか1項に記載の方法。 The method according to any one of claims 7 to 10, wherein the gel capsule is a hydrogel capsule.
  12.  前記分析に必要な薬剤が第2の増幅用試薬である、請求項5~11のいずれか1項に記載の方法。 The method according to any one of claims 5 to 11, wherein the drug required for the analysis is the second amplification reagent.
  13.  前記第1の増幅試薬と前記第2の増幅用試薬とが同一である、請求項12に記載の方法。 The method according to claim 12, wherein the first amplification reagent and the second amplification reagent are the same.
  14.  前記第1の増幅試薬と前記第2の増幅用試薬とが異なる、請求項12に記載の方法。 The method according to claim 12, wherein the first amplification reagent and the second amplification reagent are different.
  15.  前記第1の増幅試薬がゲノムDNAまたはその部分の増幅用試薬または特定配列増幅用試薬である、請求項12~14のいずれか1項に記載の方法。 The method according to any one of claims 12 to 14, wherein the first amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
  16.  前記第2の増幅試薬がゲノムDNAまたはその部分の増幅用試薬または特定配列増幅用試薬である、請求項12~15のいずれか1項に記載の方法。 The method according to any one of claims 12 to 15, wherein the second amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
  17.  前記a)において、前記液滴中に前記1つの細胞または細胞様構造物、および第1の標識を封入することを特徴とする、請求項7~16のいずれか1項に記載の方法。 The method according to any one of claims 7 to 16, wherein in the a), the one cell or cell-like structure and the first label are enclosed in the droplet.
  18.  前記B)において、前記1つの細胞または細胞様構造物および第2の標識をカプセル化することを特徴とする、請求項5~17のいずれか1項に記載の方法。 The method according to any one of claims 5 to 17, wherein in B), the one cell or cell-like structure and the second label are encapsulated.
  19.  前記第1の標識と前記第2の標識とが同一である、請求項18に記載の方法。 The method according to claim 18, wherein the first sign and the second sign are the same.
  20.  前記第1の標識と前記第2の標識とが異なる、請求項18に記載の方法。 The method according to claim 18, wherein the first sign and the second sign are different.
  21.  前記第1の標識と前記第2の標識とが核酸の量を示すことを特徴とする、請求項17~20のいずれか1項に記載の方法。 The method according to any one of claims 17 to 20, wherein the first label and the second label indicate the amount of nucleic acid.
  22.  前記C)が、前記第1の標識および/または前記第2の標識を評価することを含む、請求項17~21のいずれか1項に記載の方法。 The method according to any one of claims 17 to 21, wherein C) comprises evaluating the first label and / or the second label.
  23.  前記A)における前記細胞または細胞様構造物に含まれる核酸が、ゲノムDNAである、請求項5~22のいずれか1項に記載の方法。 The method according to any one of claims 5 to 22, wherein the nucleic acid contained in the cell or cell-like structure in A) is genomic DNA.
  24.  前記C)の分析において、種特異的な配列または特定種に保存されている配列の増幅を行うことを特徴とする、請求項6~23のいずれか1項に記載の方法。 The method according to any one of claims 6 to 23, which comprises amplifying a species-specific sequence or a sequence stored in a specific species in the analysis of C).
  25. 細胞または細胞様構造物を分析するためのシステムであって、
    [X]2つ以上の細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、カプセル化するための薬剤および/またはそれらを格納するカプセル化薬剤格納部と、
    [Y]分析に使用する所望の薬剤および/またはそれらを格納する分析薬剤格納部と、
    [Z]カプセル化のための手段と
    [W]必要に応じて、該所望の薬剤を用いて分析を行うための手段と
    を備える、システム。
    A system for analyzing cells or cell-like structures
    [X] A drug for encapsulating two or more cells or cell-like structures or a substance derived from them, or a capsule containing them, and / or an encapsulating drug storage unit for storing them.
    [Y] A desired drug used for analysis and / or an analytical drug storage unit for storing them, and
    A system comprising [Z] means for encapsulation and [W] means for performing analysis with the desired agent, if desired.
  26. 細胞または細胞様構造物を分析するためカプセルを提供するためのキットであって、
    [X]2つ以上の細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、カプセル化するための薬剤と、
    [Y]分析に使用する所望の薬剤と、
    を備える、キット。
    A kit for providing capsules for analyzing cells or cell-like structures.
    [X] An agent for encapsulating two or more cells or cell-like structures or substances derived from them, or capsules containing them.
    [Y] With the desired drug used for analysis
    A kit that includes.
  27. 細胞または細胞様構造物を分析する方法であって、
    (A)2つ以上の細胞または細胞様構造物を、核酸増幅のための薬剤とともに、細胞または細胞様構造物ごとにカプセル化する工程と、
    (B)第一の核酸増幅反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
    (C)該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに核酸増幅のための薬剤とともにカプセル化する工程と、
    (D)第二の核酸増幅反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
    (E)必要に応じて、D)で得られた、該細胞または細胞様構造物あるいはそれに由来する物質、あるいはそれらを含むカプセルを、該細胞または細胞様構造物あるいはそれに由来する物質ごとに第Xの薬剤とともにカプセル化する工程であって、Xは3以上の整数である、工程と、
    (F)必要に応じて、該第Xの薬剤に基づく反応をカプセル内で該細胞または細胞様構造物に対して行う工程と、
    (G)(E)および(F)の工程を必要に応じて繰り返す工程と、
    (H)該カプセルに含まれる核酸の配列決定を行う工程と
    を包含する、方法。
    A method of analyzing cells or cell-like structures
    (A) A step of encapsulating two or more cells or cell-like structures together with a drug for nucleic acid amplification for each cell or cell-like structure.
    (B) A step of performing the first nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and
    (C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each of the cell or cell-like structure or a substance derived thereto together with a drug for nucleic acid amplification.
    (D) A step of performing a second nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and
    (E) If necessary, the cell or cell-like structure or a substance derived from the cell or cell-like structure obtained in D), or a capsule containing them, is prepared for each cell or cell-like structure or a substance derived from the cell or cell-like structure. The step of encapsulating with the drug of X, where X is an integer of 3 or more.
    (F) If necessary, a step of carrying out a reaction based on the Xth drug on the cell or cell-like structure in a capsule, and
    A step of repeating the steps (G), (E) and (F) as necessary, and
    (H) A method comprising the step of sequencing the nucleic acid contained in the capsule.
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WO2024202886A1 (en) * 2023-03-29 2024-10-03 国立研究開発法人産業技術総合研究所 Nucleic acid detection method, hydrogel capsule, and dispersion liquid of same

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JP2015528283A (en) * 2012-08-14 2015-09-28 テンエックス・ジェノミクス・インコーポレイテッド Microcapsule composition and method
WO2017184707A1 (en) * 2016-04-19 2017-10-26 President And Fellows Of Harvard College Immobilization-based systems and methods for genetic analysis and other applications
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JP2002531056A (en) * 1998-08-07 2002-09-24 セレイ, エルエルシー Gel microdrop in gene analysis
JP2015528283A (en) * 2012-08-14 2015-09-28 テンエックス・ジェノミクス・インコーポレイテッド Microcapsule composition and method
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WO2021219567A1 (en) * 2020-04-27 2021-11-04 De Paulou Massat Sophie Microcompartment for culturing cnidarian cells
WO2024202886A1 (en) * 2023-03-29 2024-10-03 国立研究開発法人産業技術総合研究所 Nucleic acid detection method, hydrogel capsule, and dispersion liquid of same

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