WO2022045009A1 - 流体を追跡するための組成物及び流体の追跡方法 - Google Patents

流体を追跡するための組成物及び流体の追跡方法 Download PDF

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Publication number
WO2022045009A1
WO2022045009A1 PCT/JP2021/030610 JP2021030610W WO2022045009A1 WO 2022045009 A1 WO2022045009 A1 WO 2022045009A1 JP 2021030610 W JP2021030610 W JP 2021030610W WO 2022045009 A1 WO2022045009 A1 WO 2022045009A1
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Prior art keywords
nucleic acid
acid molecule
fluid
composition
liposome
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English (en)
French (fr)
Japanese (ja)
Inventor
良久 赤松
遼平 中尾
史子 今村
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Nippon Koei Co Ltd
Yamaguchi University NUC
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Nippon Koei Co Ltd
Yamaguchi University NUC
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Priority to JP2022544549A priority Critical patent/JPWO2022045009A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a composition for tracking a fluid and a method for tracking a fluid. More specifically, the present invention is a composition for tracking a fluid containing a tracker containing a nucleic acid molecule and a carrier as a component, and tracking of a fluid using a tracker containing a nucleic acid molecule and a carrier as a component. Regarding the method.
  • Fluid tracking for groundwater and surface water is carried out in surveys of environmental pollution and groundwater resources, and evaluation of ground and land use.
  • a common method for measuring water source areas such as groundwater, recharge volume, flow path, flow direction, flow velocity, etc. is to insert a tracker that can be distinguished from groundwater into a vertical hole excavated in the ground and use it as an observation point.
  • the tracker contained in the groundwater pumped from the excavated hole is measured by a hydraulic test device. The observed distribution and behavior of trackers are useful for visualizing fluid flow.
  • Patent Document 1 discloses sodium chloride used for investigating a leak in a reservoir
  • Patent Document 2 discloses a fluorescent dye such as uranin used for examining groundwater flow characteristics.
  • the use of these substances may have an adverse effect on the environment.
  • Patent Document 3 discloses a DNA tracer including a hairpin structure and a dangle ring terminal used for determining the presence of a fluid.
  • a hairpin structure or a hairpin structure can be used to withstand the ultrahigh pressure.
  • a complex DNA structure called the dungling end was essential.
  • no particular device was taken to efficiently recover the DNA tracer from the fluid.
  • Non-Patent Document 1 discloses a method of encapsulating DNA as a tracer in silica.
  • silica is easy to make canned stones, and there is a problem that there is a concern about the influence on the deterioration of water quality and distillation efficiency of the treatment apparatus in water treatment.
  • the DNA tracer does not contain fluorescent paint or sodium chloride, it has little adverse effect on the environment.
  • the DNA tracer has problems such as low stability as a tracker for moving a fluid and poor DNA recovery efficiency. Therefore, it is desired to develop a composition for tracking a fluid in which the stability and recovery efficiency of the tracker itself are improved in tracking the fluid. Therefore, it is an object of the present invention to provide a composition for tracking a fluid containing a tracker with less adverse effect on the environment and improved stability and recovery efficiency.
  • the present inventor has improved the stability of the nucleic acid molecule and improved the recovery efficiency of the nucleic acid molecule in the measurement sample by holding the nucleic acid molecule which is an organic compound in the retainer.
  • the present invention has been completed by finding that it can be used as a composition for tracking.
  • a composition for tracking a fluid which comprises a tracker containing a nucleic acid molecule and a retainer holding the nucleic acid molecule as a constituent component.
  • the nucleic acid molecule is at least one selected from the group consisting of a DNA molecule, an RNA molecule, and a chimeric nucleic acid molecule of DNA and RNA.
  • the carrier is a liposome.
  • nucleic acid molecule contains the base sequence shown in any one of SEQ ID NOs: 1 to 3.
  • the composition according to any one of the above [1] to [6], wherein the fluid is groundwater, surface water, sewage or rainwater.
  • the composition according to any one of the above [1] to [7] which contains a fluid obtained from a source into which the composition is charged.
  • Step B of charging the composition into a fluid source (3)
  • Step D of measuring the nucleic acid molecule contained in the fluid sample A method for tracking a fluid, which comprises the process of. [10]
  • the tracking method according to the above [9], wherein the measurement of the nucleic acid molecule in the step D uses a PCR method.
  • Another aspect of the present invention is a method for producing a composition for tracking a fluid, wherein the composition containing a nucleic acid molecule and a tracer containing a liposome holding the nucleic acid molecule as a constituent is 15 ° C.
  • the above method is characterized by standing or stirring for 2 hours or more.
  • the environment is highly stable in tracking the fluid, the recovery efficiency is improved, and the environment does not contain fluorescent paint or sodium chloride. It is possible to provide a composition containing a tracker having less adverse effect on. Further, by using the composition, it is possible to provide a method for tracking a fluid having less adverse effect on the environment.
  • FIG. 1 is a diagram showing the stability of the amount of DNA contained in liposomes.
  • A It shows the stability of DNA in purified water.
  • B It shows the stability of DNA in groundwater.
  • FIG. 2 is a schematic explanatory view of a channel test for confirming the movement of a composition for tracking a fluid.
  • A The test equipment used in the waterway test is shown.
  • B The process of filling the upstream water tank with a fluid is shown.
  • C Shows a state in which the pipeline is filled with a fluid.
  • D The process of putting the composition for tracking a fluid into the upstream water tank is shown.
  • FIG. 3 is a diagram showing a change over time in the amount of DNA in the downstream water tank.
  • Nucleic acid molecules that have not been liposomalized are used.
  • B Liposomes containing nucleic acid molecules are used.
  • C Nucleic acid molecule-containing liposomes were prepared and then allowed to stand at 20 ° C. for about 4 hours.
  • composition for tracking a fluid according to the present invention
  • following method a method for tracking a fluid using the composition
  • the term "fluid” means a liquid, a gas, or a mixture of a liquid and a gas, which is a continuum in which shear stress does not occur in a stationary state.
  • fluids include inland water such as groundwater, rivers, lakes and springs, and sewage such as sewage flowing down a sewage pipeline, rainwater such as rainwater flowing down a pipeline, and the atmosphere. Does not contain oil.
  • source means the source of fluid movement.
  • nucleotide means a compound containing a sugar deoxyribose, ribose, a base, and a phosphate group.
  • the "base” includes purine bases containing natural compounds such as adenine, guanine and inosine, and pyrimidine bases containing thymine, cytosine and uracil.
  • composition for tracking fluid The composition is for tracking a fluid characterized by containing a nucleic acid molecule which is an organic compound and a tracker containing a retainer for the nucleic acid molecule as a constituent component. This can improve the stability or recovery efficiency of nucleic acid molecules and provide a traceable composition without polluting the environment.
  • each component constituting the composition will be described in detail. Unless otherwise specified, the content of each component is shown in the composition.
  • Nucleic acid molecules are organic macromolecular compounds in which nucleotides are linked by phosphodiester bonds.
  • the nucleic acid molecule is not particularly limited as long as it is detectable.
  • Examples of the nucleic acid molecule include a single-stranded DNA molecule, a single-stranded RNA molecule, a double-stranded DNA molecule, a double-stranded RNA molecule, a chimeric nucleic acid molecule of DNA and RNA, a locked nucleic acid, and a modified product thereof. Be done.
  • the nucleic acid molecule may be a natural type or a non-natural type. Further, it may be linear or cyclic.
  • a preferred nucleic acid molecule is a double-stranded DNA molecule synthesized from the viewpoint of stability. Further, the nucleic acid molecule may contain a nucleic acid to be measured and a nucleic acid to be non-measured. Examples of the nucleic acid to be non-measured include a plasmid vector and a viral vector.
  • Modification of the nucleic acid molecule is not particularly limited. Modifications of nucleic acid molecules include, for example, modification of sugars or phosphate skeletons constituting nucleotides. Specific examples of modification of the sugar portion of the nucleotide include, for example, 2'-O-methyl RNA, 2'-O- (2-methoxy) ethyl RNA, 2'-deoxy-2'-fluoroarabinonucleic acid, and bridged type. Nucleic acid (LNA / BNA) and the like can be mentioned.
  • nucleic acid skeleton includes a phosphorothioate form and a phosphorodithioate form.
  • nucleic acid molecules may be used alone or in combination of two or more. These nucleic acid molecules may be appropriately set from known ones according to the properties of the fluid and the like.
  • the base length of the nucleic acid molecule is not particularly limited.
  • the base length of the nucleic acid molecule is, for example, 60 bases or more and 5000 bases or less.
  • the lower limit may be 70 bases or more, 80 bases or more, 90 bases or more, 100 bases or more, 300 bases or more, 500 bases or more, and 1000 bases or more.
  • the upper limit value is 4000 bases or less and 3000 bases or less.
  • the base length of the nucleic acid to be measured by the PCR method that is, the nucleic acid amplified by the PCR method can be 30 bases or more and 1000 bases or less, which is a lower limit value. It may be 70 bases or more, 80 bases or more, and 90 bases or more.
  • the upper limit may be 500 bases or less, 300 bases or less, 150 bases or less, 130 bases or less, 110 bases or less.
  • the nucleic acid to be measured by the PCR method may be repeatedly arranged twice or more. Further, nucleic acids other than those to be measured by the PCR method may be placed at the 5'end and / or 3'end of the nucleic acid to be measured by the PCR method. By arranging such a nucleic acid, it becomes possible to easily prevent the nucleic acid to be measured by the PCR method from being decomposed even if the nucleic acid to be measured by the PCR method is decomposed in the fluid by some influence. As the lower limit of the nucleic acid length other than the measurement target by the above PCR method, 30 bases or more, 50 bases or more, 100 bases or more, 500 bases or more, 1000 bases or more, and 2000 bases or more can be mentioned. When the nucleic acid to be measured by the PCR method is amplified and prepared by a microorganism such as Escherichia coli using a vector, the nucleic acid molecule constituting the vector may be contained.
  • nucleic acid to be measured examples include the ITS1 region of the nuclear DNA of fish or the cytochrome b region of the mitochondrial DNA, and specifically, the ITS1 (Internal Transcribed Spacer1) region of the nuclear DNA of Cyprinus carpio.
  • the base sequence shown by SEQ ID NO: 1 in Table 1 the base sequence shown by SEQ ID NO: 2 in Table 1 in the cytochrome b region of mitochondrial DNA of Ayu (Plecoglossus artivelis), and the cytochrome b region of mitochondrial DNA of Zenitanago (Acheilognathius typeus).
  • examples thereof include a nucleic acid consisting of the base sequence shown by SEQ ID NO: 3 in Table 1.
  • a retainer is a substance that encapsulates, encloses, binds to, or adsorbs a nucleic acid molecule.
  • the carrier is not particularly limited as long as it can hold a nucleic acid molecule.
  • examples of the carrier include liposomes, micelles, microcapsules, virus particles, inorganic particles, soil and the like.
  • Preferred carriers are liposomes from the viewpoint of stability and recovery efficiency of nucleic acid molecules. Further, these carriers may be used alone or in combination of two or more. These retainers may be appropriately set from known ones according to the properties and fluidity of the fluid.
  • the sealing includes a state in which the entire nucleic acid molecule is present inside the retainer, or a state in which a part of the nucleic acid molecule is present inside the retainer. That is, when a part of the nucleic acid molecule is present inside the retainer, the rest of the nucleic acid molecule is present outside the retainer through the retainer or through a gap.
  • the particle size of the retainer is not particularly limited and can be appropriately adjusted according to the type of the retainer.
  • the particle size of the retainer is, for example, 10 nm or more and 700 ⁇ m or less.
  • the lower limit is more preferably 200 nm or more, further preferably 500 nm or more, particularly preferably 1 ⁇ m or more, still more preferably 5 ⁇ m or more.
  • the upper limit value is more preferably 500 ⁇ m or less, further preferably 300 ⁇ m or less, and particularly preferably 100 ⁇ m or less.
  • the average particle size of the retainer is not particularly limited and can be appropriately adjusted according to the type of retainer.
  • the average particle size of the retainer is, for example, 50 nm or more and 200 ⁇ m or less.
  • the lower limit is more preferably 500 nm or more, further preferably 1 ⁇ m or more, and particularly preferably 10 ⁇ m or more.
  • the upper limit value is more preferably 100 ⁇ m or less, further preferably 80 ⁇ m or less, and particularly preferably 60 ⁇ m or less.
  • the retainer By setting the particle size or average particle size of the retainer in the above range, the movement similar to that of a fluid can be exhibited, the retention ability for nucleic acid molecules can be enhanced, and the nucleic acid molecules can be easily recovered or concentrated as a measurement sample.
  • the retainer may have a single particle diameter, but may be a mixture of retainers having a distribution of particle diameters having a predetermined width. Such a mixture makes it possible to track the retainer even in an underground environment with a wide distribution of gap widths.
  • the lower limit of the width in the particle size distribution of the retainer is 1 ⁇ m or more, 10 ⁇ m or more, or 30 ⁇ m or more, and the upper limit can be 100 ⁇ m or less, 70 ⁇ m or less, and 50 ⁇ m or less.
  • Liposomes are spherical vesicles with a lipid bilayer membrane.
  • the liposome is not particularly limited as long as it can retain a nucleic acid molecule.
  • Examples of the liposome include giant monolayer liposomes (GUV: Giant Unilamellar Vesicle), small monolayer liposomes, multilamellar liposomes, and the like, and giant monolayer liposomes having a large particle size are preferable.
  • the method for producing a liposome encapsulating, enclosing, binding, or adsorbing a nucleic acid molecule may be performed by a method known to those skilled in the art in the presence of the nucleic acid molecule, and is not particularly limited.
  • Examples of the method for producing liposomes include an ultrasonic treatment method, a reverse phase evaporation method, a freeze-thaw method, a lipid dissolution method, and a spray drying method. Further, the liposome may be adjusted to a particle size in a specific range by passing it through a filter having a small pore size using an extruder or the like, or the variation in the particle size may not be adjusted.
  • the constituents of the liposome are not particularly limited as long as they can form vesicles having a lipid bilayer membrane.
  • Examples of the constituent components of the liposome include phospholipids, cationic lipids, cholesterols and the like. Further, these lipids and cholesterol may be modified with polyethylene glycol, modified with sugar chains, hydrogenated or the like.
  • phospholipids include, for example, phosphatidylcholines such as 1,2-dioreoil-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylcholine, and dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, and distearoylphosphatidylethanolamine.
  • DOPC 1,2-dioreoil-sn-glycero-3-phosphocholine
  • DOPC 1,2-dioreoil-sn-glycero-3-phosphocholine
  • dipalmitoylphosphatidylcholine dipalmitoylphosphatidylcholine
  • dipalmitoylphosphatidylethanolamine dipalmitoylphosphatidylethanolamine
  • distearoylphosphatidylethanolamine distearoylphosphatidylethanolamine
  • Phosphatidylethanolamines such as dipalmitoylphosphatidylserine, phosphatidylserine such as dipalmitoylphosphatidylserine, phosphatidylic acids such as dipalmitoylphosphatidylic acid, distearoylphosphatidylic acid, dipalmitoylphosphatidylinositol, distearoylphosphatidylinositol, etc.
  • Egg yolk lecithin, soy lecithin, natural phospholipids such as lysolecithin and the like.
  • cationic lipids include, for example, 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP), dioctadecyldimethylammonium chloride, N-2,3-diorailoxypropyl-N, N, N, -trimethylammonium, didodecylammonium bromide, 1,2-dimyristyloxypropyl 1-3-dimethylhydroxyethylammonium, 2,3-dioleoyloxy-N- [2 (S permincarboxamide) ethyl] -N , N-dimethyl-1-propanamium trifluoroacetate and the like.
  • DOTAP 1,2-dioleoyloxy-3-trimethylammonium propane
  • DOTAP dioctadecyldimethylammonium chloride
  • N-2,3-diorailoxypropyl-N N, N, -trimethylammonium
  • cholesterol examples include cholesterol, 3 ⁇ - [N- (dimethylaminoethane) carbamoyl] cholesterol, N-trimethylammonioethylcarbamoyl cholesterol and the like.
  • the molecular weight of the polyethylene glycol that modifies the lipid is not particularly limited, and examples thereof include 1000 Da, 2000 Da, 5000 Da, 10000 Da, 20000 Da, 30000 Da, and 40,000 Da.
  • these liposome constituents may be used alone or in combination of two or more. These liposome constituents may be appropriately set from known ones according to the properties of the nucleic acid molecule and the fluid.
  • the content of phospholipids, cationic lipids, and cholesterol in liposomes is not particularly limited.
  • the content of the phospholipid is, for example, 10 parts by mass or more and 100 parts by mass or less, assuming that the entire liposome is 100 parts by mass.
  • the lower limit is more preferably 30 parts by mass or more, further preferably 50 parts by mass or more, and particularly preferably 70 parts by mass or more.
  • the upper limit value is more preferably 95 parts by mass or less, further preferably 92 parts by mass or less, and particularly preferably 90 parts by mass or less.
  • the content of the cationic lipid is, for example, 0 parts by mass or more and 80 parts by mass or less, assuming that the entire liposome is 100 parts by mass.
  • the lower limit is more preferably 10 parts by mass or more, further preferably 20 parts by mass or more, and particularly preferably 30 parts by mass or more.
  • the upper limit value is more preferably 70 parts by mass or less, further preferably 60 parts by mass or less, and particularly preferably 50 parts by mass or less.
  • the content of cholesterol is, for example, 0 parts by mass or more and 50 parts by mass or less, assuming that the entire liposome is 100 parts by mass.
  • the lower limit is more preferably 5 parts by mass or more, further preferably 8 parts by mass or more, and particularly preferably 10 parts by mass or more.
  • the upper limit value is more preferably 40 parts by mass or less, further preferably 30 parts by mass or less, and particularly preferably 20 parts by mass or less.
  • Micelle is a spherical molecular assembly in which amphipathic substances are associated, and is not particularly limited as long as it is acceptable in technical fields such as environment, medicine, pharmaceuticals, and foods.
  • the method for producing micelles may be a method known to those skilled in the art in the presence of nucleic acid molecules, and is not particularly limited.
  • amphipathic substances include the co-weight of hydrophilic blocks such as polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylamide, and hydrophobic blocks such as polyester, polyanhydride, polyamino acid, polyorthoester, and polyphosphazene. Coalescence etc. can be mentioned.
  • amphoteric substance examples include, for example, polyethylene glycol-polyaspartic acid, polyethylene glycol-polyglutamic acid, polyethylene glycol-poly (5-aminopentyl-aspartic acid), polyethylene glycol-polylysine, polyethylene glycol-polyarginine. , Polyethylene Glycol-Polyester, Polyethylene Glycol-Polyethylene, Polyethylene Glycol-Polyacryllate, Polyethylene Glycol-Polymethacrylate and the like. Further, these amphipathic substances may be used alone or in combination of two or more. These amphipathic substances may be appropriately set from known substances according to the properties of the fluid and the nucleic acid molecule.
  • the molecular weight of the hydrophilic block is not particularly limited.
  • the molecular weight of the hydrophilic block is, for example, 200 Da or more and 50,000 Da or less.
  • the lower limit value is more preferably 500 Da or more, further preferably 800 Da or more, and particularly preferably 1000 Da or more.
  • the upper limit value is more preferably 20000 Da or less, further preferably 10000 Da or less, and particularly preferably 5000 Da or less.
  • the molecular weight of the hydrophobic block is not particularly limited.
  • the molecular weight of the hydrophobic block is, for example, 50 Da or more and 50,000 Da or less.
  • the lower limit value is more preferably 200 Da or more, further preferably 500 Da or more, and particularly preferably 1000 Da or more.
  • the upper limit value is more preferably 20000 Da or less, further preferably 10000 Da or less, and particularly preferably 5000 Da or less.
  • microcapsules are those in which a polymer compound forms a spherical film, and are not particularly limited as long as they are acceptable in technical fields such as environment, medicine, pharmaceuticals, and foods.
  • the method for producing microcapsules may be a method known to those skilled in the art in the presence of nucleic acid molecules, and is not particularly limited. For example, an interfacial polymerization method, a core selvation method, an interfacial precipitation method, or in a liquid. The drying method and the like can be mentioned.
  • polymer compound examples include ethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, polylactic acid, polyvinyl pyridine, polymethacrylic acid, polyN-isopropylacrylamide, polystyrene sulfonate, polyallylamine, polyacrylic acid, polyethyleneimine, and polydiallyldimethyl.
  • these polymer compounds may be used alone or in combination of two or more. These polymer compounds may be appropriately set from known compounds according to the properties of the fluid and the nucleic acid molecule.
  • the virus particle is a particle composed of a virus capsid containing a nucleic acid molecule, and is not particularly limited as long as it is non-pathogenic and acceptable in technical fields such as environment, medical treatment, pharmaceuticals, and foods. ..
  • virus particles include adeno-associated virus, lentivirus particles, adenovirus particles, baculovirus particles, parvovirus particles, herpesvirus particles, poxvirus particles, Sindobis virus particles, semuliki forest virus particles, vaccinia virus particles, and retro. Viruses and the like can be mentioned. Further, these virus particles may be used alone or in combination of two or more. These virus particles may be appropriately set from known ones according to the properties of the fluid and nucleic acid molecules.
  • the inorganic particles are particles of an inorganic compound having a retention ability for nucleic acid molecules, and are not particularly limited as long as they are acceptable in technical fields such as environment, medicine, pharmaceuticals, and foods.
  • examples of the inorganic particles include glass particles, hydroxyapatite particles, zeolite particles, activated carbon particles and the like. Further, these inorganic particles may be used alone or in combination of two or more. These inorganic particles may be appropriately set from known ones according to the properties of the fluid and nucleic acid molecules.
  • the soil is not particularly limited as long as it is soil such as volcanic ash soil having an ability to adsorb nucleic acid molecules.
  • Examples of the components contained in the soil include minerals of natural origin such as Andosols, bentonite, and allophane, which have little adverse effect on the environment.
  • the tracker may further contain amino acids, proteins, sugars, inorganic compounds, polymer compounds, magnetic substances and the like.
  • the composition When liposomes are used as the retainer, the composition may be used immediately after production, but from the viewpoint of stable retention of nucleic acid molecules in the liposomes, it is used after standing or stirring under certain conditions. You may.
  • the time for allowing the liposome holding the nucleic acid molecule to stand or stir is not particularly limited.
  • the time for allowing the liposome holding the nucleic acid molecule to stand or stir is, for example, 2 hours or more.
  • the lower limit is more preferably 3 hours or more, still more preferably 4 hours or more, and particularly preferably 5 hours or more.
  • the temperature at which the liposome holding the nucleic acid molecule is allowed to stand or stirred is not particularly limited.
  • the temperature at which the liposome holding the nucleic acid molecule is allowed to stand or stirred can be appropriately adjusted according to the air temperature at the time of carrying out the present invention and the temperature of the fluid, and is, for example, 15 ° C. or higher and 30 ° C. or lower. ..
  • the lower limit may be 16 ° C. or higher, 17 ° C. or higher, 18 ° C. or higher, 19 ° C. or higher, 20 ° C. or higher, 21 ° C. or higher, 22 ° C. or higher.
  • the upper limit may be 27 ° C. or lower and 25 ° C. or lower.
  • composition containing the liposome in which the nucleic acid is retained after being allowed to stand or stir under the above-mentioned constant conditions is extremely complicated, and it is technically impossible or technically impossible to analyze the structure. Performing the task of identifying its structure requires significantly excessive financial expenditure and time. Therefore, in the description of the present composition, it is so-called “impossible / non-impossible” to describe that "the liposome is left to stand or stirred at 20 ° C. or higher for 2 hours or longer after the fabrication of the liposome holding the nucleic acid molecule". There is a "practical situation”.
  • the liposomes carrying the nucleic acid molecules are allowed to stand or stir in an aqueous solution such as a phosphate buffer solution, a Triss hydrochloride buffer solution, groundwater, surface water, sewage, rainwater, water, distilled water, or a mixture thereof. You may go.
  • a buffer solution pH 4 to 9, preferably pH 6 to 8, and more preferably pH 7 to 7.7 can be mentioned.
  • Liposomes holding the nucleic acid molecules that have been left to stand or agitated may then be left to stand or stored at 15 ° C. or lower, if necessary.
  • the composition may be refrigerated or cryopreserved until use.
  • the composition may contain an aqueous solution of groundwater, surface water, sewage, rainwater, water, distilled water, or a mixture thereof, or may be dispersed in such an aqueous solution.
  • groundwater groundwater in the environment of the fluid source can be preferably mentioned.
  • the composition contains a nucleic acid molecule having a small adverse effect on the environment and a tracer containing a retainer for the nucleic acid molecule as a constituent component, thereby improving the stability and recovery efficiency of the nucleic acid molecule and improving the recovery efficiency. It is possible to obtain a composition containing a tracer having less adverse effect on the environment.
  • the fluid tracking method of the present invention includes (1) step A for preparing a composition for tracking the fluid according to the present invention, (2) step B for charging the composition into a fluid source, and (3).
  • the method is characterized by having a step C of preparing a fluid sample at an observation point different from the source, and (4) a step D of measuring a nucleic acid molecule contained in the fluid sample. This can improve the stability of fluid tracking and the recovery efficiency of nucleic acid molecules, and can provide a method that can be safely tracked without polluting the environment. First, each step constituting the tracking method will be described in detail.
  • Step A is a step for preparing a composition for tracking the fluid. This makes it possible to prepare a composition capable of tracing the fluid used in the fluid tracking method.
  • the composition for tracking the fluid is not particularly limited, and may be, for example, any composition that satisfies the above description in the section [Composition for tracking the fluid].
  • Step B is a step of charging the prepared fluid with a composition for tracking the prepared fluid into the fluid source.
  • the method of injecting a composition for tracking a fluid into a fluid source is not particularly limited and is used in technical fields such as environmental resource surveys, environmental pollution surveys, construction, civil engineering and its basic research fields. Ordinary means can be used.
  • the source of the fluid is not particularly limited as long as the composition for tracking the fluid can be charged. Examples of the fluid supply source include groundwater, surface water, sewage, rainwater, well water, spring water, and water leakage in artificial structures existing in holes excavated in the ground.
  • Step C is a step for preparing a fluid sample at an observation point different from the source. This makes it possible to secure a sample for measuring nucleic acid molecules contained in the fluid at the observation point.
  • the method of preparing a fluid sample at the observation point is not particularly limited, and ordinary means used in technical fields such as environmental resource surveys, environmental pollution surveys, construction, civil engineering and its basic research fields are used. be able to.
  • the method for recovering the fluid at the observation point is not particularly limited as long as it can store the nucleic acid molecule of the present invention. Examples of the method for recovering the fluid include pumping the fluid, collecting sediment, and extracting the fluid from the soil.
  • the nucleic acid molecule may be recovered or concentrated by treating the measurement sample with a filter or the like before the measurement. Since the tracer contained in the composition is larger than the nucleic acid molecule, the recovery efficiency is high and effective concentration treatment can be performed.
  • a filter is used for recovery or concentration of nucleic acid molecules, it can be appropriately adjusted according to the average particle size of the carrier used, but the pore size is 0.2 to 2.0 ⁇ m, specifically 0.5 ⁇ m, 0. Those with a diameter of 0.7 ⁇ m and 1.0 ⁇ m are preferable.
  • Step D is a step for measuring the nucleic acid molecule contained in the fluid sample at the observation point. This makes it possible to confirm the existence of the composition at the observation point.
  • the method for measuring a nucleic acid molecule is not particularly limited, and ordinary means used in technical fields such as environment, medicine, pharmaceuticals, and foods and its basic research field can be used. Examples of methods for measuring nucleic acid molecules include PCR (Polymerase Chain Reaction) method for amplifying the number of nucleic acids, electrophoresis method, absorption spectrometry method, fluorescence analysis method, high performance liquid chromatography isotope diluted mass spectrometry method, and inductively coupled plasma emission spectrometry.
  • PCR Polymerase Chain Reaction
  • nucleic acid molecules examples include a method using an analysis method, an inductively coupled plasma mass spectrometry method, and the like.
  • these nucleic acid molecules may be measured alone or in combination of two or more.
  • the method for measuring these nucleic acid molecules may be appropriately set from known ones according to the properties of the nucleic acid molecules and the like.
  • the Tracking Method uses a nucleic acid molecule that has a small adverse effect on the environment and a tracker that contains a retainer for the nucleic acid molecule as a constituent component, thereby improving the stability and recovery efficiency of the nucleic acid molecule and the environment. It is possible to provide a traceable method without contaminating the nucleic acid.
  • Example 1 nucleic acid molecule-encapsulated liposomes used in the waterway test of Example 2 described later were prepared. In addition, the characteristics of the prepared nucleic acid molecule-encapsulated liposomes were analyzed.
  • Samples containing nucleic acid molecule-encapsulated liposomes include nucleic acid molecules and 20 mL of 20 mM phospholipids (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC): 1,2-dioleoyloxy-3- (trimethylammonium)).
  • DOPC 1,2-dioleoyloxy-3- (trimethylammonium)
  • Propane (DOTAP) 2: 1 (molar ratio)
  • phosphate buffer pH 7.2
  • the nucleic acid molecule is a nucleic acid consisting of the base sequence shown by SEQ ID NO: 1 in Table 2 in the ITS1 (Internal Tranceribed Spacer1) region of 3.8 ng of Cyprinus carpio nuclear DNA (synthesized by Eurofin Genomics Co., Ltd.).
  • ITS1 Internal Tranceribed Spacer1
  • the encapsulation rate of nucleic acid molecules in liposomes is measured by measuring the absorbance of the dialed sample and the pre-dialysis sample at 260 nm with a spectrophotometer (U-2800A, Hitachi High-Tech Science) (measured by Hashimoto Electronics Co., Ltd.) as follows. It was calculated as a percentage from Equation 1 of.
  • the collected analytical sample was filtered through a glass filter (GF / F 47 mm, Global Life Science Technologies Japan, Inc.) with a particle retention diameter of 0.7 ⁇ m (GF / F) and treated with 5 ⁇ l of a 10% benzalconium aqueous solution. ..
  • the amount of DNA contained in the sample for analysis was determined by amplifying the ITS1 region of Koi's nuclear DNA by the PCR method using the primer set in Table 3 and then using a DNA detector (Piko Real Real-Time PCR System, Thermo Fisher Scientific). Measured at.
  • the conditions of the PCR method are as follows: after treating at 50 ° C. for 2 minutes and 95 ° C. for 10 minutes, 55 cycles of treatment at 95 ° C.
  • a probe in which the fluorescent substance FAM was linked to the 5'end of the nucleic acid having the base sequence shown in SEQ ID NO: 6 and the quenching substance TAMRA was linked to the 3'end was used.
  • FIG. 1 shows the stability of the amount of DNA contained in liposomes in purified water or groundwater.
  • FIG. 1 (A) it was shown that the nucleic acid molecule-encapsulated liposomes added to the purified water stably retained the nucleic acid molecules for 96 hours.
  • FIG. 1 (B) it was shown that the nucleic acid molecule-encapsulated liposome added to the groundwater stably retains the nucleic acid molecule as in the purified water shown in FIG. 1 (A). rice field. From this, it was confirmed that the nucleic acid molecule-encapsulated liposome of the present invention exhibits sufficient stability regardless of whether it is purified water or groundwater containing impurities.
  • nucleic acid molecule-encapsulated liposome of the present invention has stability.
  • Example 2 In Example 2, it was examined whether the nucleic acid molecule-encapsulated liposome prepared in Example 1 could follow the fluid.
  • the test equipment consists of an acrylic resin upstream water tank that corresponds to the fluid supply source and an acrylic resin downstream water tank that corresponds to the observation point connected by an acrylic resin pipeline with an inner diameter of 8 cm and a length of 100 cm. Is.
  • the pipeline was filled with 6.9 kg of silica sand having a particle size of 2 to 3 mm.
  • the porosity of the pipeline is 52.7%.
  • the size of each water tank is 32 cm ⁇ 37 cm ⁇ 45 cm.
  • tap water was poured into the upstream water tank with the lid on the upstream end of the pipeline removed (Fig.
  • the sample for analysis is 0 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes after removing the lid at the upstream end of the conduit.
  • 100 mL of each of the three samples of the test water in the downstream water tank was sampled.
  • the sample for analysis collected was treated with 0.1 mL of a 10% aqueous solution of benzalkonium.
  • the waterway test was conducted in an environment of 10 ° C or 20 ° C. In addition, the water levels of the upstream water tank and the downstream water tank became uniform between 120 minutes and 180 minutes.
  • the method for measuring the amount of DNA contained in the sample for analysis is the same as that described in the section (Stability test of nucleic acid molecule-encapsulated liposome) in Example 1.
  • the nucleic acid molecule used as a control group was a nucleic acid (synthesized by Eurofin Genomics Co., Ltd.) consisting of the nucleotide sequence shown in SEQ ID NO: 2 in Table 4 in the cytochrome b region of mitochondrial DNA of Ayu (Plecoglossus artivelis) pMD-21.
  • a nucleic acid molecule consisting of linear DNA was used, which was incorporated into a vector, transformed into Escherichia coli JM109, cultured, and then the vector was recovered from Escherichia coli and treated with the restriction enzyme BamHI.
  • the method for measuring the amount of DNA in the control group is the same as that described in the section of Example 1 (Stability test of nucleic acid molecule-encapsulated liposome) except that the primer set shown in Table 5 was used.
  • the probe a probe in which the fluorescent substance FAM was linked to the 5'end of the base sequence shown in SEQ ID NO: 9 and the quenching substance TAMRA was linked to the 3'end was used.
  • FIG. 3 shows the change over time in the amount of DNA in the downstream water tank.
  • FIGS. 3A and 3B when the nucleic acid molecule not encapsulated in the liposome and the nucleic acid molecule-encapsulated liposome are compared, the nucleic acid molecule is encapsulated in the liposome, and the amount of DNA detected in the downstream water tank is large. At the same time, it was shown that stable detection was possible.
  • the liposome-containing solution is further diluted with water in the upstream water tank in which the liposome-containing solution is previously stored, and allowed to stand at room temperature (20 ° C.) for 4 hours. It is the result of the waterway test when the placed one was used. As shown in FIGS.
  • nucleic acid molecule-encapsulated liposome when the nucleic acid molecule-encapsulated liposome is compared with the untreated nucleic acid molecule-encapsulated liposome after the preparation of the nucleic acid molecule-encapsulated liposome, the nucleic acid molecule-encapsulated liposome is statically treated. It was shown that the amount of DNA detected in the downstream water tank increased.
  • nucleic acid molecule-encapsulated liposome of the present invention has significantly improved tracking ability for fluid as compared with the non-liposomal nucleic acid molecule.
  • composition for tracking a fluid and the method for tracking a fluid of the present invention can improve the tracking ability and environmental safety in a fluid tracking survey.
  • investigation of environmental pollution, evaluation of ground and land use, leak test of artificial structures, measurement of flow range and flow time of pipelines (sewage pipelines and rainwater pipelines) and groundwater, ventilation status and efficiency Can be effectively evaluated.

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