Selective detection, counting, and genomic analysis of living bacterium-derived nucleic acid on single-organism basis

Abstract

To provide a technique enabling the analysis of cell viability information and nucleic acid information together for each single cell in a cell population when analyzing the composition of the cell population.SOLUTION: The present disclosure discloses a method that is for analyzing the composition of a cell population and that comprises: a step for providing gel capsules in each of which a single cell of the cell population is encapsulated in a state of being labeled with a labeling agent, where the labeling agent distinctively labels a living cell and a dead cell; a step for amplifying a nucleic acid derived from each single cell of the cell population; a step for obtaining cell viability information for each single cell of the cell population; and a step for analyzing the amplified nucleic acid derived from each single cell of the cell population.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to the analysis of cell population composition and can be used in fields such as biological research, medicine, the environment, and health care. [Background technology]

[0002] Selective detection of live bacteria from the environment is used for hygienic quality evaluation in food and seed testing, and for quality assurance of microbial materials and microbial preparations. In food testing, the target bacteria are specified, and for those for which a laboratory culture method has been established, a method is generally used to measure the number of bacteria growing under certain conditions using standard agar medium. However, due to the need for culture days and the complexity of the culture, identification methods such as fluorescent staining observation of live and dead bacteria have been proposed as alternative techniques. If the sample is composed of known species, it is possible to infer the species of the observed material by judging based on images, but if the sample is composed of multiple unknown or similar bacteria, it is difficult to identify the species composition. Methods based on gene detection include methods that specifically amplify and detect the sequence of the target bacteria, and PCR, qPCR, digital PCR, LAMP, etc. are used. Methods based on gene detection are highly sensitive and can process multiple samples, but with normal methods, nucleic acids derived from dead bacteria are also detected at the same time, and there is no specificity for live bacteria. In addition, PCR and LAMP methods, which only detect the nucleic acids of live bacteria, can only be used for specific target species with known sequences. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure provides a method for analyzing the composition of a cell population, and techniques such as reagents, compositions, kits, and systems used therein. The techniques of the present disclosure are characterized by a step of obtaining live / dead information for each cell in the cell population, a step of analyzing amplified nucleic acid derived from each cell in the cell population, or the reagents, compositions, means, etc. used therefor.

[0004] In one aspect, the present disclosure relates to a method for specifically reacting with nucleic acids derived from living cells from various microorganisms, preparing amplified polynucleotides adjusted in parallel for each cell, counting the total number of living cells and comprehensively reading gene sequences that identify microbial species, and providing cell count and sequence information derived from living cells from a cell population containing a mixture of living and dead cells. The general concept is to first label dead cells and amplify their nucleic acids, and then label the amplified nucleic acids derived from living cells to specifically detect the nucleic acids derived from living cells.

[0005] Examples of embodiments of the present disclosure include the following: (Item A) 1. A method for analyzing the composition of a population of cells, comprising: obtaining viability information for each cell in the population of cells; analyzing the amplified nucleic acid from each cell in the population of cells; A method comprising: (Item AA) 1. A method for providing live and dead cell nucleic acid information on a cell-by-cell basis in a population of cells, comprising: obtaining viability information for each cell in the population of cells; analyzing the amplified nucleic acid from each cell in the population of cells; A method comprising: (Item 1) 1. A method for analyzing the composition of a population of cells, comprising: providing a gel capsule in which the cells of the cell population are encapsulated one by one in a labeled state with a labeling agent, the labeling agent differentially labels live cells and dead cells as live / dead information for each cell of the cell population; amplifying nucleic acid from each cell in the population of cells; obtaining viability information for each cell in the population of cells; analyzing the amplified nucleic acid from each cell of the population of cells; A method comprising: (Item 2) Providing a gel capsule encapsulating each of the cells of the population of cells, adding said labeling agent to the population of cells; encapsulating each cell of the cell population into a gel capsule; The method according to any of the preceding items, comprising: (Item 3) Providing a gel capsule encapsulating each of the cells of the population of cells, encapsulating individual cells of the population of cells into a gel capsule; adding said indicator to said gel capsule; The method according to any of the preceding items, comprising: (Item 4) 2. The method of any of the preceding claims, wherein analysis of the amplified nucleic acid is performed on nucleic acid from a living cell. (Item 5) The method of any of the preceding items, wherein the labeling agent specifically labels dead cells or specifically labels live cells. (Item 6) The method according to any of the preceding items, further comprising the step of adding a nucleic acid decomposition promoter to the population of cells or to the gel capsule, the nucleic acid decomposition promoter promoting the decomposition of nucleic acid derived from killed bacteria. (Item 7) The method according to any of the preceding items, wherein the nucleic acid degradation promoter is a topoisomerase inhibitor. (Item 8) The method according to any of the preceding items, wherein the nucleic acid degradation promoter comprises one or more selected from ciprofloxacin (CPFX), camptothecin (CPT), etoposide (ETP), and amsacrine (m-AMSA). (Item 9) The method according to any of the preceding items, wherein the labeling agent is a substance that permeates the membrane of dead cells. (Item 10) The method according to any of the preceding items, wherein the labeling agent is a substance that permeates the membrane of dead cells and binds to nucleic acids. (Item 11) The method according to any of the preceding items, wherein the labeling agent is a substance that inhibits amplification of bound nucleic acid. (Item 12) The method of any of the preceding items, wherein the labeling agent comprises one or more selected from ethidium monoazide (EMA), psoralen, 4'-aminomethyl-4,5'-dimethylisopsoralen [4'-AMDMIP], and propidium monoazide (PMA). (Item 13) The method according to any of the preceding items, wherein the viability information of each cell is obtained from the presence or absence of amplification of nucleic acid derived from each cell. (Item 14) The method according to any of the preceding items, wherein the presence or absence of amplification of nucleic acid derived from each cell is detected using a nucleic acid-binding fluorescent substance capable of detecting nucleic acid amplification. (Item 15) The method according to any of the preceding items, wherein the nucleic acid-binding fluorescent substance comprises one or more selected from EvaGreen, SYBR Green, SYBR Gold, SYTO9, SYTO10, Hoechst 33342, 4',6-diamidino-2-phenylindole, Acridine Orange (AO), Promidium iodide, and Ethidium homodimer-2. (Item 16) encapsulating cells of the population of cells one by one into a gel capsule, encapsulating cells of the population of cells one by one in a droplet; gelling the droplets to form gel capsules; The method according to any of the preceding items, comprising: (Item 17) 2. The method according to any of the preceding items, wherein the droplets encapsulating the cells are created by flowing a suspension of the cells through a microchannel and shearing the suspension with oil. (Item 18) 2. The method according to any of the preceding items, characterized in that the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, matrigel, collagen or a photocurable resin. (Item 19) 13. The method according to any of the preceding items, characterized in that the gel capsule is a hydrogel capsule. (Item 20) amplifying nucleic acid from each cell of the population of cells, immersing said gel capsule in one or more lysis reagents to lyse said cells, wherein polynucleotides comprising genomic DNA of said cells or portions thereof are dissolved into and retained within said gel capsule; contacting the polynucleotide with an amplification reagent to amplify the polynucleotide within the gel capsule; The method according to any of the preceding items, comprising: (Item 21) 20. The method of any of the preceding claims, further comprising the step of removing contaminants from the gel capsule. (Item 22) 3. The method according to any of the preceding items, characterized in that the lysis reagent is at least one selected from the group consisting of lysozyme, labiase, yatalase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolysin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride, urea, 2-mercaptoethanol, dithiothreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octylglucoside, octylthioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, and Zwittergent 3-12. (Item 23) The method according to any of the preceding items, further comprising the step of selecting a sample containing amplified nucleic acid to be analyzed from the samples containing amplified nucleic acid derived from each of the cells. (Item 24) The method of any of the preceding items, wherein analyzing the amplified nucleic acid comprises identifying the type of each of the cells. (Item 25) 25. The method according to any one of items 1 to 24, wherein the analysis of the amplified nucleic acid comprises a step of detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each of the cells. (Item 26) The method of any of the preceding items, wherein the step of detecting a nucleic acid having a specific sequence comprises amplifying and sequencing the nucleic acid having a specific sequence. (Item 27) 20. The method of any of the preceding claims, wherein analyzing the composition of the population of cells comprises determining the absolute number of each type of cell in the population of cells. (Item 28) 20. The method of any of the preceding claims, wherein analyzing the composition of the cell population comprises determining the absolute number of each type of cell among live cells in the cell population. (Item 29) 20. The method of any of the preceding claims, further comprising obtaining genomic sequence data for each of the cells in the population of cells from a sample comprising amplified nucleic acid from each of the cells. (Item 30) The method of any of the preceding items, wherein the population of cells is a microbiota. (Item 31) The method according to any of the preceding items for quality assessment of FMT samples. (Item 32) 2. The method according to any of the preceding items for the assessment of the hygienic quality of a food product. (Item 33) 2. The method according to any of the preceding items for the assessment of the presence of pathogens in an environment. (Item 34) The method of any of the preceding items, wherein the population of cells is a microbial preparation. (Item 35) 2. The method according to any of the preceding items for the quality assessment of a microbial preparation. (Item 36) The method according to any of the preceding items for evaluating the quality of microbial-derived feed, fermented food, or microbial-containing supplements. (Item 37) 2. The method according to any of the preceding items, for profiling viable bacteria in activated sludge. (Item 38) 2. The method according to any of the preceding items for the assessment of the presence of viable microorganisms in an environment. (Item 39) The method according to any of the preceding items for evaluation of the efficacy spectrum of an antibacterial agent. (Item 40) The method according to any of the preceding items for the evaluation of a disinfection method. (Item 41) 2. The method according to any of the preceding items, for profiling viable bacteria in a biofilm. (Item B1) adding ethidium monoazide (EMA) or propidium monoazide (PMA) to the population of cells and irradiating with light; encapsulating cells of the population of cells one by one in a droplet; gelling the droplets to form gel capsules; immersing the gel capsule in one or more lysis reagents to lyse the cells, whereby a polynucleotide comprising the genomic DNA of the cells or a portion thereof is eluted into the gel capsule; removing contaminants from the gel capsule; amplifying the polynucleotides within the gel capsule by contacting the polynucleotides with an amplification reagent, wherein EMA or PMA bound polynucleotides are not amplified; detecting the amplified polynucleotides; selecting a gel capsule containing a sample derived from live bacteria; counting the gel capsules containing the sample from the live bacteria; Analyzing the amplified polynucleotides in the gel capsule containing the sample from the live bacteria; A method comprising: (Item B2) encapsulating cells of the population of cells one by one in a droplet; gelling the droplets to form gel capsules; adding EMA or PMA to the gel capsule and irradiating it with light; washing the gel capsule and lysing the cells by immersion in one or more lysis reagents, whereby polynucleotides comprising genomic DNA or portions thereof of the cells are eluted into the gel capsule; removing contaminants from the gel capsule; amplifying the polynucleotides within the gel capsule by contacting the polynucleotides with an amplification reagent, wherein EMA or PMA bound polynucleotides are not amplified; detecting the amplified polynucleotides; selecting a gel capsule containing a sample derived from live bacteria; counting the gel capsules containing the sample from the live bacteria; Analyzing the amplified polynucleotides in the gel capsule containing the sample from the live bacteria; A method comprising: (Item C) 1. A system for analyzing a composition of a population of cells, comprising: A means for obtaining information on the viability of each cell in the population of cells; means for analyzing the amplified nucleic acid from each cell in the population of cells; Including, the system. (Item C-1) A system according to any one or more of the preceding items, comprising the features according to any one or more of the preceding items. (Item C1) 1. A system or kit for analyzing the composition of a population of cells, comprising: a labeling agent that differentially labels live and dead cells; a means for encapsulating the cells in a gel capsule; a means for amplifying nucleic acid; A means for analyzing nucleic acids; 2. A system or kit comprising: (Item C1-1) A system or kit as described in the preceding paragraphs, comprising any one or more of the features described in the preceding paragraphs. (Item C2) A composition for use in a method of analyzing the composition of a population of cells, the composition comprising a labeling agent that differentially labels live and dead cells, the method comprising the steps of obtaining live / dead information for each cell in the population of cells, and analyzing amplified nucleic acid from each cell in the population of cells. A composition comprising: (Item C2-1) A composition according to any one or more of the preceding items, comprising the features described in any one or more of the preceding items. (Item C3) A composition for use in a method for single-cell gel encapsulation and analysis of a population of cells, the composition comprising a labeling agent that differentially labels live and dead cells. (Item C3-1) A composition according to any one or more of the preceding items, comprising the features described in any one or more of the preceding items. (Item C4) The system, kit, or composition of any of the preceding items, wherein analysis of the amplified nucleic acid is performed on nucleic acid from a living cell. (Item C5) The system, kit, or composition of any of the preceding items, wherein the labeling agent specifically labels dead cells or specifically labels live cells. (Item C6) The system, kit, or composition described in any of the preceding items, further comprising a nucleic acid degradation promoter that promotes degradation of nucleic acids derived from killed bacteria, which is added to the population of cells or to the gel capsule. (Item C7) The system, kit, or composition described in any of the preceding items, wherein the nucleic acid degradation promoter is a topoisomerase inhibitor. (Item C8) The system, kit, or composition described in any of the preceding items, wherein the nucleic acid degradation promoter comprises one or more selected from ciprofloxacin (CPFX), camptothecin (CPT), etoposide (ETP), and amsacrine (m-AMSA). (Item C9) The system, kit, or composition according to any of the preceding items, wherein the labeling agent is a substance that permeates the membrane of dead cells. (Item C10) The system, kit, or composition according to any of the preceding items, wherein the labeling agent is a substance that permeates the membrane of dead cells and binds to nucleic acids. (Item C11) The system, kit, or composition according to any of the preceding items, wherein the labeling agent is a substance that inhibits amplification of bound nucleic acid. (Item C12) The system, kit, or composition of any of the preceding items, wherein the labeling agent comprises one or more selected from ethidium monoazide (EMA), psoralen, 4'-aminomethyl-4,5'-dimethylisopsoralen [4'-AMDMIP], and propidium monoazide (PMA). (Item C13) The system, kit, or composition described in any of the preceding items, wherein the live / dead information for each cell is obtained from the presence or absence of amplification of nucleic acid derived from each cell. (Item C14) The system, kit, or composition described in any of the preceding items, further comprising a means for detecting the presence or absence of amplification of nucleic acid derived from each individual cell (e.g., a nucleic acid-binding fluorescent substance capable of detecting amplification of nucleic acid). (Item C15) The system, kit, or composition described in any of the preceding items, wherein the nucleic acid-binding fluorescent substance comprises one or more selected from EvaGreen, SYBR Green, SYBR Gold, SYTO9, SYTO10, Hoechst 33342, 4',6-diamidino-2-phenylindole, Acridine Orange (AO), Promidium iodide, and Ethidium homodimer-2. (Item C16) A means for encapsulating cells of a cell population one by one into a droplet or a droplet preparation unit for encapsulating cells of a cell population one by one into a droplet; A means for gelling the droplets to generate gel capsules or a gel capsule generating unit for gelling the droplets to generate gel capsules; 20. The system, kit, or composition of any preceding item, further comprising: (Item C17) The system, kit, or composition described in any of the preceding items, wherein the suspension of cells is flowed through a microchannel and the droplets encapsulating the cells are created by shearing the suspension with oil. (Item C18) The system, kit, or composition described in any of the preceding items, characterized in that the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, Matrigel, collagen, or a photocurable resin. (Item C19) 2. The system, kit, or composition of any of the preceding items, wherein the gel capsule is a hydrogel capsule. (Item C20) a lysis reagent immersion unit for immersing a means for lysing a cell or a gel capsule in a lysis reagent, the means or the lysis reagent immersion unit being configured so that a polynucleotide containing the genomic DNA of the cell or a portion thereof is dissolved in the gel capsule and retained in the gel capsule; A means for amplifying polynucleotides in a gel capsule or an amplification reagent immersion unit for immersing the gel capsule in an amplification reagent; 20. The system, kit, or composition of any preceding item, further comprising: (Item C21) The system, kit, or composition of any of the preceding items, further comprising a means for removing contaminants from the gel capsule or a removal unit for removing contaminants from the gel capsule. (Item C22) 2. The system, kit, or composition of any of the preceding items, wherein the lysis reagent is at least one selected from the group consisting of lysozyme, labiase, yatalase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolysin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride, urea, 2-mercaptoethanol, dithiothreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octylglucoside, octylthioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, and Zwittergent 3-12. (Item C23) The system, kit, or composition described in any of the preceding items, further comprising a means for selecting a sample containing amplified nucleic acid to be analyzed from the samples containing amplified nucleic acid derived from each of the cells, or a sorting unit for selecting gel capsules and storing the gel capsules in a storage container. (Item C24) The system, kit, or composition of any of the preceding items, wherein analysis of the amplified nucleic acid includes identifying the type of each of the cells. (Item C25) The system, kit, or composition described in any of the preceding items, wherein analyzing the amplified nucleic acid includes detecting a nucleic acid having a specific sequence in a sample containing amplified nucleic acid derived from each of the cells. (Item C26) 26. The method of claim 25, wherein the step of detecting a nucleic acid having a specific sequence comprises amplifying and sequencing the nucleic acid having a specific sequence. (Item C27) The system, kit, or composition of any of the preceding items, wherein analyzing the composition of the cell population comprises determining the absolute number of each type of cell in the cell population. (Item C28) The system, kit, or composition of any of the preceding items, wherein analyzing the composition of the cell population comprises determining the absolute number of each type of cell among live cells in the cell population. (Item C29) The system, kit, or composition of any preceding item, further comprising a step of obtaining genomic sequence data for each of the cells in the population of cells from a sample containing amplified nucleic acid from the each of the cells. (Item C30) The system, kit, or composition of any of the preceding items, wherein the population of cells is a microbiota. (Item C31) A system, kit, or composition according to any of the preceding items for evaluating the quality of FMT samples. (Item C32) 2. A system, kit or composition according to any of the preceding items for the assessment of the hygienic quality of food. (Item C33) 2. A system, kit or composition according to any of the preceding items for assessing the presence of pathogens in an environment. (Item C34) 20. The system, kit, or composition of any preceding item, wherein the population of cells is a microbial preparation. (Item C35) 2. The system, kit or composition according to any of the preceding items for evaluating the quality of a microbial preparation. (Item C36) A system, kit, or composition according to any of the preceding items for evaluating the quality of microbial-derived feed, fermented food, or microbial-containing supplements. (Item C37) 2. The system, kit, or composition according to any of the preceding items, for profiling viable bacteria in activated sludge. (Item C38) 2. A system, kit, or composition according to any of the preceding items for assessing the presence of viable microorganisms in an environment. (Item C39) The system, kit, or composition according to any of the preceding items, for evaluating the efficacy spectrum of an antibacterial agent. (Item C40) The system, kit, or composition according to any of the preceding items for evaluating a disinfection method. (Item C41) A system, kit, or composition according to any of the preceding items for profiling live bacteria in a biofilm. (Item D1) Ethidium monoazide (EMA) or propidium monoazide (PMA) (reagents for distinguishing between live and dead cells) A light irradiation means; A means for encapsulating cells in droplets one by one; means for gelling the droplets to form gel capsules; a lysis reagent for the gel capsule, the lysis reagent being used to add the lysis reagent so that a polynucleotide comprising the genomic DNA or a portion thereof of the cell is dissolved in the gel capsule; a means for removing contaminants from said gel capsule; A reagent for amplifying a polynucleotide, the reagent being used to contact the polynucleotide and amplify the polynucleotide in a gel capsule, but not to amplify a polynucleotide bound with EMA or PMA; A means for detecting a polynucleotide; a means for selecting a gel capsule containing a sample derived from live bacteria; A means for counting gel capsules containing a sample derived from viable bacteria; means for analyzing the polynucleotides amplified in the gel capsule containing the sample derived from the viable bacteria; 2. A system or kit comprising: (Item D2) A composition for use in a method of analyzing the composition of a population of cells, comprising a labeling agent (e.g., ethidium monoazide (EMA) or propidium monoazide (PMA)) that differentially labels live and dead cells, the method comprising obtaining live / dead information for each cell in the population of cells, and analyzing amplified nucleic acid from each cell in the population of cells. A composition comprising:

[0006] It is contemplated that the present disclosure may provide one or more of the above features in combinations other than those specifically described. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. Effect of the Invention

[0007] Distinguishing between live and dead cells is necessary in various biological studies, and analysis of a mixed sample of multiple cell tumors has been particularly difficult in the past. The present disclosure has the characteristic that the number of live bacteria present in a sample can be evaluated regardless of whether they can be cultured or not and whether gene sequence information is available, and that whole genome sequence information can be obtained. Since DNA derived from dead bacteria is decomposed, it may become noise even in genome analysis at the level of a single organism, so it is also effective in that genome sequence information can be obtained in advance by limiting it to live bacteria. In the quality evaluation of the microbiota used in fecal transplants and microbial preparations, it is not limited to known sequences, and it is highly versatile in that various subjects can be evaluated in the same analytical format. [Brief description of the drawings]

[0008] [Figure 1]Figure 1 is a schematic diagram illustrating the flow of selective detection, counting, and genome analysis of DNA derived from live cells. In advance, the sample is treated by chemically modifying the DNA of dead cells to prevent it from being replicated by enzymatic reactions. In the gel, both live and dead cells are dissolved and the DNA is purified. However, only DNA derived from live cells increases during DNA amplification. When the amplified live cell DNA-gel is collected, the total number of live cells is counted. After that, the DNA is sequenced individually to classify and count the sequences derived from live cells. [Diagram 2] Figure 2 shows the images of gel capsules containing E. coli that were heat-treated and then treated with EMA after polynucleotide amplification (top: with EMA, bottom: without EMA). In the heat-sterilized sample, polynucleotide amplification was suppressed by EMA treatment, and fluorescence (green bright spots) was no longer observed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present disclosure will be described below while showing the best mode. Throughout this specification, the expression of the singular form should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will take precedence.

[0010] (definition, etc.) The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0011] As used herein, the term "cell" refers to any particle that contains molecules containing genetic information and is capable of replicating (whether independently or not). As used herein, the term "cell" includes all cells that can be distinguished as living or dead, including cells of single-celled organisms, bacteria, cells derived from multicellular organisms, cells in which fungi and viruses coexist, and the like.

[0012] As used herein, a "biomolecule" refers to a molecule possessed by any organism or virus. Biological molecules may include nucleic acids, proteins, sugar chains, lipids, and the like. As used herein, a "biomolecule analog" refers to a natural or non-natural variant of a biomolecule. Biological molecule analogs may include modified nucleic acids, modified amino acids, modified lipids, modified sugar chains, and the like.

[0013] As used herein, the term "population" refers to an assembly containing two or more cells.

[0014] As used herein, a "subpopulation" refers to a portion of a population having a smaller number of cells than the population.

[0015] In this specification, "gel" refers to a state in which polymeric substances or colloidal particles in a colloidal solution (sol) form a network structure as a whole due to their interactions, and the solution loses fluidity while still containing a large amount of the liquid phase, which is the solvent or dispersion medium. In this specification, "gelation" refers to changing the solution into a "gel" state.

[0016] As used herein, the term "gel capsule" refers to a gel-like particulate structure capable of retaining cells or cell-like structures therein.

[0017] In this specification, "genetic analysis" refers to examining the state of nucleic acids (DNA, RNA, etc.) in a biological sample. In one embodiment, the genetic analysis may be one that utilizes a nucleic acid amplification reaction. Examples of genetic analysis, including the above, include sequencing, genotyping and polymorphism analysis (SNP analysis, copy number polymorphism, restriction 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, etc.

[0018] As used herein, the term "single cell level" refers to processing the genetic information contained in one cell in a state where it is distinguished from the genetic information contained in other cells. Therefore, the single cell level can also be expressed as terms such as "(one) cell" or "single cell level." For example, when amplifying polynucleotides at the "single cell level," amplification is performed in a state where the polynucleotides in a certain cell and the polynucleotides in other cells can be distinguished from each other.

[0019] As used herein, the term "single cell analysis" refers to the analysis of the genetic information contained in one cell while distinguishing it from the genetic information contained in other cells.

[0020] As used herein, "nucleic acid information" refers to information about nucleic acids contained in a single cell, including the presence or absence of a particular gene sequence, the yield of a particular gene, or the total nucleic acid yield.

[0021] As used herein, "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.

[0022] As used herein, the term "microbiota" refers to the entire collection of microorganisms present in a certain physical range. Examples of a certain physical range include the intestines, skin, oral cavity, nasal cavity, or vagina of an individual, or a certain range in water bodies, soil, biological surfaces, or inside organisms in the environment. The microbiota may be a collection of microorganisms across multiple classifications, such as a combination of fungi, bacteria, archaea, unicellular animals, viruses, or individual cells of multicellular organisms, or may be a collection of microorganisms from a certain classification. One example of the microbiota is the bacterial flora.

[0023] As used herein, the "composition" of a cell population refers to information about what species are contained in the cell population or the amount of each species contained, and also includes information about whether some microbial species are contained in the cell population or their amounts.

[0024] As used herein, "labeling" refers to making a state in which a cell can be distinguished based on some characteristic. A representative example of "labeling" is a state in which a substance is specifically bound to a cell having some characteristic, making the cell distinguishable. Conversely, the term "labeling" can also refer to the creation of a state in which a cell having some characteristic is distinguishable because a substance is not bound to the cell.

[0025] As used herein, the term "labeling agent" refers to any substance that can be used to distinguish cells or nucleic acids based on some characteristic, typically a substance that can be used to differentially label and distinguish between live and dead cells.

[0026] (Summary of the Disclosure) The following is an overview of the present disclosure with reference to the embodiments. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments can be used alone or in combination.

[0027] In the present disclosure, a method for analyzing the composition of a cell population may be provided. The method may include providing a gel capsule in which cells of the cell population are encapsulated one by one in a state labeled with a labeling agent. The labeling agent may differentially label live cells and dead cells. The labeling agent may differentially label live cells and dead cells as live / dead information of each cell of the cell population. The method may include amplifying nucleic acid from each cell of the cell population, obtaining live / dead information of each cell of the cell population, and analyzing the amplified nucleic acid from each cell of the cell population. The present disclosure also provides a method for providing nucleic acid information of live and dead cells for each cell in a cell population. The method may include obtaining live / dead information of each cell in the cell population, and analyzing the amplified nucleic acid from each cell in the cell population.

[0028] The present disclosure may also provide a system, kit, composition, etc. for analyzing the composition of a cell population. The system, kit, composition may include a labeling agent for labeling cells, and a means for providing a gel capsule in which cells are encapsulated one by one, or a composition for the same. The labeling agent may differentially label live cells and dead cells. The system, kit, composition may include a composition or reagent or means for nucleic acid amplification, where the nucleic acid amplification is used to amplify nucleic acid from each cell in a cell population, and live / dead information for each cell in the cell population can be obtained. The system, kit, composition may include a means, reagent, kit, system, etc. for analyzing nucleic acid, where the amplified nucleic acid from each cell in the cell population is analyzed. The present disclosure also provides a system, kit, composition, device, drug, etc. for providing information on the nucleic acid of live cells and dead cells for each cell in a cell population. The system, kit, composition, device, drug, etc. may include a means, drug, etc. for obtaining live / dead information for each cell in a cell population, and a means, drug, etc. for analyzing amplified nucleic acid from each cell in a cell population.

[0029] The step of labeling by adding a labeling agent may be performed before or after the cells are encapsulated in a gel capsule. When labeling by adding a labeling agent after encapsulating the cells in a gel capsule, it is expected to have an effect of washing out excess labeling agent through the capsule.

[0030] The step of providing a gel capsule in which the cells of the cell population are encapsulated one by one may include a step of adding a labeling agent to the cell population, and a step of encapsulating the cells of the cell population one by one in a gel capsule. The step of providing a gel capsule in which the cells of the cell population are encapsulated one by one in a gel capsule may include a step of encapsulating the cells of the cell population one by one in a gel capsule, and a step of adding the labeling agent to the gel capsule.

[0031] By differential labeling with a labeling agent, the analysis of amplified nucleic acids can be performed on nucleic acids derived from living cells. When analyzing cells in a sample on a nucleic acid basis (e.g., metagenomic analysis), it is not possible to distinguish between information on nucleic acids derived from living cells and information on nucleic acids derived from dead cells, and it is unclear whether information on living cells can be obtained. In addition, when analyzing cells in a sample at a single cell level, cells in a dead cell state may be detected as one cell. Dead cells exist in a state in which components have completely lost their cellular form and are suspended in the sample, as well as in a state in which they maintain their shape. In the past, it was sometimes impossible to remove the latter dead cells, resulting in low-quality genome amplification. The method of the present disclosure can prevent such low-quality genome amplification by performing analysis only on nucleic acids derived from living cells. In addition, if information on which cells are living cells (e.g., live bacteria) can be obtained, it is also possible to know the breakdown of dead cells (e.g., dead bacteria) from the difference with the analysis results of a method that does not use this method.

[0032] PCR and LAMP methods have been reported to specifically amplify and detect the nucleic acids of living cells (Nogva et al., 2003; Soejima et al., 2008), but they can only be used for specific species whose sequences are known. The principle of these techniques is to detect only the DNA of living cells by decomposing the dead cell DNA using Ethidium monoazide (EMA), which efficiently modifies the DNA of dead cells, or Ciprofloxacin (CPFX), Camptothecin (CPT), or Etoposide (ETP), which are topoisomerase poisons that inhibit the activity of topoisomerase, which is necessary for DNA replication.

[0033] In the present disclosure, the step of encapsulating cells of a population of cells one by one into a gel capsule may include the steps of encapsulating cells of the population of cells one by one into a liquid droplet, and gelling the droplet to produce a gel capsule.

[0034] (Acquisition of life and death information) In the present disclosure, the labeling agent can differentially label live cells and dead cells. The labeling agent may specifically label dead cells, or may specifically label live cells, or may label live cells so as to distinguish between live cells and dead cells. The labeling agent is not particularly limited as long as it can utilize the difference in properties such as morphology between dead cells and live cells. Typically, the labeling agent is a substance that permeates the membrane of dead cells. Since the integrity of the cell membrane (and / or cell wall) of dead cells is impaired compared to that of live cells, a substance that does not permeate the cell membrane of live cells but permeates the cell membrane of dead cells can be used as a labeling agent. By detecting the presence or absence of this labeling agent in a subsequent process, information on the live or dead state of each cell can be obtained. The labeling agent may inhibit the amplification of nucleic acids, and may also generate a signal such as staining, fluorescence, luminescence, Raman scattering light, or radiation, or a change in charge.

[0035] The labeling agent may be a substance that permeates the membrane of dead cells and binds to nucleic acids. The binding to nucleic acids may be, for example, by covalent binding or crosslinking with nucleic acids, or by intercalating into the helix of nucleic acids. The presence or absence of this binding can be detected in a subsequent step to obtain information on the viability of each cell. The labeling agent may typically be a substance that inhibits the amplification of the bound nucleic acid. In a subsequent step, information on the viability of each cell can be obtained from the presence or absence of amplification of nucleic acids derived from each cell. Examples of the labeling agent include ethidium monoazide (EMA), psoralen, 4'-aminomethyl-4,5'-dimethylisopsoralen [4'-AMDMIP], propidium monoazide (PMA), and the like, and in the present disclosure, one or more selected from these may be included as the labeling agent.

[0036] In addition to the addition of a labeling agent, a nucleic acid decomposition promoter that promotes the decomposition of nucleic acid derived from killed bacteria may be added. Examples of the nucleic acid decomposition promoter include a topoisomerase inhibitor that inhibits the action of topoisomerase required for DNA replication, and a gyrase inhibitor that inhibits the action of gyrase required for DNA replication in some microorganisms, and in the present disclosure, one or more selected from these may be included as the nucleic acid decomposition promoter. Examples of the nucleic acid decomposition promoter include Ciprofloxacin (CPFX), Camptothecin (CPT), Etoposide (ETP), Amsacrine (m-AMSA), and in the present disclosure, one or more selected from these may be included as the nucleic acid decomposition promoter.

[0037] When a substance that inhibits nucleic acid amplification is used as a labeling agent, the live / dead information of each cell can be obtained from the presence or absence of amplification of nucleic acid derived from each cell. The presence or absence of amplification of nucleic acid can be detected, for example, using a nucleic acid-binding fluorescent substance that can detect the amplification of nucleic acid. Examples of nucleic acid-binding fluorescent substances include EvaGreen, SYBR Green, SYBR Gold, SYTO9, SYTO10, Hoechst 33342, 4',6-diamidino-2-phenylindole, Acridine Orange (AO), Promidium iodide, Ethidium homodimer-2, etc., and in the present disclosure, one or more selected from them may be included as a nucleic acid-binding fluorescent substance.

[0038] (Single cell analysis) In one aspect of the present disclosure, a feature of the method for analyzing the composition of a cell population is that information (live / dead information and nucleic acid information) for each individual cell can be obtained. This can be achieved by analyzing each individual cell using a method described in detail below. Although the amount of nucleic acid of a certain sequence can be specified from information on nucleic acids derived from multiple cells, the absolute amount of a specific type of cell cannot be measured because there is a difference in copy number for each type of cell (e.g., species of microorganism). However, the absolute amount of a specific type of cell can be measured based on information on nucleic acids derived from each individual cell. In addition, in the present disclosure, it is possible to measure the absolute amount, number, ratio, etc. of live cells among a specific type of cells. In particular, since the amount of nucleic acid derived from microorganisms per cell is small, even when nucleic acids derived from multiple cells are analyzed in a mixed state, it is common to use an amplification reaction. However, it is known that even when attempting to amplify the nucleic acid as a whole, such as by using random primers, the degree of amplification for each sequence is biased due to GC content, etc. When bias occurs in amplification, it is difficult to measure the relative amount of a specific type of microorganism in the microflora.

[0039] In the cell-by-cell analysis, the cells of the population of cells may be encapsulated in a gel capsule one by one. The step of encapsulating the cells of the population of cells in a gel capsule one by one may include the steps of encapsulating the cells of the population of cells in a liquid droplet one by one and gelling the droplet to generate a gel capsule.

[0040] Thus, the systems, kits, compositions, etc. of the present disclosure may include means, compositions, agents, kits, systems, etc. for encapsulating individual cells in a gel capsule.

[0041] In the present disclosure, information on each nucleic acid derived from each cell may be obtained by any method, but in order to easily obtain information on each nucleic acid derived from each cell from a large number of cells, it may be preferable to obtain amplified nucleic acids derived from each cell by a method including the steps of using a sample containing a microbiota, encapsulating cells one by one in a droplet, gelling the droplet to generate a gel capsule, immersing the gel capsule in one or more lysis reagents to lyse the cells, and contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule. In one embodiment of the present disclosure, the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule can also amplify the polynucleotide while maintaining the gel state in the gel capsule.

[0042] In one embodiment, the droplets can be created by flowing a single cell through a microchannel and encapsulating the single cell by shearing the suspension with oil. In some embodiments, the gel capsule can be a hydrogel capsule.

[0043] Materials for gel capsules include agarose, acrylamide, photocurable resin (e.g., PEG-DA), PEG, gelatin, sodium alginate, and Matrigel. TM , collagen, etc. The gelation of the droplets can be achieved by forming the droplets so as to contain the gel capsule material and cooling the droplets. Alternatively, gelation can be achieved by applying a stimulus such as light to the droplets. The gel capsule material can be included in the droplets, for example, by including the gel capsule material in a suspension of cells or cell-like structures.

[0044] The gel capsule may be a hydrogel capsule. As used herein, "hydrogel" refers to a water solvent or dispersion medium held by a network of polymeric materials or colloidal particles.

[0045] The disclosed method may include amplifying nucleic acid from each cell of a population of cells. Thus, the disclosed system, kit, composition, etc. may include a nucleic acid amplification agent that amplifies nucleic acid. The amplification step may include immersing the gel capsule in one or more lysis reagents to lyse the cells, and polynucleotides containing the genomic DNA of the cells or parts thereof are dissolved in the gel capsule and retained in the gel capsule.

[0046] The lysis reagent may be at least one selected from the group consisting of lysozyme, labiase, yatalase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolysin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride, urea, 2-mercaptoethanol, dithiothreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octylglucoside, octylthioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, and Zwittergent 3-12.

[0047] When amplifying or analyzing nucleic acids on a cell-by-cell basis for various microorganisms, it is desirable to use a lysis reagent or combination of lysis reagents that are relatively strong, for example, Gram-positive bacteria have cell walls with a thick peptidoglycan layer, so that cells may not be sufficiently lysed with only a mild lysis reagent.

[0048] If necessary, a step of removing impurities from the gel capsule after the cells are lysed may be included. Since impurities remaining in the gel capsule after the lysis operation inhibit the subsequent polynucleotide amplification, it may be desirable to pass a suitable washing solution through the gel capsule and release the inhibitory substances from the gel capsule. In order to complete these operations in the gel capsule, it may be desirable to have a hydrogel structure that achieves the penetration and release of various drug solutions and cell lysates while retaining the polynucleotide in the gel capsule. By using the gel capsule, the remaining reagents can be diluted while retaining the genetic material. This step can be repeated. By diluting the reagent to a level where inhibition is not generated, downstream operations, such as amplification reactions, can be performed smoothly.

[0049] Contaminants include non-cellular components remaining in the sample, lysed cellular components (e.g., cell membranes, intracellular proteins, polysaccharides), nucleic acid-binding proteins (e.g., histones), lysis reagents, and excess labeling agents. By using gel capsules, contaminants can be removed by immersion in a buffer solution, centrifugation, filtration, etc., while the nucleic acid to be analyzed is retained in the gel capsule. When a labeling agent is added after gel encapsulation, it is preferable to include a washing step to remove free labeling agent (e.g., EMA). In the washing step, the labeling agent bound to the nucleic acid can be maintained in a bound state while proteins that are bound to the nucleic acid and interfere with analysis are removed.

[0050] Furthermore, the amplification step may include a step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule. In this amplification step, information on cell viability may be obtained using the above-mentioned labeling agent or the like. A reaction involving heat treatment (80 degrees or higher) may cause the gel (e.g., agarose gel) to re-dissolve, which may destroy the individualized particle shape and invalidate single cell isolation. In this case, an enzyme reaction at about 60 degrees or lower is preferable in order to maintain the gel droplet shape. Isothermal strand displacement amplification reaction (multiple displacement amplification) is preferable because it can be performed within this temperature range and can amplify the entire genomic DNA. Examples of enzymes used include phi29 polymerase, Bst polymerase, Aac polymerase, and recombinase polymerase. In this case, the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule may also amplify the polynucleotide while maintaining the gel state in the gel capsule.

[0051] When PCR is performed to detect a specific cell (e.g., 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. 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 targeting RNA, the purpose is to determine the type of gene and the expression level absolutely (relatively), so it is possible to read only a part of the gene (several dozen bases) and quantify how much of each gene is expressed. When targeting genomic DNA, since in principle there is only one molecule of genomic DNA in one cell, it may be necessary to increase the sequence information of only that one molecule without missing anything in order to decipher all of the millions of bases. Processing in a gel capsule is advantageous for such amplification. In addition, it is advantageous in single-cell analysis to obtain the nucleic acid for sequencing as a whole, rather than fragmentary information from one cell at a time.

[0052] (analysis) The number of cells that can be targeted in the cell population analysis of the present disclosure is any number of 2 or more, for example, 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, 10,000 or more, 500,000 or more, 1 million or more, 5 million or more, or 10 million or more. The cell population analysis of the present disclosure can use information on each nucleic acid derived from each cell from a larger number of cells than that obtained using a conventional single cell reaction system, for example, a 0.2 mL or 1.5 mL microtube reaction system.

[0053] In the present disclosure, the composition of a cell population can be analyzed by determining the absolute number of each type of cell in the cell population.In addition, in the present disclosure, the live / dead information of each cell can be obtained, so the composition of a cell population can be analyzed by determining the absolute number of each type of cell in the live cells in the cell population.From a sample containing amplified nucleic acid from each cell in the cell population, the genome sequence data of each cell can be obtained, and this can be performed in a live cell specific manner.

[0054] In the present disclosure, prior to analysis of the total sequence information of each cell (e.g., genome sequencing), nucleic acids or other biomolecular structures or sequences prepared in parallel from various cells may be detected and selected in an individual-specific manner by referring to the structures or sequences. That is, the method may include a step of selecting a sample containing amplified nucleic acid to be analyzed from samples containing amplified nucleic acid derived from each cell. Analysis of the amplified nucleic acid may include identifying the type of each cell. Selection may be performed using cell viability information.

[0055] In one embodiment, selection can be based on the presence or absence of a specific gene sequence, the yield of a specific gene, or the total nucleic acid yield. In some embodiments, selection can be based on the presence or absence of a specific gene sequence. In some embodiments, selection can be based on the yield of a specific gene being higher or lower than a reference yield. In some embodiments, selection can be based on the total nucleic acid yield being higher or lower than a reference yield.

[0056] In certain embodiments, the presence or absence of a specific gene sequence is detected by a means selected from the group consisting of a reagent that specifically detects a specific gene sequence, agarose gel electrophoresis, microchip electrophoresis, PCR, qPCR, and gene sequencing (Sanger sequencing, NGS).In some embodiments, the reagent that specifically detects a specific gene sequence includes an antibody, a probe, a DNA-binding fluorescent dye, and a fluorescent dye-binding nucleotide.

[0057] In certain embodiments, specific gene yield or total nucleic acid yield can be measured by absorbance measurement, fluorometry, agarose gel electrophoresis, microchip electrophoresis.The method can include detecting nucleic acid having specific sequence in a sample containing amplified nucleic acid from each cell.Detecting nucleic acid having specific sequence can include amplifying and sequencing nucleic acid having specific sequence.

[0058] The method of the present disclosure may include a step of obtaining genome sequence data of each cell in a cell population from a sample containing amplified nucleic acid derived from each cell. By obtaining genome sequence data, it is possible to obtain not only sequence information but also information on the function of the sequence for each cell in the cell population.

[0059] The system, kit, composition, etc. of the present disclosure may include a means for obtaining nucleic acid sequence data. This means may be any means (e.g., a kit, a drug, etc.) that can obtain genome sequence data of each cell in a cell population from a sample containing amplified nucleic acid derived from the cell. After obtaining genome sequence data, these means may provide a function for obtaining information on the function of the sequence, not just information on the sequence, for each cell in the cell population.

[0060] It is possible to select genome sequence data to analyze from the genome sequence data of each cell. Genomic sequence data contains a large amount of information, so limiting the amount to be processed leads to reductions in labor and time.

[0061] The selection may include evaluating the presence or absence of a specific gene sequence and / or the identity with a specific gene sequence. In some embodiments, the identity with a specific gene sequence can be evaluated by using BLAST or the like. In certain embodiments, the selection may be based on the presence or absence of a specific gene sequence, and the nucleic acid information may be sorted for each cell. In other embodiments, the selection may be based on the identity between a specific gene sequence and nucleic acid information derived from two or more cells, and the nucleic acid information may be sorted for each cell. In some embodiments, the selection may be performed when the identity is equal to or greater than a certain level, or when the identity is equal to or less than a certain level. In certain embodiments, the identity may be 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. In other embodiments, the identity may be 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%.

[0062] (Cell population) In the present disclosure, the cell population may be a population of animal or plant somatic or reproductive cells, a population of microbial cells, or a combination thereof. The cell or cell-like structure of interest in the present disclosure is not particularly limited, but may include, for example, microorganisms (e.g., bacteria, fungi, unicellular animals), and cells of multicellular organisms (e.g., somatic cells, reproductive cells, cultured cells, tumor cells, animal cells, and plant cells). In the present disclosure, the cell population may be a microbiota or a microbial preparation.

[0063] Microorganisms that may be the subject of the present disclosure include, but are not limited to, eubacteria, Escherichia coli, Bacillus subtilis, cyanobacteria, cocci, bacilli, spiral bacteria, gram-negative bacteria, gram-positive bacteria, archaea, fungi, individual cells of multicellular organisms, or any combination thereof. An example of a microbiome is a bacterial flora. Since a microbiome may include multiple microorganisms with different cellular properties (e.g., the presence or absence of a cell wall, etc.), it may be preferable in the analysis of the present disclosure to employ a means that can obtain nucleic acid information for each cell or amplified nucleic acid for each cell in the same manner regardless of the microbial species.

[0064] The microbiota may be, but is not limited to, the intestinal microbiota, the skin microbiota, the oral microbiota, the nasal microbiota, the vaginal microbiota, the soil microbiota, the rhizosphere microbiota, the river / seawater microbiota, the activated sludge microbiota, the insect symbiotic microbiota, the animal symbiotic microbiota, etc. The intestinal microbiota has been widely studied for its influence on human health, and is one of the preferred subjects for analysis in the present disclosure.

[0065] In the present disclosure, it is possible to perform analysis by further combining information from analyses of nucleic acid sequence information derived from the same microflora, but not from individual cells. An example of such analysis is metagenomic analysis. Metagenomic analysis is a method of extracting and collecting the nucleic acids, genes, and DNA of microorganisms in the environment as a whole, without going through the process of culturing, and comprehensively examining their structures (base sequences). Although it is not known which microorganism each individual nucleic acid or gene originates from, it is possible to obtain information about the gene group of a collection (community) of microorganisms in the environment, and this type of method is called metagenomic analysis.

[0066] The evaluation of the microbiota composition can include determining the absolute number of each type of microorganism in the microbiota.The absolute number of each type of microorganism can be determined by identifying the microbial species for each amplified nucleic acid from each cell.The identification of the microbial species can be performed, for example, by identifying the presence or absence of a specific gene sequence.

[0067] When genome sequence data is available, the evaluation of the microbiota composition may be performed by comparing long gene sequences in each microorganism. The long sequence may be, for example, a gene sequence extracted from de novo assembly data (e.g., a 16S gene sequence or a set of common genes). Such a comparison of long gene sequences may not only increase the accuracy of the evaluation, but may also enable the evaluation of functions in the microbiota. For example, even if it is known that multiple species exist, the function of the entire microbiota cannot be understood without individual information on the function of each species based on sequence information alone. However, based on gene information, information can be obtained about the amount of species that may have a specific activity (e.g., enzyme activity) in the microbiota.

[0068] (application) The present disclosure can be used, for example, in the quality evaluation of FMT (fecal microbiota transplant) samples. Evaluations such as bacterial flora analysis and viable bacterial count evaluation may be required to ensure the stable supply and quality of FMT samples. In FMT samples, the bacterial flora varies from sample to sample, and the bacteria and number of bacteria investigated vary. The technology disclosed herein can be applied even if the composition ratio and sequence information of the target species are unknown, and is therefore considered to have technical advantages over conventional specific bacterial culture methods and observation methods.

[0069] The present disclosure may be used, for example, in the quality evaluation of microbial preparations. Evaluations such as bacterial flora analysis and viable cell count evaluation may be required for the quality assurance of the manufacturing quality, stable supply, and administration of microbial preparations. Microbial preparations are formulated as a cocktail of multiple types of bacteria. When it is necessary to distinguish closely related species of bacteria, DNA sequence information is required because microscopic observation is not possible. In addition, there is a risk that the expected properties will not be obtained due to the accumulation of mutations associated with long-term cultivation and production. It is believed that the technology disclosed herein can be effectively used in terms of lot management between preparations, as it can perform genome-level analysis of various bacterial species.

[0070] The present disclosure can be used, for example, in food microbiology testing. A general viable count (viable count) is a sanitation indicator bacterium (contamination indicator bacterium) for evaluating the hygienic quality of food. However, the official method, which requires culture, takes time and can only target specific bacteria. It is believed that the technology of the present disclosure can be advantageously applied when high-precision analysis is required.

[0071] The present disclosure can be used, for example, to investigate viable bacteria in the environment. Environmental pollution caused by pathogens in the environment can be investigated, and examples of such pathogens include Legionella, O-157, and multidrug-resistant bacteria. Official methods that require culture are time-consuming and can only target specific bacteria. It is believed that the technology of the present disclosure can be advantageously applied when high-precision analysis is required. For example, it is possible to identify and evaluate the number of multidrug-resistant bacteria that exist as viable bacteria in the environment. In addition, it is possible to identify bacteria that are active in various environments in the environment, such as the intestines, soil, and plant rhizosphere. The technology of the present disclosure is effective in specifically detecting viable bacteria that are thought to contribute particularly to the host in good soil, disease-resistant plant rhizosphere, etc., and in preferentially acquiring genome sequence information.

[0072] The methods disclosed herein can be used, for example, to evaluate the quality of microbial-derived feed and fermented foods, microbial-containing supplements, profiling of viable bacteria in activated sludge, evaluating the presence of viable microorganisms in the environment, evaluating the efficacy and spectrum of antimicrobial agents, evaluating sterilization methods, and profiling of viable bacteria in biofilms.

[0073] (system) In another aspect of the present disclosure, a system for analyzing cell population composition may be provided, which may comprise means for implementing a method or steps thereof comprising any of the features described elsewhere herein.

[0074] The system may include an apparatus for amplifying polynucleotides in cells. The apparatus may be capable of amplifying polynucleotides in cells at the single cell level, among others. The apparatus may include a droplet preparation unit for encapsulating cells in droplets one cell or structure at a time; a gel capsule preparation unit for gelling the droplets to produce gel capsules; a labeling unit for adding a labeling agent to cells or gel capsules containing cells to label them; a lysis reagent immersion unit for immersing the gel capsules in a lysis reagent; a removal unit for removing impurities from the gel capsules; and / or an amplification reagent immersion unit for immersing the gel capsules in an amplification reagent. The system or apparatus may further include a selection unit for selecting the gel capsules and storing the gel capsules in a storage container. The system or apparatus may include a detection unit for detecting the label to obtain information on the live or dead state of the cells.

[0075] The system or device may include, as necessary, reagents such as a medium for encapsulating cells, a gel capsule material, a labeling agent, a nucleic acid decomposition promoter, a lysis reagent, an amplification reagent, a nucleic acid-binding fluorescent substance capable of detecting the amplification of a nucleic acid, a reagent used for nucleic acid sequencing (e.g., a polymerase, a primer set (which may include a barcode sequence), etc.). In addition to those described elsewhere herein, reagents known in the art may be used.

[0076] The device or system may further include a sequencing unit for sequencing the nucleic acid sequence in the polynucleotide amplified in the amplification reagent immersion unit. The sequencing unit may be provided integrally with the device or as a separate device in the system. The sequencing unit may be an instrument for performing Sanger sequencing, Maxam-Gilbert sequencing, single molecule real-time sequencing (e.g., Pacific Biosciences, Menlo Park, California), ion semiconductor sequencing (e.g., Ion Torrent, South San Francisco, California), sequencing by synthesis, pyrosequencing (e.g., 454, Branford, Connecticut), sequencing by ligation (e.g., SOLiD sequencing, Life Technologies, Carlsbad, California), sequencing by synthesis and reversible terminator (e.g., Illumina, San Diego, California), nucleic acid imaging techniques such as transmission electron microscopy, nanopore sequencing, and the like.

[0077] The system or device may include a means for detecting and measuring the amplified genes. For example, a flow cytometry device suitable for handling the shape of gel capsules may be provided integrally with the device or as a separate device in the system. The means for detecting and measuring the amplified genes may include a means for performing a detection reaction (e.g., a thermal cycler and appropriate reagents) and / or a means for detecting a signal (a light sensor, a camera, and appropriate analytical means). The means for detecting and measuring the amplified genes may also function as a detection unit for detecting the label to obtain information on the viability of the cells.

[0078] The system or device may include a calculation unit that may be configured to perform any information processing described elsewhere herein. The calculation unit may be provided integrally with the device, or may be provided as a separate device (computer) in the system. In another aspect of the present disclosure, a program for performing the information processing described elsewhere herein in the calculation unit and implementing the method of the present disclosure and a storage medium having the program recorded thereon may also be provided. The calculation unit may include such a program and / or a storage medium having the program recorded thereon, as necessary.

[0079] (kit) One aspect of the present disclosure provides a kit that can be used in the method of the present disclosure. In the present disclosure, a kit for analyzing cell population composition can be provided. The kit can include a gel capsule material, and the use of the gel capsule is advantageous for amplifying nucleic acids in cells or cell-like structures at a single cell level as described elsewhere herein, and can be used as described herein for analyzing microbial composition. The kit can include, for example, a gel capsule material and, if necessary, one or more reagents. As the reagent, in addition to those described elsewhere herein, those known in the art may be used. The kit can include a labeling agent, a nucleic acid decomposition promoter, and a nucleic acid-binding fluorescent substance capable of detecting the amplification of nucleic acid, as described herein or known in the art.

[0080] A kit for analyzing microbiota composition may include a lysis reagent. The lysis reagent may comprise at least one selected from the group consisting of lysozyme, labiase, yatalase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolysin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride, urea, 2-mercaptoethanol, dithiothreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, Zwittergent 3-12. The lysis reagent is useful for obtaining amplified polynucleotides at the single cell level, particularly genome-wide amplification products.

[0081] The kit may include reagents for amplifying nucleic acids. Examples of the reagents for amplification include polymerase, a primer set (which may include a barcode sequence), a base mix, and a suitable buffer. The kit may include reagents used for sequencing nucleic acids (e.g., polymerase, a primer set (which may include a barcode sequence), and the like). For example, the kit may include reagents for amplifying and decoding specific genes in Sanger sequencing or NGS (polymerase, a primer set (which may include a barcode sequence), and others). The kit may also include reagents for detecting and measuring specific sequences, such as nucleic acid binding dyes and fluorescently labeled probes. Using these reagents, the presence or absence of specific sequences can be measured using an instrument (such as a flow cytometer) that detects and measures amplified genes.

[0082] The kit may include a means for collecting a sample. The kit may also include a means for storing the collected sample. Examples of means for collecting a sample include a syringe, a swab, a biopsy punch, a urine collection container, a stool collection container, a saliva collection container, and medical tape. Examples of means for storing the sample include a cooling agent (e.g., an ice pack, dry ice, liquid nitrogen) and a preservation solution (e.g., a solution containing any of guanidine hydrochloride, ammonium sulfate, ethanol, etc.).

[0083] In one aspect of the present disclosure, a kit for analyzing cell population composition may be provided that includes a specimen collection container containing a preservation solution. The preservation solution may include, for example, a guanidine solution (e.g., FS-0007 or FS-0008, Techno Suruga Lab) or ethanol (e.g., OMR-200 or OM-501, DNA genotek). By using a kit containing such a preservation solution, even if the sample is stored at room temperature for a certain period of time in a hospital facility or the like, effects such as a good genome sequencing rate (completion rate) and identification of many biological lineages in the sample can be achieved in subsequent single cell level analysis. In addition, the convenience is remarkably high compared to the commonly performed method of collecting and preserving stool as frozen stool.

[0084] A preservation solution is generally used to enhance the stability of nucleic acids. For example, a guanidine solution and alcohols are used to cause protein denaturation and inhibit the action of enzymes that promote the decomposition of nucleic acids. In the method of the present disclosure, it may be preferable to maintain the morphological stability of cells in the preservation of samples. This is because cells that are broken down are difficult to encapsulate in droplets, or because frequent occurrence of cell debris makes it difficult to obtain data. Therefore, it was expected that a preservation solution that causes protein denaturation would be disadvantageous to the stability of cell morphology, but in the examples of the present specification, it was unexpectedly found that many of the cells maintained their morphology well after storage in the preservation solution, and that this is particularly suitable for the method of the present disclosure.

[0085] (Preferred embodiment) The preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments can be used alone or in combination.

[0086] A reagent containing a selective membrane-permeable dye (e.g., EMA) is added to the sample. Then, EMA and nucleic acid are bound by light irradiation. EMA penetrates into the inside of dead bacteria with damaged membranes and selectively modifies DNA. Unstained DNA from live cells is amplified by PCR, but DNA from dead cells bound to EMA cannot be amplified. A sample is prepared by removing the treatment solution containing EMA from the sample, and gel capsules containing amplified and retained polynucleotides derived from a single cell genome are prepared by the method described herein. At this time, the polynucleotides are selectively amplified and prepared in the gel capsules containing live bacteria. The gel capsules are stained with a DNA-binding fluorescent dye or the like, and fluorescent-positive capsules are counted by flow cytometry. The number of fluorescent-positive capsules in a certain volume corresponds to the total number of live bacteria introduced.

[0087] Next, the gel capsules are separated and collected on a microplate or the like for each cell using the method described herein. Furthermore, a whole genome amplification reaction is carried out in the plate using the polynucleotides in each gel capsule as a template, and this is used as a library master plate. Next, a portion of the reaction solution in the library master plate is taken out to prepare a replica plate. Using this replica as a template, amplification is carried out with a primer set specific to the target gene. Next, the sequence of the amplified product is identified by Sanger sequencing or the like, and the microbial species of each sample is identified.

[0088] More effectively, the above primer set contains a barcode sequence, and an amplification reaction is performed in which a different barcode is assigned to each sample, and the PCR products from multiple replica plates are pooled and next-generation sequencing is performed. After that, the plate / well number is identified by the barcode sequence, and the sequence of the PCR product is identified, thereby identifying the microbial species of hundreds to thousands of samples at once. The above primer set may be a mixture of primer sets targeting multiple gene regions. More specifically, the primer set is selected from primer sets targeting the v3-v4 and v1-v2 regions of the 16S rRNA gene, and primer sets for distinguishing bacteria, archaea, fungi, etc., such as 18S rRNA and ITS. If multiple types of the above primer sets are used simultaneously, bacteria, archaea, and fungi can be detected simultaneously.

[0089] In addition, when it is difficult to design a primer set for the target gene sequence or to determine the target gene region with high accuracy, a method of searching for the target gene region from the digital sequence information by performing whole genome sequencing is effective. Alternatively, whole genome sequencing may be performed for the purpose of determining the genome of an unknown live bacteria. Whole genome sequencing is performed from a sample on a replica plate, and gene sequences are extracted from the digital sequence information obtained from next-generation sequencing, and data for identifying bacterial species and genes is obtained. Candidates for genes to be searched and extracted include gene regions such as 16S rRNA genes, 18S rRNA genes, and ITS that distinguish bacteria, archaea, fungi, etc., as well as specific enzymes and gene groups involved in secondary metabolite production.

[0090] By using any of the above methods, the microbial species contained in each gel capsule dispensed onto a well plate or the like can be identified and counted, and by combining this with the total number of live bacteria measured initially, the number of live cells of each microbial species in the sample can be counted. In this reaction, all PCR reactions are performed separately, and the microbial species is identified and counted for each barcode, so digital count data reflecting the number of cells can be obtained. In other words, data can be obtained that avoids bias due to the presence or absence of known sequence information, cultivability, and gene copy number, which were problems in the past. In addition, it is possible to compare the actual total number of rare cells with a relative ratio of 1% or less.

[0091] One embodiment of the method of the present disclosure includes the steps of adding EMA to a population of cells and irradiating with light, encapsulating cells of the population of cells one by one in a droplet, gelling the droplet to form a gel capsule, immersing the gel capsule in one or more lysis reagents to lyse the cells, wherein a polynucleotide comprising the genomic DNA of the cell or a portion thereof is dissolved into the gel capsule, removing contaminants from the gel capsule, contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule, wherein polynucleotides bound by EMA are not amplified, detecting the amplified polynucleotides, selecting gel capsules containing a sample derived from a live bacterium, counting gel capsules containing a sample derived from a live bacterium, and analyzing the polynucleotides amplified in the gel capsules containing a sample derived from a live bacterium. Other embodiments of the disclosed methods include a method comprising the steps of encapsulating cells of the cell population one by one in a droplet, gelling the droplet to form a gel capsule, adding EMA to the gel capsule and irradiating with light, washing the gel capsule, lysing the cells by immersing in one or more lysis reagents, wherein a polynucleotide comprising the genomic DNA of the cell or a portion thereof is eluted into the gel capsule, removing contaminants from the gel capsule, contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule, wherein polynucleotides bound by EMA are not amplified, detecting the amplified polynucleotides, selecting gel capsules comprising a sample derived from a live bacterium, counting gel capsules comprising a sample derived from a live bacterium, and analyzing the polynucleotides amplified in the gel capsules comprising a sample derived from a live bacterium. It will be understood by those skilled in the art that only a portion of the steps in these methods may be used as appropriate, and that equivalent techniques described herein or known in the art may be substituted or additionally used, so long as the object of this disclosure is achieved. (Other embodiments)

[0092] In the present disclosure, a system for analyzing the composition of a cell population may be provided. The system may include a means for obtaining live / dead information for each cell in the cell population, and / or a means for analyzing amplified nucleic acid from each cell in the cell population. In an embodiment of the present disclosure, a system or kit for analyzing the composition of a cell population may be provided, and the system or kit may include a labeling agent that differentially labels live cells and dead cells, a means for encapsulating cells in a gel capsule, a means for amplifying nucleic acid, and / or a means for analyzing nucleic acid. In the present disclosure, a composition including a labeling agent that differentially labels live cells and dead cells may be provided, and the composition may be for use in a method for analyzing the composition of a cell population. The method may include a step of obtaining live / dead information for each cell in the cell population, and a step of analyzing amplified nucleic acid from each cell in the cell population. In the present disclosure, a composition for use in a method for encapsulating and analyzing a cell population in a gel capsule for each cell may also be provided, the composition including a labeling agent that differentially labels live cells and dead cells.

[0093] In the present disclosure, a system or kit is provided, which may include a reagent for distinguishing between live and dead cells (e.g., ethidium monoazide (EMA) or propidium monoazide (PMA)), a light irradiation means, a means for encapsulating cells one by one in a droplet, a means for gelling the droplet to generate a gel capsule, a lysis reagent for the gel capsule, which is used to add the lysis reagent so that a polynucleotide containing the genomic DNA of the cell or a portion thereof is eluted into the gel capsule, a means for removing contaminants from the gel capsule, a reagent for amplifying a polynucleotide, which is used to contact the polynucleotide to amplify the polynucleotide in the gel capsule but not to amplify a polynucleotide bound to EMA, a means for detecting a polynucleotide, a means for selecting gel capsules containing a sample derived from a live bacterium, a means for counting gel capsules containing a sample derived from a live bacterium, and / or a means for analyzing the polynucleotide amplified in the gel capsules containing a sample derived from a live bacterium.

[0094] A composition for use in a method for analyzing the composition of a population of cells may be provided, the composition comprising a labeling agent that differentially labels live and dead cells (e.g., a reagent that distinguishes between live and dead cells, such as ethidium monoazide (EMA) or propidium monoazide (PMA)), the method comprising obtaining live / dead information for each cell in the population of cells, and analyzing amplified nucleic acid from each cell in the population of cells.

[0095] The system for analysis of the present disclosure includes a sample providing unit that provides a sample containing amplified nucleic acid derived from each cell in a sample, and a composition evaluating unit that evaluates the composition of the sample from the sample containing amplified nucleic acid derived from each cell in the sample. Here, the sample providing unit includes a droplet encapsulating unit that uses a sample to encapsulate cells in droplets one by one, a gel capsule generating unit that gels the droplets to generate gel capsules, and a cell lysis unit that immerses the gel capsules, which contain one or more lysis reagents for lysing the cells, in one or more lysis reagents to lyse the cells, the cell lysis unit being configured so that a polynucleotide containing the genomic DNA of the cell or a portion thereof is dissolved in the gel capsule and is retained in the gel capsule in a state in which a substance binding to the genomic DNA or a portion thereof has been removed, and a polynucleotide amplification reagent for amplifying the polynucleotide in the gel capsule.

[0096] All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth.

[0097] 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 illustrative purposes only and are not provided 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 this specification, but is limited only by the scope of the claims. EXAMPLES

[0098] Examples of the present disclosure are described below. The specific reagents that can be used are those described in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC, etc.) can also be used.

[0099] (Example 1: Experimental Example) A culture solution of E. coli K12 strain is prepared, and a model sample of dead bacteria is prepared by heat treatment (95°C, 5 min) or alcohol treatment (suspension in 70% ethanol). An untreated (live bacteria) sample, a model sample of dead bacteria, and a mixed sample of live bacteria and dead bacteria are prepared, and the bacterial pellet is suspended in 500 μL of phosphate-buffered saline (PBS) and processed according to the protocol using Takara Bio's Viable Bacteria Selection Kit for PCR, including the addition of EMA, infiltration, and light irradiation. The bacteria are then collected by centrifugation at 15,000 × g for 3 minutes. The bacterial pellet is centrifuged twice with PBS for centrifugation and washed, and then suspended in PBS to obtain an E. coli suspension. The cell concentration in the prepared cell suspension is measured, and ultra-low melting point agarose is added to a final concentration of 1.5%, to prepare the E. coli suspension to be used for gel capsule production (final cell concentration: 1.5 × 10 3 cells / μL).

[0100] Escherichia coli is encapsulated in microdroplets, the microdroplets are gelled, and whole genome amplification is carried out within the gel capsule as follows.

[0101] Using a microchannel made by ourselves using polydimethylsiloxane (Sylgard 184: Dow Corning), microdroplets are produced and E. coli is encapsulated in the microdroplets. In this example, a microchannel consisting of a first channel, a second channel, a third channel, and a fourth channel is used, with adjacent channels arranged at right angles, but it is also possible to use a microchannel connected in a roughly T-shape. In this example, a microchannel with a width of 34 μm and a height of 50 μm is used, but the size of the microchannel can be changed as appropriate depending on the size of the microdroplets to be produced and the size of a single cell to be encapsulated.

[0102] Next, an E. coli suspension is introduced from the first flow path (aqueous phase inlet), and Pico-Surf1 (2% in Novec7500) (Sphere Fluidics) (hereinafter referred to as "oil") is introduced from the second and fourth flow paths (oil phase inlets) to shear the enteric microorganism suspension, producing microdroplets with a diameter of 50 μm, which are then run through the third flow path 7 and collected in a tube with a capacity of 0.2 mL. Approximately 450,000 microdroplets are produced at a rate of 500 droplets / second. The cell concentration in the microdroplets is 0.1 cells / droplet.

[0103] In this embodiment, the diameter of the microdroplets is uniformly set to 50 μm, so that cells are easily encapsulated in the microdroplets one by one. Considering the size of a single cell, the diameter of the microdroplets is, for example, 1 to 250 μm, and preferably 20 to 200 μm. The diameter of the droplets may be about 1 to 250 μm, more preferably about 10 to 200 μm, and for example, the diameter of the droplets may be 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 50 μm, about 80 μm, about 100 μm, about 150 μm, about 200 μm, or about 250 μm.

[0104] A number of microdroplets and oil are contained in the tube, and since the microdroplets have a lower specific gravity than the oil, they accumulate in the upper layer.

[0105] Next, the tube is cooled on ice for 15 minutes, and the microdroplets are gelled with ultra-low melting point agarose. The gelled microdroplets are gel capsules. Since the diameter of the microdroplets is 50 μm, the diameter of the gel capsule is also 50 μm. The diameter of the gel capsule may be about 1 to 250 μm, more preferably about 10 to 200 μm, for example, 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 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 droplets to be prepared, but the diameter may change during gelling. In addition, the diameter of the gel capsule is preferably 1 to 250 μm. By making the diameter of the gel capsules uniform, the rate of penetration of the lysis reagent, which will be described later, into each gel capsule can be made more uniform.

[0106] Next, 20 μL of 1H,1H,2H,2H-perfluoro-1-octanol (SIGMA-ALDRICH) is added to the tube, and the oil in the lower layer is removed. Then, acetone (FUJIFILM Wako Pure Chemical Industries, Ltd.) (500 μL) and isopropanol (FUJIFILM Wako Pure Chemical Industries, Ltd.) (500 μL) are added in that order, and centrifugal washing is performed to remove the oil. In this example, the oil that has permeated the gel capsules is also included in the impurities. Furthermore, 500 μL of PBS is added and centrifugal washing is performed three times, and the gel capsules are suspended in the aqueous layer (PBS). The gel capsules accumulate in the lower layer because they have a higher specific gravity than the aqueous layer.

[0107] Next, the gel capsules are successively immersed in a bacteriolysis reagent as a dissolution reagent, and the parts of the cells other than the target material to be collected, such as the cell walls, are dissolved inside the gel capsule, and genomic DNA is eluted into the gel capsule.

[0108] Specifically, lysozyme (10 U / μL) (R1804M, Epicentre), a type of lysis reagent, is added to the tube to lyse the cells. Next, achromopeptidase (850 U / mL) (015-09951, Fujifilm Wako Pure Chemical) is added to the tube, a type of lysis reagent. Next, protease K (1 mg / mL) (MC5005, Promega) and sodium dodecyl sulfate (SDS) 0.5% (71736-100ML, SIGMA-ALDRICH), a type of lysis reagent, are added to the tube to lyse the cells, and then centrifugal washing is performed five times to remove components (contaminants) other than the protease and the genomic DNA of the lysed cells from the tube. Next, the gel capsule is immersed in Buffer D2 (QIAGEN), an aqueous solution containing potassium hydroxide, a type of lysis reagent, to dissolve the remaining components and denature the genomic DNA. The lysis reagent used in this example is lysozyme, achromopeptidase, protease K, sodium dodecyl sulfate, and Buffer D2, as described above. Potassium hydroxide is also used in the normal DNA amplification reaction process, but since it also has a lysis effect, it was used as one of the lysis reagents in this example. Since the gel capsule is immersed in the lysis reagent for a short time, the eluted genomic DNA is not shed outside the gel capsule by the lysis reagent, but is retained within the gel capsule. In this example, the lysis reagent that has permeated the gel capsule is also included in the impurities.

[0109] In this example, lysozyme, achromopeptidase, and protease K are added in sequence, sodium dodecyl sulfate is added to lyse the cells, and then centrifugation is performed only before adding Buffer D2. This allows for a sufficient washing effect. However, centrifugation may be performed after the cells are lysed with each lysis reagent.

[0110] In this way, the target genomic DNA can be collected by lysing the cells using multiple types of lysis reagents, and by immersing the cells in the lysis reagents and then centrifugal washing, contaminants such as components other than the polynucleotides of the lysis reagents and the lysed cells can be removed, allowing the genomic DNA to be purified without inhibiting the subsequent genomic DNA amplification reaction.

[0111] Amplification reagent is added to a tube containing a gel capsule that holds genomic DNA denatured in potassium hydroxide solution (Buffer D2), and the gel capsule is immersed in the amplification reagent. Specifically, the MDA (Multiple Displacement Amplification) method using phi29 DNA polymerase, a strand-displacement DNA polymerase, is used. Here, the gel capsule is immersed in the whole genome amplification reaction reagent REPLI-g Single Cell Kit (QIAGEN), and a whole genome amplification reaction is performed for 3 hours (S1000 Thermal Cycler, Bio-Rad). The amplification reagent (REPLI-g Single Cell Kit) contains a component that neutralizes the potassium hydroxide solution (Buffer D2).

[0112] Furthermore, the gel capsules were stained with a fluorescent DNA intercalator, and fluorescent gel capsules were counted and collected individually using a microscope and a flow cytometer. As a result, no fluorescent gel capsules were observed in the dead bacteria model sample, and about half of the untreated (live bacteria) samples in the mixed sample showed a fluorescent signal. This ratio corresponds to the mixture ratio of live bacteria to dead bacteria.

[0113] After dissolving the gel capsules by heating the individual gel capsules at 65 degrees, secondary amplification by the MDA method is performed in the wells of each plate to create a library master plate containing 10 μL of DNA amplified product per well. Dispense 39 μL of nuclease-free water into each well of a new plate, add 1 μL of the DNA amplified product in the library master plate, and prepare a 40-fold diluted solution of the library master plate. Next, 1 μL of the diluted solution is used to quantify the DNA concentration using a Qubit fluorometer (Thermo Fisher Scientific). In addition, PCR is performed targeting the V3V4 region of the 16S rRNA gene using 1 μL of the diluted solution as a template. After confirming the presence or absence of PCR products by agarose gel electrophoresis, sequence analysis is performed using the Sanger method on some of the samples in which amplification was observed. A homology search is performed using BLAST on the obtained sequence information, and the 16S rRNA sequence derived from E. coli is confirmed as expected from the created library master plate.

[0114] (Example 2: Acquisition and sequence of vitality information for each gel capsule) A culture solution of E. coli K12 strain was prepared, and a killed bacteria model sample was prepared by heat treatment (95°C, 5 min). The bacterial pellet was suspended in 500 μL of phosphate-buffered saline (PBS). A portion was treated according to the protocol using Takara Bio's Viable Bacteria Selection Kit for PCR, and the treatments included the addition of EMA, infiltration, and light irradiation. The bacteria were then collected by centrifugation at 15,000 × g for 3 minutes. The bacterial pellet was washed twice with PBS by centrifugation, and then suspended in PBS to obtain an E. coli suspension. The cell concentration in the prepared cell suspension was measured, and ultra-low melting point agarose was added to a final concentration of 1.5%, to prepare an E. coli suspension to be used for gel capsule production (final cell concentration: 1.5 × 10 3 Another portion of the killed bacteria model sample was prepared as a suspension of the same concentration without EMA treatment.

[0115] As described below, E. coli was encapsulated in microdroplets, and gelation of the microdroplets and whole genome amplification in the gel capsules were performed. Using a microchannel made by ourselves using polydimethylsiloxane (Sylgard 184: Dow Corning), microdroplets were produced and E. coli was encapsulated in the microdroplets. In this example, a microchannel was used that consisted of a first channel, a second channel, a third channel, and a fourth channel, and adjacent channels were arranged at right angles. In this example, a microchannel with a width of 34 μm and a height of 50 μm was used.

[0116] Next, an E. coli suspension was introduced from the first flow path (aqueous phase inlet), and Pico-Surf1 (2% in Novec7500) (Sphere Fluidics) (hereinafter referred to as "oil") was introduced from the second and fourth flow paths (oil phase inlets) to shear the intestinal microorganism suspension, producing microdroplets with a diameter of 50 μm, which were then flowed through the third flow path 7 and collected in a tube with a capacity of 0.2 mL. Approximately 450,000 microdroplets were produced at a speed of 500 droplets / second. The cell concentration in the microdroplets was 0.1 cells / droplet. In this example, the diameter of the microdroplets was 50 μm. Next, the tube was cooled on ice for 15 minutes, and the microdroplets were gelled with ultra-low melting point agarose. The gelled microdroplets were gel capsules.

[0117] Next, 20 μL of 1H,1H,2H,2H-perfluoro-1-octanol (SIGMA-ALDRICH) was added to the tube, and the oil in the lower layer was removed. Then, acetone (FUJIFILM Wako Pure Chemical Industries, Ltd.) (500 μL) and isopropanol (FUJIFILM Wako Pure Chemical Industries, Ltd.) (500 μL) were added in that order, and the tube was centrifuged to remove the oil. In this example, the oil that had permeated the gel capsules was also included in the impurities. Furthermore, 500 μL of PBS was added, and centrifugal washing was performed three times, leaving the gel capsules suspended in the aqueous layer (PBS). The gel capsules have a higher specific gravity than the aqueous layer, so they accumulate in the lower layer.

[0118] Next, the gel capsules were sequentially immersed in a lysis reagent as a lysis reagent, and the parts of the cells other than the collected object such as the cell walls were dissolved inside the gel capsule, and the genomic DNA was eluted into the gel capsule. Specifically, lysozyme (10 U / μL) (R1804M, Epicentre), a type of lysis reagent, was added to the tube to lyse the cells. Next, achromopeptidase (850 U / mL) (015-09951, Fujifilm Wako Pure Chemicals), a type of lysis reagent, was added to the tube. Next, protease K (1 mg / mL) (MC5005, Promega) and sodium dodecyl sulfate (SDS) 0.5% (71736-100ML, SIGMA-ALDRICH), a type of lysis reagent, were added to the tube to lyse the cells, and then centrifugal washing was performed five times to remove components (contaminants) other than the protease and the genomic DNA of the lysed cells from the tube. Next, the gel capsule was immersed in Buffer D2 (QIAGEN), an aqueous solution containing potassium hydroxide, a type of lysis reagent, to dissolve remaining components and denature the genomic DNA.

[0119] Amplification reagent was added to a tube containing a gel capsule that holds genomic DNA denatured in potassium hydroxide solution (Buffer D2), and the gel capsule was immersed in the amplification reagent. Specifically, the MDA (Multiple Displacement Amplification) method using phi29 DNA polymerase, a strand-displacement DNA polymerase, was used. Here, the gel capsule was immersed in the whole genome amplification reaction reagent REPLI-g Single Cell Kit (QIAGEN), and a whole genome amplification reaction was performed for 3 hours (S1000 Thermal Cycler, Bio-Rad). The amplification reagent (REPLI-g Single Cell Kit) contained a component that neutralized the potassium hydroxide solution (Buffer D2).

[0120] In addition, the gel capsules were stained with a fluorescent DNA intercalator, and fluorescent gel capsules were counted and individually collected using a microscope and a flow cytometer. As a result, no fluorescent gel capsules were observed in the dead bacteria model sample (Figure 2).

[0121] As described above, live and dead bacteria are distinguished from a population in which live and dead bacteria are mixed, and then the number of cells and sequence information derived from live bacteria are obtained. Specifically, among the gel capsules obtained as described above, gel capsules that emit fluorescence indicating live bacteria are treated according to the procedure shown in Example 1, and a part or all of the genome amplification products obtained from the gel capsules are sequenced. This makes it possible to selectively obtain sequence information derived only from live bacteria, not including dead bacteria, and evaluate the bacterial flora composition and total number limited to live bacteria from the type and number of cells.

[0122] Sequencing of single-species samples after EMA As described above, fluorescent gel capsules were collected from live E. coli K12 model samples that had been subjected to EMA treatment. Each gel capsule was dissolved by heating at 65°C (S1000 thermal cycler, Bio-Rad), and secondary amplification was performed in each well of the plate by the MDA method (REPLI-g Single Cell Kit, 150345, QIAGEN) to create a library master plate containing 10 μL of DNA amplification product per well.

[0123] For the 12 samples, library preparation was performed using the Nextera XT DNA sample prep kit (Illumina, FC-131-1096), and 2 × 75 bp paired-end reads were obtained by whole genome sequencing using Miseq (Illumina, SY-410-1003). Sequence data were assembled using SPAdes (Bankevich A et al., J Comput Biol. 2012 May;19(5):455-77. http: / / doi.org / 10.1089 / cmb.2012.002 1) to create contigs. In addition, the genome sequencing rate (completion rate) and the degree of contamination were evaluated using CheckM (Parks et al., Genome Res. 2015. 25: 1043-1055, doi:10.1101 / gr.186072.114) under default conditions. As a result, the average completion rate was calculated to be 29.5% and the average contamination rate was calculated to be 0.9% for the EMA-treated live bacteria model. This result suggests that this method can be used to select live bacteria by EMA treatment and obtain genome data.

[0124] Viable bacterial sequencing from multi-species samples A dead bacteria model sample was prepared by heat treatment (95°C, 5 min) using NBRC microbial cocktail (Cell-Mock-001) (a mixed sample of 15 types of bacterial strains). A non-heat-treated (live bacteria) sample and a dead bacteria model sample were prepared, and the bacterial pellet was suspended in 150 μL of phosphate-buffered saline (PBS) and treated with Biotium's PMA 20 mM in H20 according to the protocol, and the process up to EMA addition, infiltration, and light irradiation was performed. The bacteria were then collected by centrifugation at 15,000 × g for 3 minutes. The bacterial pellet was centrifuged twice with PBS for centrifugation and then suspended in PBS to obtain an E. coli suspension. The cell concentration in the prepared cell suspension was measured, and ultra-low melting point agarose was added to a final concentration of 1.5% to prepare an E. coli suspension to be used for gel capsule production (final cell concentration: 1.5 × 10 3Then, the same procedure as in Example 1 was carried out. Afterwards, staining with a photoactive DNA intercalator was performed, and fluorescent gel capsules were counted using a microscope and a flow cytometer, and were collected individually. As a result, no fluorescent gel capsules were observed in the dead bacteria model sample, while fluorescent gel capsules were observed in the live bacteria sample without PMA treatment and the live bacteria sample with PMA treatment. After dissolving the gel capsules by heating the collected gel capsules at 65°C (S1000 thermal cycler, Bio-Rad), secondary amplification was performed in the wells of each plate by the MDA method (REPLI-g Single Cell Kit, 150345, QIAGEN), and a library master plate containing 10 μL of DNA amplified product per well was created. For the above samples, library preparation was performed using the Nextera XT DNA sample prep kit (Illumina, FC-131-1096), and 2 × 75 bp paired-end reads were obtained by whole genome sequencing using Miseq (Illumina, SY-410-1003). Sequencing data was assembled using SPAdes (Bankevich A et al., J Comput Biol. 2012 May;19(5):455-77. http: / / doi.org / 10.1089 / cmb.2012.002 1) to generate contigs. In addition, genome completion rate and contamination were evaluated using CheckM (Parkset al., Genome Res. 2015. 25: 1043-1055, doi:10.1101 / gr.186072.114) under default conditions. As a result of phylogenetic annotation using 16S rRNA sequences or GTDB-tk, 11 bacterial genome data were obtained from the microbial cocktail sample without PMA treatment (analysis of 48 cells), including Acinetobacter radioresistens, Staphylococcus epidermidis, Lactobacillus delbrueckii, Clostridium butyricum, Cutibacterium acnes, Bacillus subtilis, Escherichia coli, Corynebacterium striatum, Comamonas terrigena, and Parabacteroides distasonis, whereas 7 bacterial genome data were obtained from the microbial cocktail sample with PMA treatment (analysis of 96 cells), including Staphylococcus epidermidis, Clostridium butyricum, Bacillus subtilis, Escherichia coli, Bacteroides uniformis, Parabacteroides distasonis, and Comamonas terrigena (Table 1).Acinetobacter radiosistens, Lactobacillus delbrueckii, Cutibacterium acnes, and Corynebacterium striatum were eliminated by PMA treatment during the selection stage of the single-cell amplified genomic library, suggesting that many of them existed as dead bacteria in the microbial cocktail. In addition, Escherichia coli and Comamonas terrigena were detected more frequently in the live bacteria samples than in the other samples, suggesting that many cells existed as live bacteria in the microbial cocktail. [Table 1]

[0125] (Example 3: Application Example) Example of quality assessment of FMT specimens or microbiological preparations In a collection of microorganisms including one or more kinds of microorganisms, such as a fecal microbial transplant (FMT) specimen, intended for the prevention and treatment of a disease, the present invention can be applied to bacterial flora analysis and viable bacterial count evaluation when the purpose is to evaluate the stability (activity and number) of the microorganisms for the stable supply and quality assurance in the transplant specimen. In an FMT specimen, the bacterial flora changes from specimen to specimen, and the investigated bacteria and number of bacteria are different. The disclosed technology can be applied even if the composition ratio and sequence information of the target species are unknown, so it is suitable for bacterial flora analysis and viable bacterial count evaluation. As a specific procedure, the FMT specimen sample is treated according to the procedure shown in Example 1, and a part or all of the genome amplification product obtained from the gel capsule is sequenced to selectively obtain only sequence information derived from the viable bacteria, and the composition and total number of the viable bacteria are evaluated based on the type and number of the bacteria.

[0126] Example of quality evaluation of microbial preparations In a collection of microorganisms including one or more kinds of microorganisms, such as a microbial preparation, intended for the prevention and treatment of disease or for health promotion, the technology of the present invention can be applied to bacterial flora analysis and viable cell count evaluation when the purpose is to evaluate the stability (activity and number) of the microorganisms for the stable supply and quality assurance of the preparation product. In particular, the expected properties may not be obtained due to the accumulation of mutations associated with long-term cultivation and production, but the technology disclosed herein can perform genome-level analysis of various bacterial species even in terms of lot management between preparations. As a specific procedure, the sample is treated according to the procedure shown in Example 1, and a part or all of the genome amplification product obtained from the gel capsule is sequenced to selectively obtain only sequence information derived from viable bacteria, and the composition and total number of viable bacteria can be evaluated from the type and number of the genome amplification product. In addition, the obtained genome sequence is compared with a reference genome to evaluate quality deterioration due to contamination generation during the manufacturing process and accumulation of gene mutations.

[0127] Example of use in food microbiology testing General viable bacteria count (viable bacteria count) is a sanitation indicator bacterium (contamination indicator bacterium) for evaluating the hygienic quality of food. However, the official method, which requires culturing, takes time and can only target specific bacteria. It is believed that the technology disclosed herein can be advantageously applied when high-precision analysis is required. As a specific procedure, the sample is treated according to the procedure shown in Example 1, and a part or all of the genome amplification product obtained from the gel capsule is sequenced to selectively obtain sequence information derived from the viable bacteria, and the number of viable bacteria present, such as coliform bacteria, E. coli, Salmonella, Enterobacteriaceae, Staphylococcus aureus, and Vibrio parahaemolyticus, which are the target organisms of current food microbiology testing, is evaluated from the type and number of the sequence information. In addition, the obtained genome sequence is compared with a reference genome to identify the bacterial species at the strain level.

[0128] Example of use in investigating live bacteria in the environment The technology disclosed herein is considered to be advantageously applicable when the purpose is to identify and specifically detect or obtain genome sequence information of multidrug-resistant bacteria that exist as viable bacteria in the environment, microorganisms that are active in various environments such as the intestines, soil, and plant rhizosphere, microorganisms that form biofilms, or microorganisms that are thought to contribute particularly to the host in good soil, disease-resistant plant rhizosphere, etc. As a specific procedure, the sample is treated according to the procedure shown in Example 1, and a part or all of the genome amplification products obtained from the gel capsule are sequenced to selectively obtain sequence information derived from the viable bacteria, and the composition and total number of the microbial flora limited to the viable bacteria are evaluated from the type and number of the bacteria, and the novelty and genes possessed are evaluated from the genome sequence information.

[0129] (Example 4: When you want to selectively obtain data on cells with specific characteristics from among a variety of cells) When the purpose is to obtain genome data of one or more specific microorganisms that are of interest to a person skilled in the art among animal symbiotic microorganisms such as enterobacteria or marine and soil microorganisms, the presence or absence of gene fragments of the target microorganism in advance in gel capsules containing polynucleotides or amplified nucleic acids recovered from the gel capsules can be confirmed in advance to reduce the acquisition of unnecessary gene sequence data and the associated costs. Examples of measurement targets include comparative analysis of microorganisms of the same lineage (e.g., analysis of subspecies in a microbial lineage related to a disease, etc.), searching for microorganisms having specific genes (e.g., secondary metabolites or enzymes produced by microorganisms), or individually selecting and analyzing bacteria, archaea, fungi, and other eukaryotic cells from among organisms of multiple lineages. In particular, it is expected to specifically detect enterobacteria and oral bacteria that have specific metabolic functions from genes, or to detect subspecies of skin bacteria, for the purpose of evaluating the intestinal environment, oral cavity, and skin environment of the host human.

[0130] (Example 5: When a large amount of host DNA, etc. is present) When the analysis sample is feces, saliva, sputum, swabs from the skin, oral cavity, nasal cavity, ears, genitals, etc., surgical washings, or tissue extracts or blood, and the microorganisms contained in the sample are the subject of analysis, the sample contains many cells, intracellular organelles, and nucleic acids derived from the host animal. Some of these may also be encapsulated inside the gel capsule to perform polynucleotide amplification. For this reason, by confirming in advance the presence or absence of gene fragments of the target microorganism or gene fragments derived from the host in the gel capsule containing the amplified polynucleotide, it is possible to avoid obtaining unnecessary gene sequence data including polynucleotides derived from the host, and to reduce the costs involved. Examples of measurements include the detection of microorganisms from human samples, such as the search for pathogenic microorganisms from blood or sputum, and the analysis of microorganisms coexisting inside tissues.

[0131] Alternatively, by actively analyzing host-derived polynucleotides, this method can be applied to comprehensive analysis of the microbiota.

[0132] For example, it is known that the intestinal flora in the digestive tract and the host animal have a symbiotic relationship in which the host provides an anaerobic environment for the colonization of the gut flora, while the intestinal flora influences the health of the host. For example, the major influences on the host's health include the production of nutrients and the development of the immune system and defense against infectious diseases. Another example is inflammatory bowel disease, which is a disease caused by a combination of genetic predisposition and abnormalities in intestinal environmental factors, including intestinal bacteria.

[0133] For such pathologies, it is necessary to analyze the genetic information of the host itself, to perform an integrated analysis of the intestinal microbiota, i.e., bacteria, viruses, and fungi, and to clarify the mechanism of action on the host's physiological functions, and the technology disclosed herein can contribute to these. In addition, the relationship between the intestinal microbiota and the pathologies of various diseases and functional analysis centered on metabolic products can be further analyzed by analyzing the genetic information of the host itself.

[0134] In the intestinal tract, intestinal bacteria and the host compete for nutrients, while intestinal bacteria also metabolize and break down nutrients for the host. The functions of metabolites derived from intestinal bacteria are determined by obtaining data from host genetic analysis, metabolomic analysis, biochemical analysis, etc., while the functions of intestinal bacteria are inferred from bacterial genome sequence information, and various data such as the metabolites produced and metabolome are obtained. By analyzing data from both the host and intestinal bacteria in a combined manner, the relationship between the two can be understood.

[0135] Example 6: Evaluation of gene mutations in single cells It is possible to evaluate the mutation diversity in genome sequences at the single-cell level, and to track the genomic heterogeneity of each microorganism in the microbiome and the occurrence and progress of mutation lineages. By selectively amplifying and detecting the target gene mutation site of the target cell in the gel capsule containing the amplified polynucleotide, it is possible to specifically obtain only the gene sequence data to be analyzed, reducing the cost involved. It can be used to detect genetically mutated microorganisms and plasmid infections.

[0136] (Example 7: Evaluation of conservation of specific biological species) When the purpose is to prove or reject the inclusion of a specific biological species in a drug, pesticide, food, etc., the disclosed technology can be used to detect and select a specific organism from a gel capsule containing an amplified polynucleotide, thereby indicating its content. In addition, detailed analysis of the obtained genetic information makes it possible to evaluate the degree of identity with a standard at the genome level, storage stability, etc. It is expected to be used for quality assurance of microbial preparations, etc.

[0137] Specifically, the procedure involves (1) performing a homology search of gene sequences from a single organism using BLAST or hmmer for the target gene, or (2) mapping of sequence data from a single organism using BWA or bowtie2 to detect differences between the gene sequence from a single organism and the target gene sequence, and identifying genetic mutations.

[0138] (Example 8: Detection of specific biological species) The analysis sample may be feces, saliva, sputum, swabs from the skin, oral cavity, nasal cavity, ears, genitals, etc., surgical washings, tissue extracts, blood, etc., and the presence of one or more specific microorganisms may be detected to determine drug efficacy and drug resistance or evaluate the metabolic ability of food. By using the disclosed technology, it is possible to detect and select specific organisms from gel capsules containing amplified polynucleotides, thereby indicating their content. In addition, by analyzing the obtained genetic information in detail, it is possible to estimate the function and storage stability at the genome level.

[0139] For example, when single-cell sequence data of mouse intestinal microorganisms are obtained, a homology search of single-organism gene sequences can be performed based on genes that metabolize dietary fiber inulin or gene markers that identify the microbial Bacteroides species, to identify the microorganisms and gene groups responsible for decomposing the food inulin, and their functions can be estimated from the homology of known genes and three-dimensional structure prediction based on amino acid sequences. It was also possible to evaluate the proportion (content) of the microbial species in the single-cell sequence data of multiple intestinal microorganisms.

[0140] Example 9: Evaluation of soil bacteria When soil or seawater is used as the analysis sample, the technology disclosed herein can be used to detect and select various specific organisms living in soil or seawater from gel capsules containing amplified polynucleotides, thereby identifying their habitat and indicating their population. In addition, detailed analysis of the obtained genetic information can be used to evaluate at the genome level cultivated varieties, livestock or aquaculture varieties, etc. that are suitable for the soil or seawater. It is also expected that the technology will be used in pesticide-free manufacturing methods using soil bacteria or marine bacteria.

[0141] Specifically, 10 g of soil was collected in a 50 mL tube (2342-050, Iwaki) (n=3), and phosphate-buffered saline (DPBS) (Dulbecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) was added to a total volume of 40 mL to thoroughly suspend the soil. After the suspension, the tube was left on ice for 5 minutes, and the supernatant was transferred to a new 50 mL tube. The supernatant was filtered using a 5 μm filter (SMWP04700, Sigma-Aldrich), centrifuged at 10,000 × g for 5 minutes (75004263, Thermo Fisher Scientific), and the supernatant was removed. The pellet was resuspended in 10 mL of PBS, dispensed in 1 mL portions into 1.5 mL tubes (MCT-150-C, Axygen), and centrifuged at 10,000 × g for 5 minutes to collect the soil bacteria. The bacterial pellets were collected in a single 1.5 mL tube, washed twice with PBS by centrifugation, and then suspended in PBS to obtain a soil bacterial cell suspension. The cell concentration in the prepared cell suspension was measured (microscope: CKX41, OLYMPUS, bacterial counting chamber A161, 2-5679-01, AS ONE), and ultra-low melting point agarose (A5030-10G, Sigma-Aldrich) was added to a final concentration of 1.5% to prepare the soil bacterial suspension to be used for gel capsule production (final cell concentration: 1.5 × 10 3 Next, analysis was carried out in the same manner as in Example 1 or 2, and evaluation was performed.

[0142] (Example 10: Analysis of aquatic microbial composition) In the experiment, 4 L of seawater or freshwater was collected in a sterilized plastic tank, filtered through a 5 μm filter (SMWP04700, Sigma-Aldrich), and then filtered through a 0.22 μm filter (GSWP04700, Sigma-Aldrich) to trap the bacterial fraction on the filter. The 0.22 μm filter was placed in a 25 mL tube (2362-025, Iwaki) containing 10 mL of phosphate-buffered saline (DPBS) (Dulbecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) and thoroughly suspended. The 10 mL of bacterial suspension was dispensed into 1.5 mL tubes (MCT-150-C, Axygen), and the bacteria from the seawater or freshwater were collected by centrifugation at 10,000 × g for 5 minutes. The bacterial pellets were collected in a single 1.5 mL tube, washed twice with PBS by centrifugation, and then suspended in PBS to obtain a cell suspension of bacteria derived from seawater or freshwater. The cell concentration in the prepared cell suspension was measured (microscope: CKX41, OLYMPUS, bacterial counting chamber A161, 2-5679-01, AS ONE), and ultra-low melting point agarose (A5030-10G, Sigma-Aldrich) was added to a final concentration of 1.5% to prepare a bacterial suspension derived from seawater or freshwater for use in making gel capsules (final cell concentration: 1.5 × 10 3 Next, analysis was carried out in the same manner as in Example 1 or 2, and evaluation was performed. [Industrial Applicability]

[0143] The present disclosure can be used in fields such as biological research, medicine, the environment, and health care. When the purpose is to prove or reject that a specific biological species is contained in a living state in a drug, pesticide, food, etc., the technology disclosed herein can be used to detect and select a specific organism from a gel capsule containing an amplified polynucleotide to indicate its content. In addition, by analyzing the obtained genetic information in detail, the degree of match at the genome level with the specimen, preservability, etc. can be evaluated. It is expected to be used for quality assurance of microbial preparations, etc. This application claims priority to patent application No. 2019-85836 filed on April 26, 2019 and patent application No. 2019-201525 filed on November 6, 2019 to the Japan Patent Office, and it is understood that the contents of the application itself should be incorporated by reference into this specification as specifically described herein.

Claims

[Claim 1] The invention described in the specification.

Images