Method for recovering nucleic acid and kit used therefor

Abstract

Provided is a method for recovering a nucleic acid present in an environmental liquid sample, the method comprising: a preparation step for preparing the environmental liquid sample; a suspension step for adding a scavenger together with a cationic compound to the prepared environmental liquid sample to produce a suspension; and a recovery step for recovering the scavenger contained in the suspension. The scavenger contains glass fibers or a silica carrier, and the cationic compound has a secondary amino group or a quaternary amino group.

Description

Method for recovering nucleic acids and kit used therefor

[0001] The present invention relates to a method for recovering nucleic acids and a kit used therefor.

[0002] Techniques for examining nucleic acids contained in environmental water (river water, sewage, seawater, etc.) are widely used in fields such as sewage surveillance and environmental DNA analysis.

[0003] For example, Japanese Patent Laid-Open Publication No. 2023-068351 (Patent Document 1) discloses a method for recovering nucleic acids in a specimen, which includes a step of adsorbing nucleic acids in the specimen to glass fibers suspended in water, and a step of recovering the glass fibers with the adsorbed nucleic acids by filtering and separating them using a sheet having a fine mesh structure.

[0004] Furthermore, the Environmental DNA Society (2020) Environmental DNA Survey and Experiment Manual ver. 2.2 (Non-Patent Document 1) discloses methods for filtering, separating, and extracting environmental DNA from environmental water.

[0005] Japanese Patent Application Laid-Open No. 2023-068351

[0006] Environmental DNA Society (2020) Environmental DNA Survey and Experiment Manual ver. 2.2

[0007] However, the methods disclosed in Patent Document 1 and Non-Patent Document 1 have limitations on the yield of nucleic acids that can be recovered, and further improvements are desired.

[0008] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for recovering nucleic acids with improved yields.

[0009] As a result of extensive research, the present inventors have found that the yield of nucleic acids can be improved by using a collection agent such as glass fiber in combination with a specific cationic compound, and have thus completed the present invention.

[0010] A first aspect of the present invention is a method for recovering nucleic acids present in an environmental liquid sample, comprising: a preparation step of preparing the environmental liquid sample; a suspension step of adding a collector together with a cationic compound to the prepared environmental liquid sample to form a suspension; and a recovery step of recovering the collector contained in the suspension, wherein the collector includes a glass fiber or silica carrier, and the cationic compound has a secondary amino group or a quaternary amino group.

[0011] A second aspect of the present invention is a kit for recovering nucleic acids, comprising a composition used to recover nucleic acids contained in a liquid sample, the composition comprising: a collection agent including a glass fiber or silica carrier; and a cationic compound having a secondary amino group or a quaternary amino group.

[0012] According to the present invention, it is possible to provide a method for recovering nucleic acids with improved yield.

[0013] FIG. 1A is a diagram showing an example of the configuration of a nucleic acid isolation kit. FIG. 1B is a diagram showing an example of the configuration of a nucleic acid isolation kit. FIG. 2A is a diagram explaining an example of a procedure for isolating nucleic acid using a nucleic acid isolation kit. FIG. 2B is a diagram explaining an example of a procedure for isolating nucleic acid using a nucleic acid isolation kit. FIG. 3 is a block diagram showing the configuration of a management system according to an embodiment. FIG. 4 is a diagram showing an example of the contents of a database. FIG. 5 is an example of a display screen displayed on a display terminal device. FIG. 6 is a diagram showing a flowchart of an example of processing executed by the management system. FIG. 7 is a diagram explaining information and items transferred in the provision system. FIG. 8 is a sequence diagram explaining processing executed in the provision system. FIG. 9 is a schematic diagram explaining the expected binding mode of a collector, a cationic compound, and a nucleic acid in the nucleic acid isolation method according to this embodiment. FIG. 10 is a photograph showing that the fractured membranes float when BAC, fractured membranes, PSI, and a polymer flocculant are added to river water. FIG. 11 is a photograph showing that the fractured membranes float when BAC, fractured membranes, PSI, and a polymer flocculant are added to river water. FIG. 12 is a photograph showing that adding BAC, fractured membranes, PSI, and triiron tetroxide to river water causes the fractured membranes to aggregate with a magnet. FIG. 13 is a graph showing the yield of nucleic acid that can be recovered when a collector and BAC are added to river water. The horizontal axis indicates the presence or absence of a collector and BAC used, and the vertical axis indicates the average yield (average concentration) of recovered nucleic acid. FIG. 14 is a graph showing the yield of nucleic acid that can be recovered when a collector and BAC are added to river water. The horizontal axis indicates the concentration of BAC used, and the vertical axis indicates the average yield (average concentration) of recovered nucleic acid. FIG. 15 is a graph showing the yield of nucleic acid that can be recovered when a collector and a cationic compound are added to river water. The horizontal axis indicates the cationic compound used and its concentration, and the vertical axis indicates the average yield (average concentration) of recovered nucleic acid. FIG. 16A is a graph showing the yield of nucleic acid that can be recovered when a collector and BAC are added to river water. The horizontal axis indicates the capture agent used and the presence or absence of BAC, and the vertical axis indicates the average yield (average concentration) of recovered nucleic acid.FIG. 16B is a graph showing the yield of nucleic acids that can be recovered when a capture agent and BAC are added to river water. The horizontal axis indicates whether a capture agent and BAC are used, and the vertical axis indicates the average concentration of recovered nucleic acids. FIG. 17 is a graph showing the yield of nucleic acids that can be recovered (left graph) and the ratio of recovered DNA and RNA (right graph) when a capture agent, BAC, and flocculant are added to river water. The horizontal axis indicates whether a capture agent, flocculant, and BAC are used, and the vertical axis indicates the average yield of recovered nucleic acids (left graph) or the ratio of DNA and RNA (right graph). FIG. 18 is a graph showing the yield of nucleic acids that can be recovered (left graph) and the ratio of recovered DNA and RNA (right graph) when a capture agent and BAC are added to river water. The horizontal axis indicates whether a capture agent and BAC are used, and the vertical axis indicates the average yield of recovered nucleic acids (left graph) or the ratio of DNA and RNA (right graph). FIG. 19 is a graph showing the yield of nucleic acid that can be recovered when a collector and BAC are added to river water. The horizontal axis shows the time allowed to stand after adding the collector and BAC, and the vertical axis shows the average yield (average concentration) of recovered nucleic acid. FIG. 20 is a graph showing the yield of nucleic acid that can be recovered when a collector, cationic compound, and flocculant are added to sewage. The horizontal axis shows the cationic compound and collector used and their concentrations, and the vertical axis shows the average yield (average concentration) of recovered nucleic acid. FIG. 21 is a graph showing the yield of nucleic acid that can be recovered when a collector, cationic compound, and flocculant are added to sewage. The horizontal axis shows the cationic compound and collector used and their concentrations, and the vertical axis shows the average yield (average concentration) of recovered nucleic acid. FIG. 22 is a graph showing the yield of nucleic acid that can be recovered when a collector and BAC are added to river water. The horizontal axis shows the shape of the collector used, and the vertical axis shows the average yield (average concentration) of recovered nucleic acid. 23 is a graph showing the yield of nucleic acid that can be recovered when a collection agent and a cationic compound are added to river water. The vertical axis shows the cationic compound used (final concentration 0.01%), and the horizontal axis shows the percentage of nucleic acid recovered relative to the control.Figure 24 is a graph showing the stability of nucleic acids in TBS buffer (left), and a graph showing the yield of nucleic acids that can be recovered when a collection agent and BAC are added to a TBS buffer containing dissolved nucleic acids (right). In the left graph, the horizontal axis indicates the time allowed to stand after adding nucleic acids to the TBS buffer, and the vertical axis indicates the concentration of nucleic acids in the TBS buffer. In the right graph, the horizontal axis indicates the time allowed to stand after adding nucleic acids to the TBS buffer, and the vertical axis indicates the yield of recovered nucleic acids. Figure 25 is a graph showing the yield of nucleic acids that can be recovered when a collection agent and BAC are added to a clinical sample (urine). The horizontal axis indicates the presence or absence of a collection agent and BAC used, and the vertical axis indicates the average concentration of recovered nucleic acids.

[0014] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0015] <<Method for recovering nucleic acids present in an environmental liquid sample>> A first aspect of this embodiment is a method for recovering nucleic acids present in an environmental liquid sample, comprising: a preparation step of preparing the environmental liquid sample; a suspension step of adding a collector together with a cationic compound to the prepared environmental liquid sample to form a suspension; and a recovery step of recovering the collector contained in the suspension, wherein the collector includes a glass fiber or silica carrier, and the cationic compound has a secondary amino group or a quaternary amino group.

[0016] <Preparation Step> In this step, an environmental liquid sample is prepared. In this embodiment, an "environmental liquid sample" refers to a liquid sample derived from something present in a natural or artificial environment. Examples of substances present in a natural environment include river water, seawater, lake water, soil, compost, and sludge. Examples of substances present in an artificial environment include sewage, industrial wastewater, wastewater from livestock facilities, oral drainage, and drinking water. When the substance present in the natural or artificial environment is a liquid, that liquid can be used as is. When the substance present in the natural or artificial environment is a solid, a suspension obtained by suspending it in distilled water or the like can be used as the environmental liquid sample. A sample collected from a natural or artificial environment may be used as is, or a sample that has been pretreated, such as by removing insoluble components, may be used.

[0017] In one aspect of this embodiment, the environmental liquid sample may include at least one selected from the group consisting of river water, seawater, lake water, and sewage. The environmental liquid sample may also consist of at least one selected from the group consisting of river water, seawater, lake water, and sewage. The amount of the environmental liquid sample is not particularly limited, but may be, for example, 10 mL to 2000 mL, or 400 mL to 1000 mL.

[0018] In another aspect of this embodiment, in the preparation step, the environmental liquid sample may not contain a cationic compound, because the cationic compound is used in this embodiment to promote binding between the collection agent and nucleic acids.

[0019] The environmental liquid sample may contain a nucleic acid to be tested. The nucleic acid may be DNA, RNA, or both. The nucleic acid to be tested is not particularly limited and may be derived from plants, animals (e.g., fish), or microorganisms.

[0020] <Suspension Step> In this step, a suspension is generated by adding a collector together with a cationic compound to the prepared environmental liquid sample. In this embodiment, "adding a collector together with a cationic compound" means that the collector and the cationic compound are used together in this step, but not in other steps. The order in which the collector and the cationic compound are added is not particularly limited. The collector may be added to the environmental liquid sample first, and then the cationic compound may be added. The cationic compound may be added to the environmental liquid sample first, and then the collector may be added. Alternatively, the collector and the cationic compound may be added to the environmental liquid sample simultaneously. In one aspect of this embodiment, a composition (mixture) containing the collector and the cationic compound may be added to the environmental liquid sample.

[0021] This process may further include mixing (e.g., by inversion) the resulting suspension.

[0022] In one aspect of this embodiment, the suspension step and the recovery step described below are preferably performed before degradation of nucleic acids in the environmental liquid sample progresses, particularly immediately after the environmental liquid sample is collected and prepared. From this perspective, the time from start to finish of the suspension step (from the start of the suspension step to the start of the recovery step) may be 1 minute to 10 minutes or less, or 1 minute to 60 minutes or less.

[0023] In this embodiment, the term "collection agent" refers to a reagent that adsorbs and recovers nucleic acids contained in an environmental liquid sample. The collection agent includes glass fiber or a silica carrier. The collection agent may contain glass fiber or may be made of glass fiber.

[0024] Examples of the material of the glass fiber include borosilicate glass, soda-lime glass, and quartz glass. In one aspect of the present embodiment, the material of the glass fiber may contain or consist of borosilicate glass.

[0025] The glass fibers may be in the form of a powder, a sheet like filter paper, or small pieces. In one aspect of this embodiment, the collector may contain or consist of powdered glass fibers. The arithmetic mean fiber length of the powdered glass fibers may be 20 μm or more and 500 μm or less, or 100 μm or more and 300 μm or less. The arithmetic mean fiber length can be determined, for example, by measurement using a Valmet FS5 (manufactured by Valmet).

[0026] The glass fibers may be produced by known methods or may be purchased commercially. Examples of commercially available glass fibers include the products described in the Examples below. Powdered glass fibers can be obtained, for example, by crushing commercially available glass filter paper using stainless steel beads (see the Examples below).

[0027] The silica carrier may be in the form of a powder, a sheet, a gel, or a fiber. The average particle diameter of the powdered silica carrier may be 5 μm or more and 200 μm or less. The average particle diameter of the gelled silica carrier may be 5 μm or more and 100 μm or less. The arithmetic mean fiber length of the fibrous silica carrier may be 20 μm or more and 500 μm or less, or 100 μm or more and 300 μm or less. The arithmetic mean fiber length can be measured, for example, using a Valmet FS5 (manufactured by Valmet).

[0028] The silica carrier may be produced by a known method or may be purchased as a commercially available product. Examples of commercially available silica carriers include the products described in the Examples below. A fibrous silica carrier can be obtained, for example, by crushing commercially available silica filter paper using stainless steel beads (see Examples below).

[0029] The concentration of the collector may be 0.0005 w / v % or more and 1 w / v % or less, or 0.0025 w / v % or more and 0.1 w / v % or less, based on the suspension.

[0030] In this embodiment, the term "cationic compound" refers to a compound that behaves as a cation when dissolved in water. The cationic compound has a secondary amino group or a quaternary amino group. In this embodiment, the two substituents bonded to the nitrogen atom of the quaternary amino group may be bonded to each other to form a ring structure (e.g., an imidazole ring or a pyridine ring). That is, in this embodiment, the "quaternary amino group" may include an imidazole group and a pyridinium group. In one aspect of this embodiment, the cationic compound may have an imino group.

[0031] In one aspect of this embodiment, the cationic compound may further include a hydrophobic group (e.g., a linear alkyl group having 10 to 18 carbon atoms) or an aromatic ring. In another aspect of this embodiment, the cationic compound may further include a linear alkyl group having 10 to 18 carbon atoms.

[0032] In another aspect of this embodiment, the cationic compound may include a compound having a quaternary amino group and a linear alkyl group having 10 or more carbon atoms (preferably a linear alkyl group having 10 to 18 carbon atoms), or a compound having a secondary amino group and having a bactericidal effect. Here, "bactericidal effect" means the action of killing microorganisms.

[0033] Examples of cationic compounds having a secondary amino group include chlorhexidine gluconate, alkyldiaminoethylglycine hydrochloride, polyhexamethylene biguanide, N-methyldodecylamine, etc. Examples of cationic compounds having a quaternary amino group include benzalkonium chloride, domiphen bromide, hexadecyltrimethylammonium bromide, 1-decyl-3-methylimidazolium chloride, 1,3-didecyl-2-methylimidazolium, tetradecyltrimethylammonium, cetylpyridinium, dodecyltrimethylammonium, trimethyloctadecylammonium, benzyldimethyldodecylammonium, 1-dodecylpyridinium chloride, benzyldimethylhexadecylammonium chloride, cetylpyridinium bromide, and didecyldimethylammonium, etc. Examples of cationic compounds having an imino group include chlorhexidine gluconate.

[0034] In one aspect of this embodiment, the cationic compound may include at least one selected from the group consisting of benzalkonium chloride, chlorhexidine gluconate, alkyldiaminoethylglycine hydrochloride, polyhexamethylene biguanide, N-methyldodecylamine, domiphen bromide, hexadecyltrimethylammonium bromide, 1-decyl-3-methylimidazolium chloride, 1,3-didecyl-2-methylimidazolium, tetradecyltrimethylammonium, cetylpyridinium, dodecyltrimethylammonium, trimethyloctadecylammonium, benzyldimethyldodecylammonium, 1-dodecylpyridinium chloride, benzyldimethylhexadecylammonium chloride, cetylpyridinium bromide, and didecyldimethylammonium. Furthermore, the cationic compound may include or consist of benzalkonium chloride.

[0035] The concentration of the cationic compound may be from 0.0001 w / v% to 0.05 w / v%, from 0.0005 w / v% to 0.05 w / v%, or from 0.001 w / v% to 0.01 w / v%, based on the suspension. When the cationic compound contains benzalkonium chloride, the concentration of the benzalkonium chloride is preferably from 0.0005 w / v% to 0.05 w / v%, and more preferably from 0.001 w / v% to 0.01 w / v%, based on the suspension.

[0036] It has been known that benzalkonium chloride is added to environmental liquid samples as a disinfectant. However, it was not previously known that adding a cationic compound such as benzalkonium chloride to an environmental liquid sample together with a collection agent significantly improves the yield of nucleic acids, a finding first discovered by the present inventors. While the specific mechanism by which nucleic acid yield is improved is unclear, the present inventors believe that the positively charged cationic compound first forms an ionic bond with the negatively charged bases of the nucleic acid or with the surface of the collection agent, and then the multiple cationic compounds that have formed ionic bonds form hydrophobic bonds or π-π interactions with each other (forming a bridge structure), thereby improving the amount of nucleic acid adsorption to the collection agent (Figure 9). Given this effect of cationic compounds, the cationic compounds can also be considered "promoters that promote the binding between the collection agent and nucleic acids."

[0037] Furthermore, the nucleic acid recovery method according to the present embodiment can be considered to improve the recovery rate of nucleic acids. Therefore, it is possible to recover even trace amounts of nucleic acid species contained in environmental samples, which could not be recovered using conventional techniques. By making it possible to recover even trace amounts of nucleic acid species contained in environmental samples, it is expected that the number of biological species that can be identified by metabarcoding will increase and that microorganisms with low abundance will be identified.

[0038] In one aspect of this embodiment, the mass ratio of the scavenger to the cationic compound in the suspension (mass of scavenger / mass of cationic compound) may be 0.25 to 250, or 1.25 to 62.5. In another aspect of this embodiment, the cationic compound is benzalkonium chloride, and the mass ratio of the scavenger to the benzalkonium chloride in the suspension is preferably 0.25 to 250, and more preferably 1.25 to 62.5.

[0039] In one aspect of this embodiment, the pH of the suspension is not particularly limited as long as the effects of the present invention are achieved. The pH of the suspension may be 5.3 or more and 9.3 or less, or 6 or more and 8 or less. A pH adjuster may be added in the suspension step to adjust the pH of the suspension to the above range.

[0040] In one aspect of this embodiment, the suspension step may further include adding a flocculant. Here, "flocculant" refers to a reagent that flocculates the collector when added to the suspension. By adding the flocculant, the collector floats to the top of the suspension in an aggregated state (e.g., Figures 10 and 11). Therefore, when the collector is recovered by filtration, clogging of the filter tends to be suppressed, and a decrease in filtration rate tends to be suppressed.

[0041] Examples of the flocculant include polyaluminum chloride (PAC), polysilica iron compounds (PSI), and polymer flocculants. In this embodiment, the flocculant may contain at least one selected from the group consisting of polyaluminum chloride, polysilica iron compounds, and polymer flocculants. Examples of the polymer flocculant include N-131 (nonionic, molecular weight 1600), A-102T (weakly anionic, molecular weight 1700), A-108T (medium anionic, molecular weight 1900), and A-133 (strong anionic, molecular weight 700), all manufactured by Taki Chemical Industry Co., Ltd. The flocculant may be produced by a known method, or a commercially available product may be purchased.

[0042] The flocculant may be in powder form or liquid form.

[0043] The concentration of the PAC or PSI may be 0.001% to 0.2% v / v, or 0.002% to 0.0125% v / v, based on the suspension. The concentration of the polymer flocculant may be 0.00001% to 0.001% w / v, or 0.00005% to 0.0005% w / v, based on the suspension.

[0044] In one aspect of this embodiment, when a flocculant is added, the suspension step may further include adding triiron tetroxide. By adding the triiron tetroxide, the collector flocculates together with the triiron tetroxide. Therefore, by applying a magnetic force, the collector can be collected at a predetermined position (e.g., FIG. 12).

[0045] The above-mentioned triiron tetroxide may be in the form of a powder or a liquid.

[0046] The concentration of the triiron tetroxide may be 0.0005 w / v % or more and 0.05 w / v % or less, or 0.0025 w / v % or more and 0.025 w / v % or less, based on the suspension.

[0047] <Recovery Step> In this step, the collector contained in the suspension is recovered. The method for recovering the collector from the suspension is not particularly limited, and a known method may be used. In one aspect of the present embodiment, the recovery step may involve filtering the suspension to recover the collector. The filtration method may be gravity filtration, suction filtration, or pressure filtration. Any filter paper may be used to filter the suspension as long as the collector does not pass through the filter paper. In one aspect of the present embodiment, the mesh size of the filter paper may be, for example, 1 μm or more and 200 μm or less, 1 μm or more and less than 180 μm, or 1 μm or more and 175 μm or less. In another aspect of the present embodiment, the mesh size of the filter paper may be, for example, 20 μm or more and 200 μm or less. In another aspect of the present embodiment, when a flocculant and triiron tetroxide are added in the suspension step, the collector may be recovered by applying a magnetic force. At this time, the collector forms flocs together with the coagulant and the triiron tetroxide.

[0048] <Other Steps> In one aspect of this embodiment, the nucleic acid recovery method may further include an extraction step of extracting the nucleic acid from the collection agent recovered in the recovery step. The method for extracting the nucleic acid from the collection agent is not particularly limited, and the nucleic acid can be recovered by a known method. The extraction step may be performed, for example, using a commercially available nucleic acid extraction kit used in the Examples described below.

[0049] <Kit Used in the Method for Isolating Nucleic Acid> A kit used to carry out the method for isolating nucleic acid described above will now be described.

[0050] <Kit Configuration 1> Fig. 1A is a diagram showing an example of the configuration of a nucleic acid isolation kit. The nucleic acid isolation kit 100 includes a composition 11 contained in a container 1, a filter unit 2, a sampling bag 3, and a cap 4. By using the nucleic acid isolation kit 100, a user can isolate nucleic acids from a liquid sample (e.g., an environmental liquid sample) by the method described above.

[0051] Container 1 contains composition 11. Composition 11 includes a collector and a cationic compound. The collector is the same as the collector used in the nucleic acid recovery method described above, and refers to a reagent that adsorbs and recovers nucleic acids contained in a liquid sample. The collector includes glass fiber or a silica carrier. The collector may contain glass fiber or may be made of glass fiber. The collector may be in powder form, sheet form, or gel form. The cationic compound is the same as the cationic compound used in the nucleic acid recovery method described above, and refers to a compound that behaves as a cation when dissolved in water. The cationic compound has a secondary amino group or a quaternary amino group.

[0052] The filter unit 2 includes a filter 21 and a housing 23. In the filter unit 2, the filter 21 is provided inside the cylindrical housing 23. The filter 21 has holes of a size that allows the liquid to pass through but not the collector. One end of the housing 23 has an opening 23A for introducing the liquid. The liquid introduced through the opening 23A passes through the filter 21 and is discharged from an opening 23B provided at the other end of the housing 23. An identifier 22 is attached to the outside of the side wall of the housing 23. The identifier 22 is, for example, a QR code (registered trademark) or a barcode. Identification information used to uniquely distinguish a specific filter unit from other filter units is recorded in the identifier 22. The identification information is, for example, a code combining letters, numbers, and / or symbols.

[0053] The sampling bag 3 includes a main body 31, an opening 32, and a cap 33. A user can pour a liquid sample and a composition 11 into the sampling bag 3 and mix them. When the opening 32 is closed with the cap 33, the inside of the sampling bag 3 becomes airtight. The opening 32 can be connected to the opening 23A of the filter unit 2.

[0054] The caps 4 include caps 4A and 4B. Cap 4A has a shape that allows it to be connected to opening 23A of filter unit 2, and cap 4B can be connected to opening 23B of filter unit 2. When openings 23A and 23B are closed by caps 4A and 4B, the inside of filter unit 2 becomes airtight. Furthermore, when cap 4A is coupled to opening 23A, a space of a predetermined size is formed inside filter unit 2 by cap 4A, filter 21, and housing 23.

[0055] In the nucleic acid isolation kit 100, the composition 11 may be provided in a state where it is contained in the container 1, or may be provided in a state where it is contained inside the sampling bag 3.

[0056] In another embodiment of the nucleic acid isolation kit, the kit includes a composition 11 contained in a container 1. According to this kit, by mixing a liquid sample contained in a general container (e.g., a beaker) with the composition 11, the nucleic acids in the liquid sample bind to the collector via the cationic compound. The user can then recover the collector to which the nucleic acids have bound from the suspension of the liquid sample and the composition using a general filtration device.

[0057] In another embodiment of the kit for carrying out the above-described nucleic acid extraction method, the nucleic acid extraction kit includes a filter unit 2, a sampling bag 3, and a cap 4. For example, consider a case where a user is provided with an amount of composition 11 greater than the amount required for a single extraction step. In such a case, the user prepares filter units 2, sampling bags 3, and caps 4 according to the number of samples. Therefore, a nucleic acid extraction kit including a filter unit 2, a sampling bag 3, and a cap 4 may be provided to the user separately from composition 11 as a kit used in the above-described nucleic acid extraction method.

[0058] <Kit Configuration 2> Figure 1B is a diagram showing an example of a nucleic acid isolation kit having a different configuration from the nucleic acid isolation kit shown in Figure 1A. The nucleic acid isolation kit 101 includes a sampling bag 3, a collector 51 contained in a container 5, an additive liquid 61 contained in a container 6, a filter unit 7, a discharge unit 8, a seal 24, and a storage bag 41. By using the nucleic acid isolation kit 101, a user can isolate nucleic acids from a liquid sample (e.g., an environmental liquid sample) by the method described above. Note that the same or corresponding parts as those in the nucleic acid isolation kit 100 are designated by the same reference numerals, and redundant description will not be repeated.

[0059] The collector 51 contained in the container 5 is the same as the collector used in the nucleic acid recovery method described above, and refers to a reagent that adsorbs and recovers nucleic acids contained in a liquid sample. The collector 51 includes glass fiber or a silica carrier. The collector 51 may contain glass fiber or may be made of glass fiber. The collector 51 may be in the form of a powder, a sheet, or a gel.

[0060] The additive solution 61 contained in the container 6 contains a cationic compound. The cationic compound is the same as the cationic compound used in the nucleic acid recovery method described above, and refers to a compound that behaves as a positive ion when dissolved in water. The cationic compound has a secondary amino group or a quaternary amino group.

[0061] The additive liquid 61 may further contain a flocculant. Here, the "flocculant" refers to a reagent that flocculates the collector 51 when added to the suspension. By adding the flocculant, the collector 51 floats in an aggregated state at the top of the suspension (see, for example, FIGS. 10 and 11 ). Therefore, when the collector 51 is recovered by filtration, clogging of the filter 72 tends to be suppressed, and a decrease in the filtration rate tends to be suppressed.

[0062] The filter unit 7 includes a housing 71 and a filter 72. In the filter unit 7, the filter 72 is provided inside the cylindrical housing 71. The filter 72 has holes of a size that allows the liquid to pass through but not the adsorbent 51. The liquid introduced into one end of the cylindrical housing 71 passes through the filter 72 and is discharged from an opening provided at the other end of the housing 71. The adsorbent 51 collected on the filter 72 is placed together with the filter unit 7 in a storage bag 41 and stored in a refrigerator.

[0063] The discharge unit 8 is used to discharge the liquid inside the sampling bag 3. The discharge unit 8 includes a storage section 81, a perforated cap 82, and a nozzle section 83. The storage section 81 is configured to be able to store the filter unit 7. The perforated cap 82 has a shape that allows it to be connected to the opening 32 of the sampling bag 3. When the sampling bag 3 and the discharge unit 8 are connected together, the liquid inside the sampling bag 3 is discharged from the opening 32, passes through the storage section 81, and is discharged from the nozzle section 83.

[0064] An identifier is printed on the seal 24. The identifier is, for example, a QR code (registered trademark) or a barcode. The seal 24 is attached to the storage bag 41 in a state in which the identifier is readable. Identification information used to uniquely distinguish the filter units in a specific storage bag from filter units in other storage bags is recorded in the identifier. The identification information is, for example, a code that combines letters, numbers, and / or symbols.

[0065] In the nucleic acid isolation kit 101, the collection agent 51 may be provided in a state where it is contained in a sampling bag 3.

[0066] In another embodiment of the kit for carrying out the nucleic acid recovery method described above, a nucleic acid recovery kit 101 includes a filter unit 7, a sampling bag 3, and a storage bag 41. For example, consider a case where the user is provided with more amounts of the capture agent 51 and the additive liquid 61 than are required for a single extraction step. In such a case, the user prepares the filter unit 7, the sampling bag 3, and the storage bag 41 according to the number of samples. Therefore, a nucleic acid recovery kit including the filter unit 7, the sampling bag 3, and the storage bag 41 may be provided to the user as a kit for use in the nucleic acid recovery method described above, separately from the capture agent 51 and the additive liquid 61.

[0067] <<Method of Using the Nucleic Acid Extraction Kit>> Next, a method of extracting nucleic acids from a liquid sample using the nucleic acid extraction kit will be described.

[0068] <Method of Use 1> FIG. 2A is a diagram illustrating the procedure for extracting nucleic acid using the nucleic acid extraction kit 100. As shown in FIG.

[0069] In step S1, the user places the collected liquid sample and composition 11 into a sampling bag 3 to form a suspension. The collector is insoluble in water, and the cationic compound is soluble in water. The user then closes the opening 32 with the cap 33. After closing the opening 32, the user mixes the suspension. This operation causes the nucleic acids in the liquid sample to bind to the collector via the cationic compound.

[0070] In step S3, the user removes the cap 33 from the sampling bag 3 and connects the opening 23A of the filter unit 2 to the opening 32 of the sampling bag 3. After connecting the filter unit 2 to the sampling bag 3, the user discharges the contents of the sampling bag 3 to the outside of the sampling bag 3 through the filter unit 2. Because the filter 21 allows liquid to pass through but does not allow the collector to pass through, the collector to which the nucleic acids are bound remains on the filter 21.

[0071] In step S5, the user removes filter unit 2 and sampling bag 3 while the collector is still attached to filter 21, and closes openings 23A and 23B of filter unit 2 with caps 4A and 4B, respectively. By this operation, the collector to which nucleic acids are bound is contained in a predetermined space inside filter unit 2.

[0072] In step S7, the user stores filter unit 2 containing the capturing agent in storage 410. Filter unit 2 and cap 4 form a container that contains the capturing agent to which nucleic acids are bound.

[0073] <Method of Use 2> Next, a method of extracting nucleic acid from a liquid sample will be described using the nucleic acid extraction kit 101. Fig. 2B is a diagram illustrating the procedure for extracting nucleic acid using the nucleic acid extraction kit 101.

[0074] In step S31, the user places the collected liquid sample and the collector 51 into the sampling bag 3 to prepare a mixed liquid. The collector is insoluble in water. The user then closes the opening 32 with the cap 33. After closing the opening 32, the user stirs the mixed liquid. This operation breaks down the collector 51 into small pieces, which are dispersed in the mixed liquid.

[0075] In step S33, the user removes the cap 33 from the sampling bag 3 and adds the additive liquid 61 to the sampling bag 3 to form a suspension. Then, the user closes the opening 32 with the cap 33. After closing the opening 32, the user mixes the suspension. This operation causes the nucleic acids in the liquid sample to bind to the collector 51 via the cationic compound contained in the additive liquid 61.

[0076] In step S35, the user removes the cap 33 from the sampling bag 3, and with the filter unit 7 stored in the storage section 81, connects the perforated cap 82 to the opening 32 of the sampling bag 3. The user then discharges the contents of the sampling bag 3 to the outside of the sampling bag 3 through the filter unit 7. The filter 72 allows liquid to pass through but does not allow the collector 51 to pass through, so the collector 51 to which the nucleic acid is bound remains on the filter 72.

[0077] In step S37, the user removes the filter unit 7 from the storage section 81 with the collector 51 collected by the filter 72, and stores the filter unit 7 in the storage bag 41. This prevents nucleic acids derived from sources other than the liquid sample used from adhering to the collector 51. After storing the filter unit 7 in the storage bag 41, a seal 24 is attached to the storage bag 41.

[0078] In step S39 , the user stores the storage bag 41 accommodating the filter unit 7 including the collection agent 51 in the storage cabinet 410 .

[0079] When analyzing nucleic acids recovered using the above-described nucleic acid recovery kit 100 and nucleic acid recovery kit 101, the user adds the collection agent removed from filter unit 2 and the collection agent 51 retained on filter 72 of filter unit 7 to an elution solution. The elution solution is, for example, water and a buffer solution. The nucleic acids bound to the collection agent via the cationic compound dissolve in the elution solution, so the user can provide the elution solution in which the nucleic acids have been dissolved for the desired analysis.

[0080] By using the above-described nucleic acid isolation kit 100 and nucleic acid isolation kit 101, the user can isolate nucleic acids without using a device such as a centrifuge. Therefore, the user can isolate nucleic acids using the kit at the same location where the liquid sample was isolated. This reduces the labor and costs associated with transporting the liquid sample.

[0081] Furthermore, the collected nucleic acids are stored in a state bound to the collection agent. Because substances other than nucleic acids can be removed from the liquid sample before storage, the collection agent can be stored in a smaller space than the liquid sample.

[0082] Furthermore, in the recovery method using the above-described kit, the user can store the collection agent dispersed in the liquid sample in filter unit 2 and filter unit 7 without directly touching the collection agent. Therefore, nucleic acids other than nucleic acids contained in the liquid sample can be prevented from binding to the collection agent.

[0083] Furthermore, the storage bag 41 containing the filter unit 2 and filter unit 7 of the kit is provided with an identifier indicating identification information. In other words, the collection agent bound to nucleic acid is contained in a container with an identifier. The user can efficiently manage the collected nucleic acid and its information by registering the identification information and information about the liquid sample in association with each other on the server. A method for managing the collection agent contained in the filter unit 2 and the collection agent 51 contained in the storage bag 41 together with the filter unit 7 will be described later.

[0084] In the above-described steps S3 and S35, the contents of the sampling bag 3 may be discharged by gravity, or the user may crush the sampling bag 3 to apply pressure to the inside of the sampling bag 3. Alternatively, the contents of the sampling bag 3 may be aspirated from outside the sampling bag 3. For example, in this case, the nozzle portion 83 of the discharge unit 8 may have a connection port shaped to be connectable to a vacuum-reducing suction filter bottle, or the discharge unit 8 may not include the nozzle portion 83, and the storage portion 81 may have a connection port shaped to be connectable to a vacuum-reducing suction filter bottle. Alternatively, the opening 23B of the filter unit 2 may be shaped to be connectable to a vacuum-reducing suction filter bottle. Discharging the contents of the sampling bag 3 by suction can shorten the time required to recover nucleic acids.

[0085] <Sample Management System> In research on environmental samples, researchers may need to travel to the site to collect the samples, which requires time and money for researchers to travel to the site.

[0086] Furthermore, when researchers use the analysis results of a sample in their research, they may need information about the sample. Sample information may include, for example, at least one of the location where the sample was collected, the date and time the sample was collected, and the person who collected the sample. However, if the sample and the sample information are not stored in association with each other, researchers may not be able to use the analysis results of the sample in their research.

[0087] Furthermore, not only researchers but also ordinary citizens may collect environmental samples and send them to laboratories, where the analysis results are fed back to the general public, thereby carrying out environmental conservation activities. In such cases, technology is needed that allows even citizens who are unfamiliar with handling samples to easily obtain sample information and store it together with the sample.

[0088] Therefore, in the container management system according to the present embodiment, samples and information about the samples are managed by linking them with the identification information of the identifier attached to the container the sample is stored in. With this management system, researchers can easily obtain information about the target samples.

[0089] Furthermore, the container management system according to this embodiment can centrally store collected samples and information about those samples. Therefore, by using this management system, researchers can access samples and information about those samples that have been stored in the management system by others. This allows researchers to obtain samples collected at a desired location and their information and use those samples in their research without actually going to the location.

[0090] In the description of this embodiment, the sample is a collector to which nucleic acids are bound via a cationic compound. In this case, the sample information refers to information about a liquid sample containing nucleic acids bound to the collector. When nucleic acids are recovered using the above-described nucleic acid recovery kit 100, the filter unit 2 and cap 4 serve as a container for storing the sample. When nucleic acids are recovered using the above-described nucleic acid recovery kit 101, the storage bag 41 containing the collector 51 recovered on the filter 72 of the filter unit 7 corresponds to the container for storing the sample in this embodiment.

[0091] A system for managing nucleic acid samples with guaranteed collection methods will be described. Fig. 3 is a block diagram showing the configuration of a management system 1000 according to an embodiment. The management system 1000 is generally configured to allow a user B to use samples collected by a sample collector A, sample information, and sample analysis results. As shown in Fig. 3, the management system 1000 includes a terminal device 200, a server 300, a storage unit 400, an analysis device 500, and a display terminal device 600.

[0092] The terminal device 200, the server 300, the terminal device 420 of the storage unit 400, the analysis device 500, and the display terminal device 600 are connected to each other via a network NW. The network NW includes, for example, the Internet, a WAN (Wan Area Network), or a LAN (Lan Area Network).

[0093] The terminal device 200 transmits information about the sample collected by sample collector A to the server 300 via the network NW. At that time, sample collector A reads the identification information of the container in which the sample is stored and transmits the identification information and information about the sample stored in the container to the server. The terminal device 200 has, for example, a scanner or camera for obtaining the identification information, a keyboard or touch panel for the user to input sample information, and an antenna for obtaining location information where the sample was collected.

[0094] The server 300 receives information from devices connected via the network NW, registers the information in a database, and transmits the information to the devices upon request. The server 300 includes, as its main components, a processor 301, a memory 302, and an interface 303. The components are interconnected by a data bus.

[0095] The processor 301 is an example of an electric circuit, and controls the operation of the server 300 by executing a given program. The program executed by the processor 301 may be stored in the memory 302, or may be stored in a storage device (not shown) external to the server 300. The processor 301 is, for example, a CPU (Central Processing Unit).

[0096] The memory 302 can store a database and a program executed by the processor 301. The database stores identification information of the container in which the sample is stored, information about the sample, and analysis results of the sample, all of which are associated with one another. The memory 302 includes volatile memory (e.g., RAM (Random Access Memory)) and non-volatile memory (e.g., ROM (Read Only Memory), a hard disk drive, and a solid state drive). The program may be stored in an external storage device accessible by the processor 301.

[0097] The interface (I / F) 303 is an interface for exchanging various data between the processor 301 and devices connected to the I / F 303. The I / F 303 is realized by, for example, a terminal block, a connector, and a network adapter. Data may be exchanged via the I / F 303 wirelessly, such as via Bluetooth (registered trademark) or a wireless LAN, or via a wired connection using a USB (Universal Serial Bus) or the like. The server 300 may also receive analysis results acquired by devices other than the analysis device 500 via the I / F 303.

[0098] The storage unit 400 stores containers containing samples collected by sample collector A in a predetermined area. The storage unit 400 includes a storage cabinet 410 and a terminal device 420. The storage cabinet 410 has a storage section including one or more storage areas. Each storage area is assigned an area number. In the storage unit, a container received by an operator C from a sample collector A is stored in a predetermined storage area. The terminal device 420 is equipped with a scanner or camera for acquiring identification information. When the operator C stores the received container in the predetermined storage area, the operator C acquires the identification information of the container using the terminal device 420. The operator C transmits the identification information of the container and the area number of the storage area in which the container is stored to the server 300 using the terminal device 420. The server 300, which receives the identification information of the sample container and the area number of the storage area from the terminal device 420, associates the information and registers it in a database.

[0099] The analytical device 500 analyzes the sample and generates an analysis result. The analytical device 500 analyzes the sample received from the sample collector A or the storage unit 400. At that time, the analytical device 500 acquires identification information of the container in which the sample is stored. The analytical device 500 transmits the analysis result generated by analyzing the sample and the identification information to the server 300. The server 300 receives the identification information and the analysis result from the analytical device 500 and associates them with each other and registers them in a database.

[0100] The display terminal device 600 receives sample information and / or analysis results from the database of the server 300 and displays them. User B requests the server 300 to disclose the sample information and / or analysis results recorded in the database via the display terminal device 600. The display terminal device 600 acquires the relevant information from the server 300. The display terminal device 600 displays the acquired information on a display unit. The display unit is, for example, an LCD monitor. The display terminal device 600 acquires map information and displays the sample information together with the map information.

[0101] <<Database Registration Contents>> The contents of the database recorded in the server 300 will be described. FIG. 4 is a diagram showing an example of the database contents. The database records, for example, the container identification number, the collection location of the sample contained in the container, the collection date and time of the sample contained in the container, the collector of the sample contained in the container, the area number in which the container is stored in the storage 410, and the analysis results of the sample contained in the container. The container identification number is an example of identification information and is a number that can uniquely distinguish the container from other containers. The sample collection location is identified, for example, based on the address where the sample was collected, entered by the user, or location information (e.g., GPS information) acquired by the terminal device 200. The sample collector is not limited to a personal name and may be a predetermined personal identification number. The analysis results are results obtained by analyzing the sample.

[0102] <<Display Screen>> In response to a request from user B, the display terminal device 600 displays sample information and / or analysis results registered in the database of the server 300. Figure 5 shows an example of a display screen displayed on the display terminal device 600. The display screen 700 includes a map window 710 and a sample information window 720.

[0103] The map window 710 displays, for example, a map obtained by the display terminal device 600 via the Internet. The map window 710 also displays a point Q indicating a position corresponding to the sample collection location registered in the database.

[0104] The sample information window 720 displays information about the sample collected at point Q.

[0105] According to the display screen 700 described above, user B can easily recognize the location where the sample was collected.

[0106] <<Processing Flow in Management System>> Figure 6 is a flowchart of an example of a process in which the management system 1000 manages samples. Of the processes in Figure 6, step S10 is performed by sample collector A. Of the processes in Figure 6, steps S12 and S14 are performed by the terminal device 200. Of the processes in Figure 6, steps S16, S20, S22, and S24 are realized, for example, by the server 300 executing a given program. Of the processes in Figure 6, step S18 is performed by operator C who manages containers in the storage unit 400. Of the processes in Figure 6, step S26 is performed by the display terminal device 600.

[0107] 6 , in step S10, sample collector A places a sample in a container. The container is composed of filter unit 2 and cap 4. The sample is, for example, nucleic acid bound to a collector. The container may also be storage bag 41 that stores collector 51 collected on filter 72 of filter unit 7.

[0108] In step S12, the terminal device 200 acquires information about the sample based on input by the sample collector A via a keyboard or touch panel and / or position information acquired by the antenna.

[0109] In step S14, the terminal device 200 acquires the identification information of the container containing the sample by reading the identifier 22 attached to the container using a scanner or camera provided in the terminal device 200.

[0110] In step S16, the server 300 receives the sample information and container identification information acquired by the terminal device 200, associates them, and registers them in a database.

[0111] In step S18, the operator C receives the container containing the sample and stores the container in the storage cabinet 410.

[0112] In step S20, worker C causes terminal device 420 to read the identification information of the container stored in step S18. Terminal device 420 then transmits the identification information and the zone number of the storage zone in which the container is stored to server 300. Server 300 accepts the zone number of the storage zone in which the container is stored and the identification information of the container from terminal device 420, associates them, and registers them in a database.

[0113] In step S22, the analytical device 500 analyzes the sample contained in the container and transmits the analysis results to the server.

[0114] In step S24, the server 300 receives the analysis results and the container identification information, associates them, and registers them in the database.

[0115] In step S26, the display terminal device 600 displays to User B the sample information, the zone number, and the analysis results.

[0116] According to the management system described above, a user can recognize information about a sample and information about the location where the sample is stored, and thus can easily find the sample he or she needs.

[0117] According to the management system described above, the user can easily recognize the sample information and the analysis results of the sample.

[0118] <<Provision System Using Management System>> A provision system 2000 will be described that uses the above-described management system 1000 to provide samples and / or analysis results together with sample information in response to a request from a user D. FIG. 7 is a diagram illustrating the information and items delivered in the provision system 2000. The provision system 2000 is managed by company E, which operates the system, and is composed of a server 300, a storage unit 400, and an analysis device 500. Note that the analysis device 500 is not an essential element in the provision system 2000 according to this embodiment, and company E may obtain analysis results of samples generated by an external analysis device 500.

[0119] Sample collector A, who has collected a sample, sends the sample, identification information of the sample's container, and information about the sample to the provision system 2000. The server 300 associates the identification information received from sample collector A with the sample information and registers them in a database. In the provision system 2000, the sample received from sample collector A is stored in a predetermined storage area of ​​the storage facility 410. At that time, worker C reads the identification information of the sample's container into the terminal device 420 and transmits the identification information and the area number of the predetermined storage area to the server 300. The server 300 associates the identification information with the area number and registers them in the database. When the analysis device 500 analyzes the sample, the analysis results of the sample are sent to the server 300, and the server 300 registers the analysis results in the database.

[0120] The provision system 2000, which has received a sample and information about the sample, pays a sample provision fee to the sample collector A in exchange for the sample and the sample information. The sample provision fee paid to the sample collector A from the provision system is not limited to money, but may be, for example, predetermined service points or the analysis results of the sample. The predetermined service points are a benefit of the provision system 2000 that can be used in place of money in the provision system 2000. For example, a person who receives a sample and / or analysis results from the provision system 2000 can use the predetermined service points to pay the provision fee.

[0121] User D obtains sample information and sample and / or analysis results from the provision system 2000. User D is, for example, a researcher who obtains samples and / or analysis results held by the provision system 2000 and intends to use them in research. User D receives sample information registered in the database from the provision system 2000. User D selects a sample to use based on the received sample information and receives the sample and / or analysis results of the sample from the provision system 2000. Having received the sample and / or analysis results, User D pays a provision fee to the provision system 2000 in return.

[0122] The provision system 2000 provides user D with a sample and / or analysis results of the sample. When the provision system 2000 provides a sample, it provides the sample requested by user D from among the samples stored in the repository 410. User D, who receives the sample, can subject the sample to the analysis desired by user D. When the provision system 2000 provides sample analysis results, if the analysis results of the target sample are registered in the database, the provision system 2000 provides the analysis results to user D. If the analysis results of the target sample are not registered in the database, the target sample is analyzed by the analysis device 500, and the provision system 2000 provides the obtained analysis results to user D. The provision system 2000 may analyze the sample with the analysis device 500 in advance and register the analysis results in the database, or may analyze the target sample with the analysis device 500 in response to a request from user D and obtain the analysis results. The provision system 2000 is capable of providing one analysis result to multiple users.

[0123] <Processing Flow of the Provision System> The processing performed between the above-mentioned provision system 2000, sample collector A, and user D will be described. FIG. 8 is a sequence diagram for explaining the processing executed in the provision system 2000 managed by company E, sample collector A, and user D. Referring to FIG. 8, sample collector A collects a sample and sends the sample to company E (P50). Furthermore, sample collector A obtains information about the sample and identification information about the sample container using the terminal device 200, and transmits these to the server 300 (P52). Then, sample collector A receives compensation for providing the sample and sample information.

[0124] Company E receives the sample from sample collector A, and an employee of company E stores the sample in a storage unit of the provision system 2000 (P30). The sample is stored in a storage area within the storage cabinet 410. When the terminal device 420 of the storage unit 400 receives a command to send the sample from the server 300, the employee of company E sends the target sample to user D or the analytical device 500 (P32).

[0125] In the provision system 2000, the analysis device 500 analyzes the sample received from the repository 410. The analysis device 500 transmits the generated analysis results to the server 300 (P40).

[0126] Server 300 accepts sample information from sample collector A and registers the information in the database, and company E sends payment to sample collector A (P20). When server 300 accepts a request for disclosure of information registered in the database from user D via network NW, it transmits information about the target sample to user D (P22). Server 300 accepts a request for provision of a sample and / or analysis results from user D (P24). When server 300 determines that it has accepted a request for provision of a sample from user D, it transmits a sample delivery command to terminal device 420 of storage unit 400 (P24). When server 300 determines that it has accepted a request for provision of sample analysis results from user D, and the analysis results are not registered in the database, it transmits a sample delivery command to terminal device 420 of storage unit 400 (P24). When server 300 determines that it has received a request from user D for the analysis results of the sample, and if the analysis results are registered in the database, it transmits the analysis results to user D (P26). Server 300 also receives the analysis results from analysis device 500, registers the analysis results in the database, and transmits the analysis results to user D (P26). Company E receives payment from user D for providing the sample and / or analysis results (P28).

[0127] User D requests server 300 to disclose sample information and receives the sample information from server 300 (P10). Based on the received sample information, User D requests the sample and / or analysis results from server 300, and after receiving the sample and / or analysis results, sends payment to Company E (P12).

[0128] According to the provision system described above, company E can earn profits by receiving payment from user D in exchange for samples and / or analysis results. Sample collector A can also earn profits by receiving payment from company E in exchange for samples and sample information. Furthermore, by paying a fee, user D can obtain samples and / or analysis results without having to collect samples on-site or analyze them himself.

[0129] The nucleic acid recovery method, kit used in the nucleic acid recovery method, and sample management system according to this embodiment have been described above. The nucleic acid recovery method according to this embodiment can be used not only for recovering environmental liquid samples but also for recovering nucleic acids from ordinary liquid samples. That is, the nucleic acid recovery method according to this embodiment is a method for recovering nucleic acids present in a liquid sample, and includes the following steps: preparing the liquid sample; suspending the liquid sample by adding a collector and a cationic compound to the prepared liquid sample to form a suspension; and recovering the collector contained in the suspension. The collector includes glass fiber or silica, and the cationic compound has a secondary amino group or a quaternary amino group. Examples of the liquid sample include liquid samples derived from living organisms (e.g., urine).

[0130] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In these examples, the notation "%" indicates "w / v %" unless otherwise specified.

[0131] <Experiment 1: Preparation of Collector> A collector was prepared according to the following procedure. First, four sheets (dry weight: 100 mg) of glass filter paper (product name: GA-55, Φ25 mm) manufactured by Advantec, nuclease-free water (1 mL), and stainless steel beads (size for 22 mL, number of beads added: 1) were added to a 10 mL crushing tube. The crushing tube was treated using a Multi-Beads Shocker (manufactured by Yasui Machinery Co., Ltd.) at 1500 rpm for 15 seconds to obtain a crushed film (a collector made of powdered glass fiber). The obtained crushed film was used as the crushed film for one sample in the following experiment.

[0132] Experiment 2: Method for recovering nucleic acids present in environmental liquid samples Nucleic acid recovery Nucleic acids present in environmental liquid samples were recovered according to the method described in Non-Patent Document 1 using the following procedure. (1) 400 mL of river water (or 1 L of seawater) was collected in a 500 mL bottle (or 1 L bottle) (preparation step). (2) Each additive (a cationic compound such as BAC, a flocculant, etc.) was added to the bottle and stirred thoroughly. (3) The crushed membranes (collector) from Experiment 1 were added to the bottle, stirred, and then allowed to stand for 1 minute to obtain a suspension (suspension step). (4) The resulting suspension was filtered by gravity sedimentation through a 40 μm mesh filter. The crushed membranes remaining on the mesh filter were recovered (recovery step).

[0133] <Nucleic Acid Extraction> Nucleic acids were extracted from the recovered crushed membranes using the following procedure. (1) The recovered crushed membranes were placed in a Salivet tube, and ATL buffer (400 μL) and proteinase K (proK) (40 μL) included in the Dneasy Blood & Tissue kit (Qiagen) were added. The crushed membranes were suspended while being broken down with a pipette tip. (2) The Salivet tube containing the suspension was incubated at 55°C for 30 minutes. (3) The Salivet tube was centrifuged at 3000 g for 3 minutes. (4) Nuclease-free water (220 μL) was added to the Salivet tube, and the tube was allowed to stand for 1 minute. (5) The Salivet tube was centrifuged at 3000 g for 3 minutes. (6) EtOH (400 μL) was added to the solution in the bottom of the salivet tube to obtain a mixed solution. (7) The mixed solution (400 μL) was added to the spin column of the Dneasy Blood & Tissue kit (manufactured by Qiagen) and centrifuged at 6000 g for 1 minute. (8) After discarding the flow-through, the remaining mixed solution was added and centrifuged at 6000 g for 1 minute. (9) After discarding the flow-through, WB1 (500 μL) included in the kit was added to the spin column and centrifuged at 6000 g for 1 minute. (10) After discarding the flow-through, WB2 (500 μL) included in the kit was added to the spin column and centrifuged at 6000 g for 1 minute. (11) The filter was transferred to a new tube and centrifuged at the maximum speed of the centrifuge (19,000 g) for 2 minutes. (12) The filter was removed so as not to come into contact with the wall of the tube and placed in a DNA Lobind tube (Eppendorf). (13) AE buffer (100 μL) was added to the tube with the filter placed in it and allowed to stand for 1 minute. (14) The tube was centrifuged at 6,000 g for 1 minute. (15) The concentration of nucleic acid in the solution at the bottom of the tube was measured. The tube containing the solution was then stored at -80°C.

[0134] Nucleic acid concentration measurements were performed by appropriately using either nucleic acid quantification using Nanodrop or nucleic acid quantification using Qubit. Average values ​​were calculated using N=3. Nucleic acid quantification using Nanodrop: The nucleic acid concentration was quantified from absorbance using 2 μL of purified nucleic acid with Nanodrop ND-1000. Nucleic acid quantification using Qubit: The nucleic acid concentration was quantified using a nucleic acid quantification kit (manufactured by Thermo Fisher Scientific, product names: 1×dsDNA HS and BR RNA).

[0135] Experiment 3: Effect of Benzalkonium Chloride on Nucleic Acid Yield We investigated how adding benzalkonium chloride (a cationic compound, hereafter sometimes referred to as "BAC") manufactured by Fujifilm Wako Co., Ltd. to an environmental liquid sample affected the nucleic acid yield. The collection agents used were a crushed membrane (corresponding to the Example) prepared by the method described above and uncrushed glass filter paper (four sheets, corresponding to the Comparative Example). The nucleic acid recovery method was the same as in Experiment 2. A group with BAC added (BAC+, corresponding to the Example, final concentration 0.01%) and a group without BAC added (BAC-, corresponding to the Comparative Example) were set up. River water (400 mL) was used as the environmental liquid sample. Nucleic acid quantification was performed using Nanodrop. The results are shown in Figure 13.

[0136] The results of Figure 13 demonstrate that adding crushed membranes and BAC to environmental liquid samples (crushed membranes (BAC+)) significantly improves nucleic acid yield. While adding BAC to environmental liquid samples for long-term storage and adding crushed membranes to environmental liquid samples to recover nucleic acids have been known for some time, the significant improvement in nucleic acid yield achieved by combining these methods was not previously known, and this finding was first discovered by the present inventors. Generally, nucleic acid degradation is limited over short periods of time. For example, in Experiment 21, described below, almost no degradation of nucleic acids in environmental liquid samples was observed within at least one hour (see BAC(-) in Figure 19). Furthermore, in Experiment 26, described below, almost no degradation of nucleic acids in TBS buffer was observed within at least two hours (see the left panel of Figure 24). Considering these points, the present inventors believe that cationic compounds such as BAC contribute to improved binding between crushed membranes and nucleic acids (e.g., Figure 9).

[0137] <Experiment 4: Investigation of the Order of Addition of BAC> We investigated whether changing the order in which the crushed membrane and BAC were added to the environmental liquid sample would affect the yield of nucleic acids. Aside from changing the order of addition, the same experiment as Experiment 3 was conducted. Nucleic acids were quantified using Nanodrop. The results are shown in Table 1.

[0138]

[0139] The results in Table 1 show that, regardless of the order of addition of the capture agent and cationic compound (BAC), the nucleic acid yield was improved in the group to which the cationic compound was added compared to the group to which no cationic compound was added.

[0140] <Experiment 5-1: Investigation of the ratio of crushed membrane to BAC> Using 400 mL of river water, the ratio of crushed membrane amount to BAC amount (crushed membrane amount to promoter amount ratio) (mass ratio) was investigated. The same experiment as Experiment 3 was conducted except that crushed membrane and BAC were used in the ratios shown in Table 2. Nucleic acid quantification was performed using Nanodrop. The results are shown in Table 3A.

[0141]

[0142]

[0143] The results in Tables 2 and 3A show that when samples with the same amount of disrupted membrane were compared, the group with BAC added tended to have a higher nucleic acid yield than the group without BAC added. It was also observed that the nucleic acid yield improved when the ratio of disrupted membrane amount to BAC amount was in the range of 0.25 to 250. Furthermore, it was observed that the nucleic acid yield improved even more when the ratio of disrupted membrane amount to BAC amount was in the range of 1.25 to 62.5.

[0144] <Experiment 5-2: Investigation of the ratio of crushed membrane to domiphen bromide> Using 400 mL of river water, the ratio of crushed membrane amount to domiphen bromide amount (crushed membrane amount to promoter amount ratio) (mass ratio) was investigated. The same experiment as Experiment 3 was conducted except that crushed membrane and domiphen bromide were used in the ratios shown in Table 2 above. Nucleic acid quantification was performed using Nanodrop. The results are shown in Table 3B.

[0145]

[0146] The results in Tables 2 and 3B show that when samples with the same amount of crushed membrane were compared, the nucleic acid yield tended to be higher in the group with added domiphen bromide than in the group without added domiphen bromide. It was also confirmed that the nucleic acid yield was improved when the ratio of crushed membrane amount to domiphen bromide amount was in the range of 0.25 to 250. Furthermore, it was confirmed that the nucleic acid yield was further improved when the ratio of crushed membrane amount to domiphen bromide amount was in the range of 0.625 to 125.

[0147] Experiment 6: Study of BAC Concentration The concentration of BAC added to 400 mL of river water was varied to evaluate the change in nucleic acid yield. 100 mg of crushed membranes (equivalent to four sheets of glass filter paper) were used. The same experiment as Experiment 3 was conducted except that the BAC concentration (final concentration) was varied. Nucleic acids were quantified using Nanodrop. The results are shown in Figure 14.

[0148] The results in FIG. 14 show that the nucleic acid yield was greatest when the BAC concentration was in the range of 0.001% to 0.01%.

[0149] Experiment 7: Study of the Concentration of Other Cationic Compounds The change in nucleic acid yield was evaluated by changing the concentration of cationic compounds other than BAC. 100 mg of crushed membranes (equivalent to four sheets of glass filter paper) were used. Other cationic compounds used were chlorhexidine gluconate (manufactured by Fujifilm Wako Co., Ltd.) and alkyldiaminoethylglycine hydrochloride (manufactured by Fujifilm Wako Co., Ltd.). The same experiment as Experiment 3 was conducted except that the concentration (final concentration) of the cationic compound was changed. Nucleic acid quantification was performed using Nanodrop. The results are shown in Figure 15.

[0150] 15, it was found that not only BAC, a compound having a quaternary amino group (quaternary ammonium salt), but also chlorhexidine gluconate and alkyldiaminoethylglycine hydrochloride, which are compounds having a secondary amino group (secondary ammonium salt), contribute to improving the yield of nucleic acid. It was also found that chlorhexidine gluconate and alkyldiaminoethylglycine hydrochloride are effective in improving the yield of nucleic acid at concentrations similar to those of BAC.

[0151] Experiment 8: Examination of Other Collecting Agents An investigation was conducted to determine whether the yield of nucleic acids would be improved by using glass filter paper and silica filter paper other than Advantec's glass filter paper (product name: GA-55, Φ25 mm). Using the glass filter paper or silica filter paper shown in Table 4, each crushed membrane was prepared in the same manner as in Experiment 1.

[0152]

[0153] The same experiment as Experiment 3 was carried out except that each of the prepared crushed membranes was used. Nucleic acids were quantified using Nanodrop. The results are shown in Table 5.

[0154]

[0155] The results in Table 5 show that the yield of nucleic acids is greatly improved when the crushed membranes obtained from either glass filter paper or silica filter paper are used in combination with BAC.

[0156] <Experiment 9-1: Study on the Use of Glass Powder as a Collector> The yield of nucleic acid was studied when milled fiber (product name: MF20JH1-20, glass fiber crushed to a length of 100 to 300 μm) manufactured by Asahi Fiberglass Co., Ltd. was used as a collector instead of glass filter paper manufactured by Advantec. The same experiment as Experiment 3 was conducted except that the milled fiber (150 mg) was used as the collector. Nucleic acid quantification was performed using Qubit. The results are shown in FIG. 16A.

[0157] The results in Figure 16A show that even when the milled fiber is used as a collection agent, the yield of nucleic acid is improved by using it in combination with BAC.

[0158] <Experiment 9-2: Study on the Use of Glass Powder as a Collector> The yield of nucleic acid was studied when glass fiber or silica gel (silica carrier) shown in Table 6 below was used as a collector instead of glass filter paper manufactured by Advantec. An experiment similar to Experiment 3 was conducted, except that the glass fiber or silica gel was used as a collector under the conditions shown in Table 6 below. Nucleic acid quantification was performed using Nanodrop. The results are shown in Figure 16B.

[0159]

[0160] The results in FIG. 16B show that even when the glass fiber or silica gel is used as a collection agent, the yield of nucleic acid is improved by using it in combination with BAC.

[0161] <Experiment 10: Examination of Other Surfactants> BAC is a type of cationic surfactant. We investigated whether the yield of nucleic acids would improve when other surfactants (anionic surfactants, nonionic surfactants) were used. An experiment similar to Experiment 3 was conducted, except that the components shown in Table 6 were used instead of BAC. Nucleic acids were quantified using Nanodrop. The results are shown in Table 7.

[0162]

[0163] The results in Table 7 show that the cationic compounds BAC, chlorhexidine gluconate, and alkyldiaminoethylglycine hydrochloride significantly improved the nucleic acid yield. On the other hand, the anionic surfactant lithium dodecyl sulfate only slightly increased the nucleic acid yield. Furthermore, the nonionic surfactant Tween 20 hardly affected the nucleic acid yield.

[0164] <Experiment 11: Study of Chaotropic Salts> Chaotropic salts are generally used to improve yield when binding nucleic acids to silica carriers. Therefore, we investigated how chaotropic salts affect nucleic acid yield when recovering nucleic acids using the collection agent of this example. An experiment similar to Experiment 3 was conducted, except that guanidine thiocyanate (chaotropic salt) was used instead of BAC. Nucleic acids were quantified using Nanodrop. The results are shown in Table 8.

[0165]

[0166] The results in Table 8 indicate that even when guanidine thiocyanate, which is considered to be a highly effective chaotropic salt, was used, no improvement in nucleic acid yield was confirmed. These results suggest that the improvement in nucleic acid yield achieved by using a combination of a capture agent and BAC is due to a mechanism (e.g., Figure 9) different from that of the chaotropic salts conventionally used in nucleic acid purification using spin columns.

[0167] Experiment 12: pH Study We investigated whether the pH during nucleic acid recovery affects the yield of nucleic acids. The same experiment as Experiment 3 was conducted, except that 200 mL of collected river water was adjusted to pH 5.3, 7.2, or 9.3 with a 6 mol / L hydrochloric acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.) and a 5 mol / L sodium hydroxide solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and BAC or chlorhexidine gluconate was used as the cationic compound. Nucleic acids were quantified using Nanodrop. The results are shown in Table 9.

[0168]

[0169] The results in Table 9 show that the yield of nucleic acids is improved by using a combination of a collection agent and a cationic compound at any pH.

[0170] <Experiment 13: Nucleic Acid Spiking Experiment> Environmental liquid samples contain contaminants such as cells and bacteria. However, even in liquid samples free of such contaminants, we investigated whether the effects of combining BAC with a collection agent would be observed. Specifically, a liquid sample was prepared by adding (spiking) purified nucleic acid (5000 ng) to 400 mL of TBS (20 mM Tris-HCl + 150 mM NaCl). Using the prepared liquid sample, an experiment similar to Experiment 3 was conducted, except that the conditions shown in Table 10 were used. For example, in the experiment shown by Sample No. 1 in Table 10, the liquid sample was first passed through a glass membrane (glass filter paper) in the absence of BAC (BAC(-)), and the amount of nucleic acid recovered by the glass membrane was quantified. The liquid sample (flow-through) that passed through the glass membrane was then contacted with a crushed membrane in the presence of BAC (BAC+), whereupon the nucleic acid was again recovered and quantified. The same applies to the other experiments indicated by the numbers. Nucleic acid quantification was performed using Qubit. The results are shown in Table 10.

[0171]

[0172] The results in Table 10 revealed the following. For BAC(-), the nucleic acid recovery rate was approximately 0.1% for both glass membrane and crushed membrane, but nucleic acid could be recovered by concentrating the flow-through with crushed membrane (BAC+) (Sample Nos. 1 and 2). For BAC(+), the recovery rate increased even with glass membrane, but only to about 1% (Sample No. 3). On the other hand, the crushed membrane improved the recovery rate to 22% (Sample No. 4). For the flow-through, the crushed membrane (BAC+) after glass membrane treatment had a recovery rate of 3.9%, which was lost to the flow-through (Sample No. 3). On the other hand, the nucleic acid recovery rate from the flow-through after crushed membrane (BAC+) was approximately 0.06%, indicating that a large amount of nucleic acid was recovered with crushed membrane (BAC+) (Sample No. 4). The 22% recovery rate of nucleic acids from the crushed membrane (BAC+) suggests that the remainder was either (A) not released from the capture agent during extraction, or (B) released from the capture agent but lost because it could not be adsorbed to the kit column.

[0173] Experiment 14: Evaluation of Fiber Length of Crushed Membranes The fiber length of the crushed membranes was evaluated using the following procedure. First, each glass filter paper manufactured by Advantec shown in Table 11 was added to a 10 mL tube. For wet crushing, water (1 mL) was added and the filter was crushed with stainless steel beads at 1500 rpm for 5 to 30 seconds. A Multi-Bead Shocker (manufactured by Yasui Machinery Co., Ltd.) was used for crushing. The resulting crushed membranes were measured using a Valmet FS5 (manufactured by Valmet) to determine the fiber length. The results are shown in Table 11. From the results in Table 11, it was found that the arithmetic mean fiber length of the crushed membranes (glass fiber) was 100 μm to 300 μm.

[0174]

[0175] Experiment 15: Study of Flocculants (River Water) The yield of nucleic acids was investigated when a flocculant was used in addition to a collector and a cationic compound. Combinations of BAC (+ / -) and flocculants (polyaluminum chloride: PAC, polysilica iron: PSI) were tested with river water (400 mL), and the nucleic acid yield was evaluated. The final concentrations of PAC and PSI were 0.005 v / v% and 0.0125 v / v%, respectively. The same experiment as Experiment 3 was conducted except for the use of a new flocculant. Nucleic acid quantification was performed using Qubit. The results are shown in Figure 17. The results in Figure 17 indicate that the addition of BAC improved the nucleic acid yield in all groups (BAC only, BAC + PAC, BAC + PSI). Furthermore, the addition of PSI improved the nucleic acid yield in the BAC-free group.

[0176] Experiment 16: Study of polymer flocculant (river water) The yield of nucleic acids when a polymer flocculant was used was studied. For river water (400 mL), no promoter, BAC only, BAC + PSI, BAC + Fe ３ O ４ The nucleic acid yield was evaluated by examining the combinations of BAC + PSI, and BAC + PSI + polymer flocculant (N-131 or A-102T). The same experiment as in Experiment 3 was conducted except for using the above combinations. Quantification of nucleic acids was performed using Qubit. The results are shown in Table 12 and Figures 10 to 12.

[0177]

[0178] The results in Table 12 show that the yield of nucleic acids can be improved by combining a flocculant (PSI, PAC, polymer flocculant) with a collection agent and a cationic compound. Furthermore, the photographs in Figures 10 and 11 show that the collection agent can be suspended in an aggregated state by using a combination of flocculants. The photograph in Figure 12 shows that the collection agent can be recovered in an aggregated state with a magnet by using a flocculant and triiron tetroxide.

[0179] Experiment 17: Study of flocculant (seawater) An experiment similar to Experiment 16 was carried out, except that seawater (1000 mL) was used instead of river water (400 mL). Nucleic acids were quantified using Nanodrop. The results are shown in Table 13.

[0180]

[0181] The results in Table 13 show that the addition of a flocculant (PSI) also improved the yield of nucleic acid in the BAC(-) group. Furthermore, the BAC(+) group showed improved yield of nucleic acid except when a glass membrane was used. In particular, the combination of BAC+PSI improved the yield of nucleic acid.

[0182] <Experiment 18: Evaluation of Silica Carrier> It was investigated whether the yield of nucleic acids would improve when a silica carrier was used as a collector. The same experiment as Experiment 3 was carried out, except that the silica carrier and crushed membrane shown below were used as collectors. Nucleic acids were quantified using Nanodrop. The results are shown in Table 14. Silica carriers used (all manufactured by EVONIK) CARPLEX #80: general-purpose crushed product (average particle size 15 μm) CARPLEX BS-306: gel-type product (average particle size 24 μm) CARPLEX BS-510BX: gel-type product (average particle size 10.5 μm)

[0183]

[0184] The results in Table 14 show that the yield of nucleic acids is improved when a silica carrier is used as a collection agent in combination with BAC. In particular, when CARPLEX BS-306 was used as a collection agent, the yield of nucleic acids was improved by about 7 times.

[0185] Experiment 19: Evaluation of the diversity of recovered nucleic acids We evaluated whether the types of nucleic acids recovered included not only dsDNA but also RNA. The same experiment as Experiment 3 was conducted, except that river water was used as the environmental liquid sample and glass filter paper and crushed membrane were used as the collection agents. dsDNA and RNA were quantified using Qubit nucleic acid quantification. The results are shown in Figure 18.

[0186] The results in Figure 18 show that when crushed membranes and BACs are used in combination, the yield of nucleic acids increases, but the DNA:RNA ratio does not change significantly compared to when crushed membranes are used alone.

[0187] <Experiment 20: Investigation of Other Cationic Compounds (1)> It was investigated whether the yield of nucleic acid would be improved when cationic compounds other than those used in the previous experiments were used. The same experiment as Experiment 3 was conducted, except that the cationic compounds shown in Table 15 were used. Nucleic acid quantification was performed using Qubit. The results are shown in Table 15. Regarding the cationic compounds listed in Table 15, domiphen bromide manufactured by Sigma-Aldrich Corporation was used. The other cationic compounds used were manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0188]

[0189] The results in Table 15 show that any cationic compound can improve the yield of nucleic acids when used in combination with a collection agent. In particular, domiphen bromide and hexadecyltrimethylammonium bromide were found to have the same effect as BAC.

[0190] Experiment 21: Study of the Standing Time After Addition of Fractured Membranes, etc. We investigated whether the yield of nucleic acids would change by changing the standing time after adding the fractured membranes and cationic compound to the environmental liquid sample. The same experiment as Experiment 3 was conducted, except that BAC (final concentration 0.01%) and fractured membranes were added to river water (400 mL), stirred, and then the standing time was set to 1 minute, 10 minutes, 30 minutes, and 60 minutes, respectively. Nucleic acids were quantified using Nanodrop. The results are shown in Figure 19.

[0191] The results in Figure 19 indicate that changing the incubation time does not significantly affect the yield of nucleic acids. In other words, the nucleic acid recovery method according to this example enables processing in a short time by setting the incubation time to 1 minute. Furthermore, the results for BAC(-) suggest that nucleic acids in environmental liquid samples are hardly decomposed within at least 1 hour.

[0192] Experiment 22: Investigation of Nucleic Acid Recovery from Sewage We investigated whether the yield of nucleic acids would be improved if sewage was used as the environmental liquid sample. The same experiment as Experiment 3 was conducted, except that sewage (100 mL) was used as the environmental liquid sample, BAC, chlorhexidine gluconate, and alkyldiaminoethylglycine hydrochloride were used as the cationic compounds, and PAC and PSI were used as the flocculants. Nucleic acids were quantified using Nanodrop. The results are shown in Figures 20 and 21. The results in Figures 20 and 21 demonstrate that the yield of nucleic acids is also improved when sewage is used as the environmental liquid sample.

[0193] Experiment 23: Premixing of a Collector and a Cationic Compound In the procedure of Experiment 2, the collector and cationic compound were added separately to the environmental liquid sample. The collector and cationic compound were premixed to form a composition (mixture), and then the addition of this composition to the environmental liquid sample was examined to determine whether the nucleic acid yield would change. The recovery method using this composition (samples listed as "Premixed" in Table 16) was performed as follows: (1) The crushed membrane and BAC were mixed in a sample container to obtain a composition (1 h). (2) The environmental liquid sample was added to the sample container containing the composition, stirred, and then allowed to stand for 1 minute. (3) The collector was recovered using a filter, and nucleic acids were extracted and quantified using the same method as in Experiment 2. Nucleic acid quantification was performed using a Nanodrop.

[0194]

[0195] The results are shown in Table 16. In Table 16, for samples that do not have "pre-mixed" written on them, nucleic acids were recovered by performing an experiment similar to Experiment 3. The results in Table 16 show that even when the collection agent and cationic compound were mixed in advance and the environmental liquid sample was added to the mixture, the yield of nucleic acids was improved in both optimal concentration ranges (0.001%, 0.01%) compared to the control experiment (collection agent without BAC added).

[0196] <Experiment 24: Investigation of the surface area of ​​the collector> It was investigated whether changing the surface area of ​​the collector would affect the yield of nucleic acids. The same experiment as Experiment 3 was carried out, except that the following four types of collectors were used and nucleic acids were quantified using Qubit dsDNA. Nucleic acids were quantified using Qubit. The results are shown in Figure 22. Collectors used (all made from Advsntec GA-55 (25 mm) filter paper) Glass filter paper (uncut) Glass filter paper cut into quarters Glass filter paper cut into 5 mm squares Crushed membrane crushed using the method of Experiment 1

[0197] The results in Figure 22 show that even with the same amount of glass filter paper, the yield of nucleic acid tends to improve as the size decreases (as the surface area increases). Furthermore, for all collection agents, the yield of nucleic acid improved with the addition of BAC.

[0198] Experiment 25: Examination of Other Cationic Compounds (2) We investigated whether the yield of nucleic acid would be improved if cationic compounds other than those used in the previous experiments were used. The same experiment as Experiment 3 was conducted, except that the cationic compounds shown below were used and the final concentration of the cationic compounds was set to 0.01%. Nucleic acid was quantified using Nanodrop. The recovery ratio was calculated based on the amount of nucleic acid recovered in a control experiment (control) conducted without using a cationic compound. The results are shown in Figure 23. In Figure 23, the vertical axis represents the cationic compound used (final concentration 0.01%), and the horizontal axis represents the nucleic acid recovery ratio relative to the control. Note that in Figure 23, the names of the cationic compounds have been partially abbreviated due to space limitations. (List of Cationic Compounds Used) Polyhexamethylene biguanide hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.), hexadecyltrimethylammonium bromide (Fujifilm Wako Pure Chemical Industries, Ltd.), 1,3-didecyl-2-methylimidazolium chloride (Sigma-Aldrich), alkyldiaminoethylglycine hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.), domiphen bromide (Sigma-Aldrich), benzalkonium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), chlorhexidine gluconate (Fujifilm Wako Pure Chemical Industries, Ltd.), tetradecyltrimethylammonium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), N-methyldodecylamine (Sigma-Aldrich), cetylpyridinium (Fujifilm Wako Pure Chemical Industries, Ltd.) Fluor Wako Pure Chemical Industries, Ltd.), 1-dodecylpyridinium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), dodecyltrimethylammonium chloride (Sigma-Aldrich), trimethyloctadecylammonium chloride (Sigma-Aldrich), benzyldimethyldodecylammonium chloride (Sigma-Aldrich), 1-decyl-3-methylimidazolium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), tetramethylammonium chloride (Sigma-Aldrich), hexylamine (Sigma-Aldrich), benzyldimethyloctylammonium chloride (Sigma-Aldrich), benzyltrimethylammonium hydroxide (Fujifilm Wako Pure Chemical Industries, Ltd.), N,N-dimethylhexylamine (Sigma-Aldrich), tetrabutylammonium chloride (Sigma-Aldrich), N-hexylmethylamine (Sigma-Aldrich), tetrabutylphosphonium chloride (Sigma-Aldrich), benzyldimethylhexylammonium chloride (Sigma-Aldrich), N,N-dimethyldodecylamine (Sigma-Aldrich), dodecylamine (Sigma-Aldrich), tetradecylamine (Sigma-Aldrich), hexadecylamine (Sigma-Aldrich) , Octadecylamine (Sigma-Aldrich), Hexyltrimethylammonium (Sigma-Aldrich), Benzyldimethylhexylammonium (Sigma-Aldrich), Octylamine (Fujifilm Wako Pure Chemical Industries, Ltd.), N-Octyltrimethylammonium (Sigma-Aldrich), Benzyldimethylhexadecylammonium chloride (Sigma-Aldrich), Cetylpyridinium bromide hydrate (Sigma-Aldrich), Didecyldimethylammonium (Fujifilm Wako Pure Chemical Industries, Ltd.). ,

[0199] The results in Figure 23 show that when polyhexamethylene biguanide, hexadecyltrimethylammonium bromide, 1,3-didecyl-2-methylimidazolium, alkyldiaminoethylglycine hydrochloride, domiphen bromide, benzalkonium chloride, chlorhexidine gluconate, tetradecyltrimethylammonium, N-methyldodecylamine, cetylpyridinium, 1-dodecylpyridinium chloride, dodecyltrimethylammonium, trimethyloctadecylammonium, benzyldimethyldodecylammonium, 1-decyl-3-methylimidazolium chloride, benzyldimethylhexadecylammonium chloride, cetylpyridinium bromide, and didecyldimethylammonium are used in combination with a collector to improve the yield of nucleic acids by 1.5-fold or more compared to the control experiment.

[0200] <Experiment 26: Stability of Nucleic Acids in TBS Buffer> The stability of nucleic acids was examined by varying the incubation time after adding nucleic acids to TBS buffer. Specifically, 500 ng of nucleic acid was first added to 1 mL of 1x TBS buffer (20 mM Tris-HCl + 150 mM NaCl). The nucleic acid concentration was quantified using Qubit 0 to 120 minutes after addition. The results are shown in Figure 24 (left panel). The results in the left panel of Figure 24 indicate that the nucleic acid in TBS buffer did not change significantly in concentration up to 120 minutes after addition, and was stable without decomposition.

[0201] The yield of nucleic acids recovered from TBS buffer was also examined using the same method as in Experiment 3. Specifically, 5000 ng of nucleic acid was added to 1x TBS buffer (400 mL). The same experiment as Experiment 3 was performed, except that BAC (final concentration: 0% or 0.01%) and crushed membranes were added, stirred, and then allowed to stand for 1 minute, 5 minutes, 30 minutes, and 60 minutes, respectively. Nucleic acid quantification was performed using Qubit. The results are shown in Figure 24 (right). The results in the right panel of Figure 24 indicate that changing the standing time did not significantly affect the yield of nucleic acid. In other words, the nucleic acid recovery method of this example demonstrated that processing could be completed in a short time by setting the standing time to 1 minute.

[0202] Experiment 27: Investigation of Nucleic Acid Recovery from Clinical Samples We investigated whether the yield of nucleic acids would improve when a clinical sample (urine) was used as a liquid sample. The same experiment as Experiment 3 was conducted, except that a pooled human urine sample (100 mL) was used as the liquid sample, BAC and alkylaminodiethylglycine hydrochloride were used as the cationic compounds, and PAC (10 μL) and PSI (25 μL) were used as the agglutinants. Nucleic acids were quantified using Qubit. The results are shown in Figure 25. The results in Figure 25 demonstrate that the yield of nucleic acids is improved when a clinical sample (urine) is used as the liquid sample.

[0203] Experiment 28: Study of Sheet-Type Collector An investigation was conducted to determine whether the use of a sheet-type collector would improve the yield of nucleic acids. A sheet-type collector was prepared as follows. First, four sheets (dry weight: 100 mg) of glass filter paper (product name: GA-55, Φ25 mm) manufactured by Advantec, nuclease-free water (1 mL), and stainless steel beads (size for 22 mL, number of beads added: 1) were added to a 10 mL crushing tube. The crushing tube was treated using a Multi-Bead Shocker (manufactured by Yasui Machinery Co., Ltd.) at 1500 rpm for 15 seconds to obtain a crushed membrane (a collector made of powdered glass fiber). The obtained crushed membrane was dried at 40°C for 3 days and formed into a sheet-type collector. An experiment similar to Experiment 3 was conducted, except that the obtained sheet-type collector was used as a collector for one sample. The results are shown in Table 17. The crushed membranes in Table 17 were prepared in accordance with Experiment 1.

[0204]

[0205] The results in Table 17 show that the yield of nucleic acids is improved even when a sheet-shaped collector is used.

[0206] Aspects It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0207] (Item 1) A method for recovering nucleic acids according to one embodiment is a method for recovering nucleic acids present in an environmental liquid sample, comprising: a preparation step of preparing the environmental liquid sample; a suspension step of adding a collector together with a cationic compound to the prepared environmental liquid sample to form a suspension; and a recovery step of recovering the collector contained in the suspension, wherein the collector includes a glass fiber or a silica carrier, and the cationic compound has a secondary amino group or a quaternary amino group. The method for recovering nucleic acids according to item 1 can provide a method for recovering nucleic acids with improved yields.

[0208] (Item 2) In the method for recovering nucleic acids according to item 1, the collector contains powdered glass fibers. According to the method for recovering nucleic acids according to item 2, the yield of nucleic acids is further improved.

[0209] (Item 3) In the method for recovering nucleic acid according to item 2, the powdered glass fibers have an arithmetic mean fiber length of 20 μm or more and 500 μm or less. According to the method for recovering nucleic acid according to item 3, the yield of nucleic acid is further improved.

[0210] (4) In the method for recovering nucleic acid according to any one of the above items 1 to 3, the material of the glass fiber contains borosilicate glass.

[0211] (Item 5) In the method for recovering nucleic acid according to any one of items 1 to 4, the cationic compound further has a linear alkyl group having 10 to 18 carbon atoms. According to the method for recovering nucleic acid according to item 5, the yield of nucleic acid is further improved.

[0212] (Item 6) In the method for recovering nucleic acid according to any one of Items 1 to 5, the cationic compound comprises a compound having a quaternary amino group and a linear alkyl group having 10 or more carbon atoms, or a compound having a secondary amino group and having a bactericidal effect. The method for recovering nucleic acid according to Item 6 further improves the yield of nucleic acid.

[0213] (Item 7) In the method for recovering nucleic acid according to any one of Items 1 to 6, the cationic compound comprises at least one selected from the group consisting of benzalkonium chloride, chlorhexidine gluconate, alkyldiaminoethylglycine hydrochloride, polyhexamethylene biguanide, N-methyldodecylamine, domiphen bromide, hexadecyltrimethylammonium bromide, 1-decyl-3-methylimidazolium chloride, 1,3-didecyl-2-methylimidazolium, tetradecyltrimethylammonium, cetylpyridinium, dodecyltrimethylammonium, trimethyloctadecylammonium, benzyldimethyldodecylammonium, 1-dodecylpyridinium chloride, benzyldimethylhexadecylammonium chloride, cetylpyridinium bromide, and didecyldimethylammonium. The method for recovering nucleic acid according to Item 7 further improves the yield of nucleic acid.

[0214] (Item 8) In the method for recovering nucleic acid according to item 7, the cationic compound contains benzalkonium chloride, and the concentration of the benzalkonium chloride is 0.0005 w / v % or more and 0.05 w / v % or less, based on the suspension. According to the method for recovering nucleic acid according to item 8, the yield of nucleic acid is further improved.

[0215] (Item 9) In the method for recovering nucleic acid according to any one of Items 1 to 8, the mass ratio of the collector to the cationic compound in the suspension is 0.25 to 250. According to the method for recovering nucleic acid according to Item 9, the yield of nucleic acid is further improved.

[0216] (Item 10) In the method for recovering nucleic acid according to any one of Items 1 to 9, the cationic compound is added in the suspension step to promote binding between the capturing agent and the nucleic acid. According to the method for recovering nucleic acid according to Item 10, the cationic compound is added for the purpose of promoting binding between the capturing agent and the nucleic acid.

[0217] (Item 11) The method for recovering nucleic acid according to any one of Items 1 to 10, further comprising adding a flocculant in the suspension step, wherein the flocculant comprises at least one selected from the group consisting of polyaluminum chloride, polysilica iron compounds, and polymer flocculants. The method for recovering nucleic acid according to Item 11 facilitates recovery of the collection agent to which nucleic acid has been adsorbed.

[0218] (Item 12) The nucleic acid recovery method according to Item 11, further comprising adding triiron tetroxide in the suspension step. According to the nucleic acid recovery method according to Item 12, the capturing agent can be collected at a predetermined position by applying a magnetic force.

[0219] (Item 13) In the method for recovering nucleic acids according to any one of Items 1 to 12, the environmental liquid sample comprises at least one selected from the group consisting of river water, seawater, lake water, and sewage. The method for recovering nucleic acids according to Item 13 is suitably used for river water, seawater, lake water, sewage, etc.

[0220] (Item 14) In the method for recovering nucleic acid according to any one of Items 1 to 13, the recovery step involves filtering the suspension to recover the collection agent. According to the method for recovering nucleic acid according to Item 14, it is easy to recover the collection agent to which nucleic acid has been adsorbed.

[0221] (15) In the method for recovering nucleic acid according to any one of items 1 to 14, in the preparation step, the environmental liquid sample does not contain the cationic compound.

[0222] (Item 16) In one aspect, a nucleic acid recovery kit includes a composition used to recover nucleic acids contained in a liquid sample, the composition including a collection agent containing a glass fiber or silica carrier, and a cationic compound having a secondary amino group or a quaternary amino group. The recovery kit described in Item 16 can improve the recovery efficiency of nucleic acids contained in a liquid sample.

[0223] (Item 17) A nucleic acid recovery kit according to one embodiment includes: a sampling bag having an opening; a filter unit that engages with the opening and has a filter; and a cap configured to form a cavity between the filter and the cap by engaging with the filter unit. The recovery kit described in Item 17 can reduce the burden on a user required to recover nucleic acids contained in a liquid sample.

[0224] (Item 18) The recovery kit according to Item 16 may further include the sampling bag, filter unit, and cap according to Item 17. The recovery kit according to Item 18 provides items necessary for carrying out the nucleic acid recovery method.

[0225] (Item 19) An identifier indicating identification information may be attached to the filter unit included in the collection kit according to either item 17 or 18. According to the collection kit according to item 19, the collected nucleic acid can be identified based on the identification information.

[0226] (Item 20) A management system according to one aspect is a management system for managing samples contained in containers bearing identifiers indicating identification information and information about the samples, the management system comprising a terminal device, a server, and a storage unit configured to be able to communicate information with each other via a network, the storage unit being capable of containing the containers, the terminal device including an acquisition unit that acquires information about the samples and the identification information, and the server storing the identification information and the sample information in association with each other. According to the management system described in Item 20, the server stores containers bearing identifiers and information about the samples contained in the containers in association with each other. Therefore, a user can easily obtain information about the samples contained in the containers based on the sample containers.

[0227] (Item 21) In the management system described in Item 20, the sample may be at least one of river water, sewage, seawater, and a biological sample. According to the management system described in Item 21, a sample that is at least one of river water, sewage, seawater, and a biological sample can be managed together with sample information.

[0228] (Clause 22) In the management system described in paragraph 20 or 21, the acquisition unit has a receiver for acquiring location information, the sample information includes a location where the sample was collected, and the location where the sample was collected is identified by the location information acquired by the receiver. According to the management system described in paragraph 22, information regarding the location where the sample was collected, which is identified by the location information received by the terminal device, can be managed together with identification information.

[0229] (Item 23) The management system according to any one of Items 20 to 22 further includes an analytical device, wherein the analytical device analyzes the sample and obtains an analysis result, and the server stores the analysis result in association with the identification information. According to the management system according to Item 23, a user can easily obtain sample information and the analysis result of the sample based on the identification information.

[0230] (24) The management system according to any one of the above items 20 to 23 further includes a display terminal device, and the display terminal device displays information about the sample. According to the management system according to the above item 24, a user can easily recognize the sample information.

[0231] (Item 25) In the management system according to any one of Items 20 to 24, the sample information includes a location where the sample was collected, and the display terminal device maps and displays the sample information on a map based on the acquired map information and the location where the sample was collected. According to the management system according to Item 25, a user can recognize the location where the target sample was collected on the map.

[0232] (Item 26) An information provision system according to one aspect provides information about the sample using the management system according to any one of Items 20 to 25, wherein the management system receives a request for the provision of the sample information from a user, provides the requested sample information to the user, and receives payment from the user for the provision of the sample information. According to the information provision system according to Item 26, the user can obtain the sample information held by the management system in exchange for the payment.

[0233] (Item 27) In the information provision system described in Item 26, the management system further receives the sample and information about the sample from the collector, and sends payment for the sample and the information about the sample to the collector. According to the information provision system described in Item 27, the sample collector can receive payment in exchange for the sample and the information about the sample.

[0234] (Clause 28) In the information provision system described in clause 26 or 27, the management system further receives a request for the provision of the sample from the user, provides the requested sample to the user, and receives payment from the user for the provision of the sample. The information provision system described in clause 28 can receive payment in exchange for sending the sample to the user. Furthermore, users of the information provision system can obtain the sample by paying the payment.

[0235] (Item 29) In the information provision system described in any one of Items 26 to 28, the management system further receives a request from the user for the provision of the analysis results of the sample, provides the requested analysis results of the sample to the user, and receives payment from the user for the provision of the analysis results of the sample. The information provision system described in Item 29 can receive payment in exchange for sending the analysis results of the sample to the user. Furthermore, users of the information provision system can obtain the analysis results of the sample by paying the payment.

[0236] (Clause 30) An information provision system according to one aspect provides information on the sample using the management system described in Clause 23, wherein the management system: accepts a request from a user for the provision of analysis results of the sample; generates the requested analysis results of the sample using the analytical device; provides the requested analysis results to the user; and accepts payment from the user for the provision of the requested analysis results of the sample. The information provision system described in Clause 30 analyzes the sample in response to the user's request and sends the obtained analysis results to the user. Therefore, the user can obtain the analysis results of the sample even if the analysis results of the sample are not registered in the server.

[0237] (Item 31) A method for recovering nucleic acids according to one embodiment is a method for recovering nucleic acids present in a liquid sample, the method comprising: a preparation step of preparing the liquid sample; a suspension step of adding a collection agent together with a cationic compound to the prepared liquid sample to form a suspension; and a recovery step of recovering the collection agent contained in the suspension, wherein the collection agent comprises glass fiber or silica, and the cationic compound has a secondary amino group or a quaternary amino group. The method for recovering nucleic acids according to item 31 can provide a method for recovering nucleic acids with improved yield.

[0238] (Item 32) A nucleic acid recovery kit according to one embodiment includes: a sampling bag having an opening; a filter unit that engages with the opening and has a filter; and a storage bag capable of containing the filter unit. The recovery kit described in Item 32 can reduce the burden on a user required to recover nucleic acids contained in a liquid sample.

[0239] (Item 33) The recovery kit according to Item 16 may further include the sampling bag, filter unit, and storage bag according to Item 32. The recovery kit according to Item 33 provides items necessary for carrying out the nucleic acid recovery method.

[0240] (Item 34) The collection kit according to either item 32 or 33 may further include a sticker for attaching an identifier indicating identification information to the storage bag. According to the collection kit according to item 34, the collected nucleic acid can be identified based on the identification information.

[0241] (Item 35) The collection kit according to any one of Items 32 to 34 may further include a discharge unit having a storage section capable of storing the filter unit and configured to be connectable to the opening. According to the collection kit according to Item 35, the solution in the sampling bag 3 is discharged via the discharge unit.

[0242] (Item 36) In the recovery kit described in Item 35, the discharge unit may include a connection port that can be connected to a suction filter bottle. According to the recovery kit described in Item 36, the solution in the sampling bag 3 is discharged by suction, thereby shortening the time required to recover nucleic acid.

[0243] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0244] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include meanings equivalent to the claims and all modifications within the scope of the claims.

[0245] 1, 5, 6 Container, 2, 7 Filter unit, 3 Sampling bag, 4 Cap, 8 Discharge unit, 11 Composition, 21, 72 Filter, 22 Identifier, 23, 71 Housing, 23A Opening, 23B Opening, 24 Seal, 31 Main body, 32 Opening, 33 Cap, 42 Storage bag, 100, 101 Nucleic acid recovery kit, 200 Terminal device, 300 Server, 301 Processor, 302 Memory, 303 Interface, 400 Storage unit, 410 Storage, 420 Terminal device, 500 Analysis device, 600 Display terminal device, 700 Display screen, 1000 Management system, 2000 Provision system.

Claims

1. A method for recovering nucleic acids present in an environmental liquid sample, comprising: a preparation step of preparing the environmental liquid sample; a suspension step of adding a collector together with a cationic compound to the prepared environmental liquid sample to form a suspension; and a recovery step of recovering the collector contained in the suspension, wherein the collector includes a glass fiber or silica carrier, and the cationic compound has a secondary amino group or a quaternary amino group.

2. The method for recovering nucleic acids according to claim 1, wherein the collecting agent comprises powdered glass fiber.

3. The method for recovering nucleic acids according to claim 2, wherein the powdered glass fibers have an arithmetic mean fiber length of 20 μm or more and 500 μm or less.

4. A method for recovering nucleic acids according to claim 1 or claim 2, wherein the material of the glass fiber contains borosilicate glass.

5. The method for recovering nucleic acids according to claim 1 or 2, wherein the cationic compound further has a linear alkyl group having 10 to 18 carbon atoms.

6. A method for recovering nucleic acids according to claim 1 or claim 2, wherein the cationic compound comprises a compound having a quaternary amino group and a linear alkyl group having 10 or more carbon atoms, or a compound having a secondary amino group and having bactericidal activity.

7. The method for recovering nucleic acid according to claim 1 or 2, wherein the cationic compound comprises at least one compound selected from the group consisting of benzalkonium chloride, chlorhexidine gluconate, alkyldiaminoethylglycine hydrochloride, polyhexamethylene biguanide, N-methyldodecylamine, domiphen bromide, hexadecyltrimethylammonium bromide, 1-decyl-3-methylimidazolium chloride, 1,3-didecyl-2-methylimidazolium, tetradecyltrimethylammonium, cetylpyridinium, dodecyltrimethylammonium, trimethyloctadecylammonium, benzyldimethyldodecylammonium, 1-dodecylpyridinium chloride, benzyldimethylhexadecylammonium chloride, cetylpyridinium bromide, and didecyldimethylammonium.

8. The method for recovering nucleic acids according to claim 7, wherein the cationic compound contains benzalkonium chloride, and the concentration of the benzalkonium chloride is 0.0005 w / v% or more and 0.05 w / v% or less, based on the suspension.

9. A method for recovering nucleic acids according to claim 1 or claim 2, wherein the mass ratio of the collecting agent to the cationic compound in the suspension is 0.25 or more and 250 or less.

10. A method for recovering nucleic acids according to claim 1 or claim 2, wherein the cationic compound is added in the suspension step to promote binding between the collection agent and the nucleic acid.

11. A method for recovering nucleic acids according to claim 1 or claim 2, further comprising adding a flocculant in the suspension step, wherein the flocculant comprises at least one selected from the group consisting of polyaluminum chloride, polysilica iron compounds, and polymer flocculants.

12. The method for recovering nucleic acids according to claim 11, further comprising adding triiron tetroxide in the suspension step.

13. A method for recovering nucleic acids according to claim 1 or claim 2, wherein the environmental liquid sample comprises at least one selected from the group consisting of river water, seawater, lake water and sewage.

14. A method for recovering nucleic acids according to claim 1 or 2, wherein in the recovery step, the collection agent is recovered by filtering the suspension.

15. A nucleic acid recovery kit comprising a composition used to recover nucleic acids contained in a liquid sample, the composition comprising: a collection agent including a glass fiber or silica carrier; and a cationic compound having a secondary amino group or a quaternary amino group.

16. A nucleic acid recovery kit comprising: a sampling bag having an opening; a filter unit that engages with the opening and has a filter; and a cap configured to engage with the filter unit to form a cavity between the filter and the cap.

17. The nucleic acid recovery kit according to claim 15, further comprising the sampling bag according to claim 16, a filter unit, and a cap.

18. A nucleic acid recovery kit according to claim 16 or 17, wherein the filter unit is provided with an identifier indicating identification information.

19. A method for recovering nucleic acids present in a liquid sample, comprising: a preparation step of preparing the liquid sample; a suspension step of adding a collector together with a cationic compound to the prepared liquid sample to form a suspension; and a recovery step of recovering the collector contained in the suspension, wherein the collector includes glass fiber or silica, and the cationic compound has a secondary amino group or a quaternary amino group.

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