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  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F120/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
  • C08F120/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
  • C08F120/10—Esters
  • C08F120/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
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  • 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]
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F130/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
  • C08F130/02—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
  • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
  • C08F220/04—Acids; Metal salts or ammonium salts thereof
  • C08F220/06—Acrylic acid; Methacrylic acid; Metal salts or ammonium salts thereof
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
  • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
  • C08F220/10—Esters
  • C08F220/12—Esters of monohydric alcohols or phenols
  • C08F220/16—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms
  • C08F220/18—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms with acrylic or methacrylic acids
  • C08F220/1804—C4-(meth)acrylate, e.g. butyl (meth)acrylate, isobutyl (meth)acrylate or tert-butyl (meth)acrylate
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F230/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
  • C08F230/02—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F30/00—Homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
  • C08F30/02—Homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
• • 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
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

• the present invention relates to a sensitizer for nucleic acid amplification, a composition for nucleic acid amplification, and a test kit.
• the nucleic acid amplification method is a method for amplifying a target nucleic acid from several copies to tens of thousands of times or more, and is used in a wide variety of fields for genetic testing, microbial testing, and virus testing.
• a representative method of nucleic acid amplification is the polymerase chain reaction (PCR) method.
• PCR polymerase chain reaction
• amplification products are detected by visualizing them with a fluorescent compound or the like or measuring the turbidity of the solution at the reaction end point (end point) after a predetermined reaction cycle.
• a typical PCR method is a method of amplifying DNA, but it can also be applied to the amplification of RNA. is called).
• cDNA complementary DNA
• PCR is performed using this cDNA as a template to amplify RNA.
• a method for determining the amount of initial nucleic acid based on the amount of amplified product obtained by the PCR method is also known, and such a PCR method is a quantitative polymerase chain reaction method (hereinafter abbreviated as “qPCR method”). is called).
• qPCR method refers to a real-time PCR method, which is a method of visualizing the amount of DNA amplified in each cycle of PCR using a fluorescent DNA staining reagent or fluorescent probe.
• the method using a fluorescent probe is known as a highly reliable method in that it can specifically detect the amplification of the target nucleic acid.
• RT-qPCR method A reverse transcription-quantitative PCR method (hereinafter sometimes abbreviated as "RT-qPCR method”) that combines the RT-PCR method and the qPCR method is also known.
• the RT-qPCR method is further divided into a 2-step method in which cDNA synthesis and qPCR are performed in separate vessels, and a 1-step method in which these are performed as a series of reactions in the same vessel.
• the 1-step method is superior in terms of ease of operation, little contamination from outside the system, and high detection sensitivity (hereinafter sometimes referred to as "sensitivity") for the nucleic acid to be measured. ing.
• dPCR method digital PCR method
• the PCR method is also used for the purpose of determining the base sequence of the target nucleic acid (sequencing).
• sequencing PCR methods including the Sanger method, all of which are based on the endpoint PCR method.
• Patent Document 1 discloses a mutant PCNA (proliferation nuclear antigen) monomer as a highly versatile DNA replication promoting factor (additive) for promoting the elongation reaction of DNA.
• PCNA proliferation nuclear antigen
• additive highly versatile DNA replication promoting factor
• An object of the present invention is to provide a sensitizer for nucleic acid amplification that is superior in mass productivity and storage stability compared to additives composed of proteins.
• a sensitizer for nucleic acid amplification which is a polymer containing a structural unit derived from a monomer represented by: [2] The sensitizer for nucleic acid amplification according to [1] above, which is a homopolymer consisting of one structural unit derived from the monomer represented by formula (1) above. [3] Formula (2):
• R 4 represents a hydrogen atom or a methyl group
• R 5 represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.
• R 6 represents a hydrogen atom or a methyl group
• R 7 represents a C 3-6 alkyl group having two or more hydroxy groups.
• a composition for nucleic acid amplification comprising the sensitizer for nucleic acid amplification according to any one of [1] to [5] above.
• a primer is an oligonucleotide having a length of 10 to 40 bases.
• the primer concentration is 0.1 to 3.0 ⁇ M.
• test kit comprising the composition for nucleic acid amplification according to any one of [6] to [11].
• a nucleic acid amplification method comprising using the polymer according to any one of [1] to [5] above as a sensitizer.
• Preparing a reaction solution for nucleic acid amplification by mixing the composition for nucleic acid amplification according to any one of [6] to [11] above with a sample containing a nucleic acid to be amplified.
• Nucleic acid amplification methods including.
• the sensitizer for nucleic acid amplification of the present invention can improve the nucleic acid detection sensitivity in the nucleic acid amplification method.
• the sensitizer for nucleic acid amplification of the present invention is a synthetic polymer, it is excellent in mass productivity and storage stability as compared with additives composed of proteins.
• (meth)acryloyloxy group basically means “acryloyloxy group or methacryloyloxy group”.
• (meth)acryloyloxy group means "acryloyloxy group and/or methacryloyloxy group”.
• Other terms similar to "(meth)acryloyloxy group” have the same meaning as "(meth)acryloyloxy group”.
• stepwise numerical ranges are described in this specification, the lower and upper limits of each numerical range can be combined.
• “preferably 10 to 100, more preferably 20 to 90” is described, “preferable lower limit: 10” and “more preferred upper limit: 90” can be combined (i.e., “10 to 90” is also within the scope of this specification).
• the sensitizer for nucleic acid amplification of the present invention (hereinafter sometimes referred to as “the sensitizer of the present invention”) has the following formula (1):
• a polymer (hereinafter sometimes abbreviated as “the polymer of the present invention") containing a structural unit derived from a monomer represented by (hereinafter sometimes abbreviated as “monomer (1)”) be.
• the sensitizer for nucleic acid amplification means an additive for improving the nucleic acid detection sensitivity in the nucleic acid amplification method.
• Nucleic acid amplification methods include, for example, Polymerase Chain Reaction (PCR) method, Loop mediated isothermal amplification (LAMP) method, Transcription Mediated Amplification (TMA) method, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (IICAN) method, Strand Displacement Amplification (SDA) method, Ligase Chain Reaction (LCR) method, Nucleic Acid Sequence-Based Amplification (NASBA) method, and the like.
• the nucleic acid amplification method is preferably PCR method. That is, the sensitizers of the present invention are preferably used in polymerase chain reaction methods.
• the above-mentioned reverse transcription polymerase chain reaction method is preferable. That is, the sensitizer of the present invention is preferably used in the reverse transcription polymerase chain reaction method.
• the aforementioned quantitative polymerase chain reaction method is preferable. That is, the nuclear sensitizer of the present invention is preferably used in a quantitative polymerase chain reaction method.
• the sensitizer of the present invention may be used alone or in combination of two or more. Moreover, the sensitizer of the present invention may be used in combination with other additives.
• structural unit (1) is the carbon-carbon double structure of the (meth)acryloyl group contained in the monomer (1) It means a structural unit having a structure formed by a bond reaction.
• Structural units derived from other monomers have the same meanings as structural units derived from monomer (1).
• the sensitizer of the present invention may be a homopolymer consisting of one type of structural unit (1) or a copolymer containing two or more types of structural units (1).
• the copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.
• the sensitizer of the present invention is preferably a homopolymer consisting of one type of structural unit (1), more preferably two It is a homopolymer composed of a structural unit derived from -(meth)acryloyloxyethylphosphorylcholine, more preferably a homopolymer composed of a structural unit derived from 2-methacryloyloxyethylphosphorylcholine.
• X 1 is preferably a (meth)acryloyloxy group, more preferably a methacryloyloxy group.
• L 1 in formula (1) represents an alkylene group having 2 to 4 carbon atoms which may have one hydroxy group, or an alkyleneoxyalkylene group having 2 to 4 carbon atoms.
• the alkylene group may be linear or branched.
• Examples of the alkylene group having 2 to 4 carbon atoms which may have one hydroxy group include -C 2 H 4 -.
• Examples of the alkyleneoxyalkylene group having 2 to 4 carbon atoms include -C 2 H 4 -O-C 2 H 4 -.
• L 1 is preferably -C 2 H 4 - or -C 2 H 4 -O-C 2 H 4 -, more preferably -C 2 H 4 - (that is, ethylene group is).
• R 1 to R 3 in formula (1) each independently represent an alkyl group having 1 to 3 carbon atoms.
• the alkyl group may be linear or branched.
• Examples of the alkyl group having 1 to 3 carbon atoms include methyl group, ethyl group and propyl group. From the viewpoint of raw material availability, R 1 to R 3 are both preferably methyl groups.
• Preferred monomers (1) are those in which X 1 is a (meth)acryloyloxy group, L 1 is -C 2 H 4 - or -C 2 H 4 -O-C 2 H 4 -, and A monomer in which R 1 to R 3 are methyl groups.
• a more preferred monomer (1) is a monomer in which X 1 is a (meth)acryloyloxy group, L 1 is an ethylene group, and R 1 to R 3 are methyl groups (that is, 2- (meth)acryloyloxyethylphosphorylcholine).
• a more preferred monomer is 2-methacryloyloxyethylphosphorylcholine.
• Monomer (1) can use a commercial item.
• the polymer of the present invention has the formula (2):
• Monomer (2) may be used alone or in combination of two or more. That is, the polymer of the present invention may be a copolymer containing one or more structural units (1) and one or more structural units (2). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.
• R4 in formula (2) represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of the polymer, R4 is preferably a methyl group.
• R 5 in formula (2) represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.
• the alkyl group may be linear or branched.
• alkyl groups having 1 to 20 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group
• R 5 is preferably an alkyl group having 1 to 20 carbon atoms, more preferably an alkyl group having 2 to 20 carbon atoms, and still more preferably an alkyl group having 12 to 18 carbon atoms. and particularly preferably straight-chain alkyl groups having 12 to 18 carbon atoms.
• R 5 is preferably an alkyl group having 3 to 6 carbon atoms.
• the monomer (2) include (meth)acrylic acid, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, pentyl (meth)acrylate, hexyl ( meth)acrylate, heptyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, undecyl (meth)acrylate, dodecyl (meth)acrylate, tridecyl (meth)acrylate, tetradecyl (meth)acrylate Acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate, heptadecyl (meth)acrylate, stearyl (meth)acrylate and the like.
• Monomer (2) can use a commercial item.
• (meth)acrylic acid ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, heptyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, undecyl (meth)acrylate, dodecyl (meth)acrylate, tridecyl (meth)acrylate, tetradecyl (meth)acrylate, pentadecyl ( meth)acrylate, cetyl (meth)acrylate, heptadecyl (meth)acrylate and stearyl (meth)acrylate are preferred, butyl (meth)acrylate, dodecyl (meth)acrylate, tridecyl (meth)acrylate,
• a total of 100 mol of the structural unit (1) and the structural unit (2) (that is, the The amount of the structural unit (1) (that is, the monomer (1) used in the polymerization) is preferably 30 to 99 with respect to the total of 100 mol of the monomer (1) and the monomer (2).
• mol, more preferably 30 to 90 mol, still more preferably 50 to 90 mol, particularly preferably 75 to 90 mol, and the amount of the structural unit (2) (that is, the monomer (2) used in the polymerization) is , preferably 1 to 70 mol, more preferably 10 to 70 mol, still more preferably 10 to 50 mol, particularly preferably 10 to 25 mol.
• the polymer of the present invention has the formula (3):
• structural unit (3) derived from a monomer represented by (hereinafter sometimes abbreviated as “monomer (3)”) good too.
• Monomer (3) may be used alone or in combination of two or more. That is, the polymer of the present invention may be a copolymer containing one or more structural units (1) and one or more structural units (3), or one or more structural units (1 ), one or more structural units (2) and one or more structural units (3).
• the copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.
• R6 represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of the polymer, R6 is preferably a methyl group.
• R 7 in formula (3) represents a C 3-6 alkyl group having two or more hydroxy groups.
• the number of hydroxy groups in R 7 is preferably 2-5.
• the alkyl group may be linear or branched. Examples of alkyl groups having 3 to 6 carbon atoms include propyl, butyl, pentyl and hexyl groups.
• the monomer (3) include glycerol mono(meth)acrylate, threitol mono(meth)acrylate, erythritol mono(meth)acrylate, xylitol mono(meth)acrylate, arabitol mono(meth)acrylate, mannitol. mono(meth)acrylate, galactitol mono(meth)acrylate, sorbitol mono(meth)acrylate and the like.
• glycerin mono(meth)acrylate and xylitol mono(meth)acrylate are preferred, glycerin mono(meth)acrylate is more preferred, and glycerin monomethacrylate is even more preferred.
• monomer (3) A commercially available product may be used as the monomer (3), or it may be produced by a known method.
• monomer (3) can be produced by an esterification reaction between (meth)acrylic acid or its derivative (eg, acid chloride) and a polyhydric alcohol having 3 or more hydroxy groups.
• the esterification reaction is well known, and a person skilled in the art can set the conditions appropriately and perform the reaction.
• a total of 100 mol of the structural unit (1) and the structural unit (3) (that is, the The amount of the structural unit (1) (that is, the monomer (1) used in the polymerization) is preferably 30 to 80 with respect to the total of 100 mol of the monomer (1) and the monomer (3).
• mol, more preferably 30 to 70 mol, more preferably 30 to 60 mol, and the amount of the structural unit (3) (that is, the monomer (3) used in the polymerization) is preferably 20 to 70 mol, more preferably is 30 to 70 mol, more preferably 40 to 70 mol.
• the polymer of the present invention contains the structural unit (1), the structural unit (2) and the structural unit (3), from the viewpoint of the sensitizing effect, the structural unit (1), the structural unit (2) and the structural unit (3) ) (i.e., total 100 mol of monomer (1), monomer (2) and monomer (3) used in polymerization), the amount of structural unit (1) (i.e.
• the monomer (1) used in the polymerization is preferably 30 to 80 mol, more preferably 30 to 70 mol, still more preferably 30 to 60 mol, and the amount of the structural unit (2) (that is, the polymerization
• the monomer (2) used in the above is preferably 10 to 60 mol, more preferably 20 to 60 mol, and still more preferably 30 to 60 mol, and the amount of the structural unit (3) (that is, used for polymerization
• the monomer (3)) is preferably 10 to 60 mol, more preferably 10 to 50 mol, still more preferably 10 to 40 mol.
• the polymer of the present invention may contain other structural units derived from monomers different from the monomers (1) to (3) as long as the effects of the present invention are not impaired.
• Other monomers may be used alone or in combination of two or more. Examples of other monomers include, but are not particularly limited to, benzyl (meth)acrylate, isobornyl (meth)acrylate, and the like.
• the amount of other structural units in the polymer of the present invention is preferably 20 mol % or less with respect to all structural units. More preferably, the polymer of the present invention does not contain other structural units.
• the polymer of the present invention is preferably a homopolymer consisting of one type of structural unit (1), a copolymer consisting of a structural unit (1) and a structural unit (2), a structural unit (1), a structural unit ( 2) and at least one selected from the group consisting of a copolymer consisting of a structural unit (3), more preferably a homopolymer consisting of one structural unit (1), a structural unit (1) and a structural unit It is a copolymer consisting of (2), or a copolymer consisting of structural unit (1), structural unit (2) and structural unit (3).
• a homopolymer consisting of one type of structural unit (1) means a homopolymer in which all of its structural units (repeating units) consist of one type of structural unit (1).
• a copolymer consisting of a structural unit (1) and a structural unit (2) refers to a copolymer in which all of its structural units (repeating units) consist of a structural unit (1) and a structural unit (2).
• “Copolymer consisting of structural unit (1), structural unit (2) and structural unit (3)” means that all structural units (repeating units) are structural unit (1), structural unit (2 ) and the structural unit (3). Other similar expressions have similar meanings.
• the weight average molecular weight of the polymer of the present invention is not particularly limited, it is preferably 10,000 to 1,000,000.
• the weight average molecular weight can be determined in terms of polyethylene glycol by gel filtration chromatography using EcoSEC system (manufactured by Tosoh Corporation) or the like.
• the polymer of the present invention can be produced by known methods (for example, the method described in International Publication No. 2018/216628).
• the amount of the sensitizer of the present invention is determined by the concentration in the composition for nucleic acid amplification described below.
• the concentration of the sensitizer of the present invention in the composition is preferably 0.00001 to 10 w/v%, more preferably 0.001 to 1 w, from the viewpoint of the sensitization effect and suppression of viscosity increase of the composition. /v%, more preferably 0.01 to 0.5w/v%.
• the concentration means the total concentration of the two or more sensitizers. Concentrations described for other ingredients below also refer to the sum of the concentrations of the two or more ingredients when two or more of the ingredients are used.
• Preferred examples of the sensitizer of the present invention include the following sensitizer of the present invention (I) (polymer (I) of the present invention) to sensitizer of the present invention (IV ) (the polymer (IV) of the present invention).
• the sensitizer (I) of the present invention is the following homopolymer (I-1), copolymer (I-2) and copolymer (I-3) It is at least one selected from the group consisting of, preferably the following homopolymer (I-1), copolymer (I-2) or copolymer (I-3): 2-(meth) homopolymer (I-1) consisting of a structural unit (1) derived from acryloyloxyethylphosphorylcholine; a structural unit (1) derived from 2-(meth)acryloyloxyethylphosphorylcholine; (Meth) acrylic acid, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, pentyl (meth) acrylate, hexyl (meth) acrylate
• a copolymer (I-2) having 99 moles and an amount of the structural unit (2) of 1 to 70 moles; and a structural unit (1) derived from 2-(meth)acryloyloxyethylphosphorylcholine, (Meth) acrylic acid, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, pentyl (meth) acrylate, hexyl (meth) acrylate, heptyl (meth) acrylate, octyl (meth) acrylate, nonyl ( meth) acrylate, decyl (meth) acrylate, undecyl (meth) acrylate, dodecyl (meth) acrylate, tridecyl (meth) acrylate, tetradecyl (meth) acrylate, pentadecyl (meth) acrylate, cetyl (meth) acrylate,
• the amount of the structural unit (1) in the copolymer (I-2) is 30 to 90 mol, and the structural unit ( The amount of 2) is 10 to 70 mol, and the amount of structural unit (1) in copolymer (I-3) is 30 to 70 mol, and the amount of structural unit (2) is 20 to 60 mol. and the amount of the structural unit (3) is preferably 10 to 50 mol.
• the amount of the structural unit in the copolymer (I-2) is based on the total amount of the structural unit (1) and the structural unit (2) of 100 mol, and the amount of the structural unit in the copolymer (I-3) is The standard amount of structural units is 100 mol in total of structural unit (1), structural unit (2) and structural unit (3).
• the amount of the structural unit (1) in the copolymer (I-2) is 50 to 90 mol, and the structural unit ( The amount of 2) is 10 to 50 mol, and the amount of structural unit (1) in copolymer (I-3) is 30 to 60 mol, and the amount of structural unit (2) is 30 to 60 mol. and the amount of the structural unit (3) is more preferably 10 to 40 mol.
• the amount of the structural unit in the copolymer (I-2) is based on the total amount of the structural unit (1) and the structural unit (2) of 100 mol, and the amount of the structural unit in the copolymer (I-3) is The standard amount of structural units is 100 mol in total of structural unit (1), structural unit (2) and structural unit (3).
• the amount of the structural unit (1) in the copolymer (I-2) is 75 to 90 mol, and the structural unit ( The amount of 2) is 10 to 25 mol, and the amount of structural unit (1) in copolymer (I-3) is 30 to 60 mol, and the amount of structural unit (2) is 30 to 60 mol. and the amount of the structural unit (3) is more preferably 10 to 40 mol.
• the amount of the structural unit in the copolymer (I-2) is based on the total amount of the structural unit (1) and the structural unit (2) of 100 mol, and the amount of the structural unit in the copolymer (I-3)
• the standard amount of structural units is 100 mol in total of structural unit (1), structural unit (2) and structural unit (3).
• the sensitizer (II) of the present invention is the following homopolymer (II-1), copolymer (II-2) and copolymer (II-3) It is at least one selected from the group consisting of, preferably the following homopolymer (II-1), copolymer (II-2) or copolymer (II-3): Homopolymer (II-1) consisting of a structural unit (1) derived from 2-methacryloyloxyethylphosphorylcholine; A structural unit (1) derived from 2-methacryloyloxyethylphosphorylcholine; derived from butyl (meth)acrylate, dodecyl (meth)acrylate, tridecyl (meth)acrylate, tetradecyl (meth)acrylate, pentadecyl (
• the amount of the structural unit (1) in the copolymer (II-2) is 50 to 90 mol, and the structural unit ( The amount of 2) is 10 to 50 mol, and the amount of structural unit (1) in copolymer (II-3) is 30 to 60 mol, and the amount of structural unit (2) is 30 to 60 mol. and the amount of the structural unit (3) is preferably 10 to 40 mol.
• the amount of the structural unit in the copolymer (II-2) is based on the total amount of the structural unit (1) and the structural unit (2) of 100 mol, and the amount of the structural unit in the copolymer (II-3) is The standard amount of structural units is 100 mol in total of structural unit (1), structural unit (2) and structural unit (3).
• the amount of the structural unit (1) in the copolymer (II-2) is 75 to 90 mol, and the structural unit ( The amount of 2) is 10 to 25 mol, and the amount of structural unit (1) in copolymer (II-3) is 30 to 60 mol, and the amount of structural unit (2) is 30 to 60 mol. and the amount of the structural unit (3) is more preferably 10 to 40 mol.
• the amount of the structural unit in the copolymer (II-2) is based on the total amount of the structural unit (1) and the structural unit (2) of 100 mol, and the amount of the structural unit in the copolymer (II-3) is The standard amount of structural units is 100 mol in total of structural unit (1), structural unit (2) and structural unit (3).
• the sensitizer (III) of the present invention is the following homopolymer (III-1), copolymer (III-2) and copolymer (III-3) At least one selected from the group consisting of preferably the following homopolymer (III-1), copolymer (III-2) or copolymer (III-3): Homopolymer (III-1) consisting of a structural unit (1) derived from 2-methacryloyloxyethylphosphorylcholine; A structural unit (1) derived from 2-methacryloyloxyethylphosphorylcholine; and the structural unit (2) derived from butyl (meth)acrylate or stearyl (meth)acrylate, and the amount of the structural unit (1) per 100 mol of the total of the structural unit (1) and the structural unit (2) is 50 to 90 mol, and the amount of the structural unit (2) is 10 to 50
• the amount of the structural unit (1) in the copolymer (III-2) is 75 to 90 mol, and the structural unit ( The amount of 2) is preferably 10 to 25 mol.
• the standard for the amount of the structural units in the copolymer (III-2) is 100 mol in total of the structural units (1) and (2).
• the sensitizer (IV) of the present invention is the following homopolymer (IV-1), copolymer (IV-2) and copolymer (IV-3) It is at least one selected from the group consisting of, preferably the following homopolymer (IV-1), copolymer (IV-2) or copolymer (IV-3): Homopolymer (IV-1) consisting of a structural unit (1) derived from 2-methacryloyloxyethylphosphorylcholine; A structural unit (1) derived from 2-methacryloyloxyethylphosphorylcholine; and a structural unit (2) derived from butyl methacrylate or stearyl methacrylate, and the amount of the structural unit (1) is 75 to 90 mol per 100 mol in total of the structural unit (1) and the structural unit (2).
• composition for nucleic acid amplification The present invention also provides a composition for nucleic acid amplification containing the sensitizer of the present invention (hereinafter sometimes referred to as "the composition of the present invention").
• the sensitizer of the present invention may be used alone or in combination of two or more.
• the composition for nucleic acid amplification means a composition used in a nucleic acid amplification method.
• the explanation of the nucleic acid amplification method is as described above.
• the nucleic acid amplification method is preferably PCR method.
• the compositions of the invention are preferably used in polymerase chain reaction methods.
• the above-mentioned reverse transcription polymerase chain reaction method is preferable.
• the compositions of the present invention are preferably used in reverse transcription polymerase chain reaction methods.
• compositions of the invention are preferably used in quantitative polymerase chain reaction methods.
• composition of the present invention can be prepared by dissolving the sensitizer of the present invention in a solvent such as water, optionally together with other components used for nucleic acid amplification. That is, the composition of the invention is preferably a composition comprising the sensitizer of the invention and water (and optionally other ingredients). A description of the concentration of the sensitizer of the invention in the composition of the invention is provided above.
• components used for nucleic acid amplification known components used for known nucleic acid amplification typified by PCR can be used. Such components include, for example, buffers, substrates, primers, DNA polymerases, fluorescent DNA staining reagents, fluorescent probes, passive references, nucleic acids and the like. Any of the other components may be used alone or in combination of two or more.
• the buffer is not particularly limited, but for example, a base such as tris(hydroxymethyl)aminomethane, tricine or bicine and an acid such as sulfuric acid, hydrochloric acid, acetic acid or phosphoric acid are mixed to adjust the pH to 6 to 9, more preferably One adjusted to about 7 to 8 can be mentioned.
• the buffer appropriately contains a magnesium salt and/or a manganese salt.
• the buffer may further contain salts such as potassium chloride and ammonium sulfate.
• the buffer may further contain a water-soluble organic solvent such as dimethylsulfoxide, dimethylformamide, formamide and glycerin.
• the buffer may further contain surfactants such as polyoxysorbitan fatty acid esters and polyoxyethylene alkylphenyl ethers.
• the buffer may further contain a protein such as bovine serum albumin.
• the substrate is not particularly limited, for example, deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), mixtures of deoxythymidine triphosphate (dCTP) (dNTPs) is mentioned.
• dATP deoxyadenosine triphosphate
• dTTP deoxythymidine triphosphate
• dGTP deoxyguanosine triphosphate
• dCTP mixtures of deoxythymidine triphosphate
• dNTPs mixtures of deoxythymidine triphosphate
• dTTP deoxyuridine triphosphate
• ddATP dideoxyadenosine triphosphate
• ddTTP dideoxythymidine triphosphate
• ddGTP dideoxyguanosine triphosphate
• ddCTP dideoxythymidine triphosphate
• primers include oligonucleotides with a length of 10 to 40 bases.
• the length of said oligonucleotide is preferably 15-30 bases, more preferably 15-25 bases.
• the oligonucleotides can be designed and prepared by known methods.
• the oligonucleotides may have fluorescent groups formed from fluorescein and the like.
• the primer concentration in the composition of the present invention is preferably 0.1-25 ⁇ M, more preferably 0.1-15 ⁇ M, still more preferably 0.5-10 ⁇ M.
• a known DNA polymerase can be used as the DNA polymerase. From the viewpoint of heat resistance, enzymes derived from thermophilic bacteria, thermophilic archaea, hyperthermia bacteria, hyperthermia archaea, and mutant enzymes thereof are preferred.
• a DNA polymerase is appropriately selected from a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, or an enzyme having both functions, depending on the purpose of nucleic acid amplification. Moreover, whether to use a DNA polymerase having nuclease activity or a DNA polymerase having no nuclease activity is appropriately selected.
• Fluorescent DNA staining reagents are not particularly limited, but include, for example, SYBR TM Green I and the like.
• fluorescent probes include, but are not limited to, TaqMan TM probes.
• a passive reference may be appropriately selected according to the purpose of nucleic acid amplification. Passive references include, for example, ROX TM Dye.
• any DNA and/or RNA may be used, for example, as an exogenous control gene, in addition to the aforementioned primers and fluorescent probes.
• the nucleic acid may be synthesized in vitro, or may be prepared from cells, microorganisms, viruses, etc. by known methods.
• the cells, microorganisms, viruses, and the like may be those collected from humans, animals and plants in the natural world or environment, or may be those isolated and cultured.
• composition of the present invention may further contain oils such as mineral oil; solid phase carriers such as glass beads and magnetic beads.
• kit-type products that combine multiple selected ingredients from the above, and master-mix (sometimes called primer mix, pre-mix, etc.) type products in which those ingredients are pre-mixed. good too.
• master-mix sometimes called primer mix, pre-mix, etc.
• the composition of the present invention can be made into a test kit by combining necessary components.
• the invention provides test kits comprising the compositions of the invention.
• the constituent members are not particularly limited, examples thereof include specimen-collecting instruments, specimen-collecting containers, specimen pretreatment reagents, calibration standards, testing instruments, consumables, measurement cassettes, instruction manuals, and the like.
• the constituent members are appropriately selected according to the mode of inspection.
• the components may be included in a test kit. Moreover, you may use a commercial item as said structural member. Also, as the constituent members, those of specified standards may be used.
• test kit of the present invention is used, for example, for genetic testing, microbial testing, and viral testing, preferably for viral testing. That is, the test target of the test reagent test kit of the present invention is preferably a virus.
• test kit of the present invention examples include tests performed in the fields of medicine, veterinary medicine, forensic medicine, drug analysis, food analysis, environmental research, etc. Among these, medical and veterinary medicine Testing performed in the field is preferred, and testing performed in the medical field is more preferred.
• the test kit of the present invention is preferably for clinical testing, more preferably for in vitro diagnosis.
• the present invention provides (i) a nucleic acid amplification method comprising using a polymer of the present invention as a sensitizer, and (ii) a nucleic acid amplification method by mixing a composition of the present invention with a sample containing the nucleic acid to be amplified. Also provided is a nucleic acid amplification method comprising preparing a reaction mixture for amplification.
• the polymer of the present invention and the composition of the present invention in the nucleic acid amplification method of the present invention are as described above.
• the explanation of the nucleic acid amplification method is also as described above, unless otherwise specified.
• the sample used in the nucleic acid amplification method of the present invention contains nucleic acids to be amplified.
• the sample is preferably a sample solution containing water and nucleic acids to be amplified.
• a sample may contain one type of nucleic acid, or may contain two or more types of nucleic acid.
• the concentration of the nucleic acid in the sample is appropriately determined according to the purpose of nucleic acid amplification, but if the concentration can be adjusted, it is preferably 1 to 10 20 copies/ ⁇ L, more preferably 1 to 10 10 copies/ ⁇ L. , more preferably 1 to 10 5 copies/ ⁇ L, particularly preferably 1 to 500 copies/ ⁇ L, and most preferably 1 to 100 copies/ ⁇ L.
• the amount of sample used is preferably a trace amount to 1 ⁇ L, more preferably 0.01 to 1 ⁇ L, more preferably 0.2 to 1 ⁇ L, per 1 ⁇ L of the composition of the present invention. 0.4 ⁇ L.
• the nucleic acid amplification method of the present invention is preferably a reverse transcription polymerase chain reaction method. Also, the nucleic acid amplification method of the present invention is preferably a quantitative polymerase chain reaction method.
• polymer 1 The weight average molecular weight of polymer 1 was 1,030,000 in terms of polyethylene glycol as measured by gel permeation chromatography (hereinafter sometimes abbreviated as "GPC") under the conditions described later.
• polymer 3 a random copolymer (hereinafter referred to as "polymer 3") was obtained.
• the weight average molecular weight of polymer 3 was 600,000 in terms of polyethylene glycol by GPC measurement under the conditions described later.
• Table 1 summarizes the monomers used in Synthesis Examples 1 to 5, their molar ratios, and the weight average molecular weights of the obtained polymers.
• Example solution Positive Control RNA, N set No.2 (N2) attached to the SARS-CoV-2 RT-qPCR Detection Kit (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), nuclease-free purified water ( Hereinafter, it may be described as "PW (nuclease-free)”) to 10 copies/ ⁇ L to prepare a sample solution.
• PW nuclease-free
• reaction solution Using the components of the kit and 50x ROX Passive Reference (manufactured by Nippon Gene Co., Ltd.), a solution having a common composition shown in Table 2 was prepared, and a predetermined amount of the polymer obtained in Synthesis Examples 1 to 5 was added thereto. A composition for nucleic acid amplification was prepared by adding a predetermined amount of PW (nuclease-free).
• 2 ⁇ L of the polymer aqueous solution having a concentration that is 10 times the final concentration of the polymer in the reaction solution was added.
• the final concentration of polymer 1 in the reaction solution is 0.1 w/v%
• 2 ⁇ L of a 1 w/v% aqueous solution of polymer 1 components having a common composition and PW (nuclease-free) solution 15 ⁇ L of the composition for nucleic acid amplification was prepared by mixing with 13 ⁇ L, and 5 ⁇ L of the sample solution was added thereto to prepare 20 ⁇ L of the reaction solution.
• reaction The PCR plate into which the reaction solution was dispensed was set in StepOnePlus TM Real-Time PCR System (manufactured by Applied Bioscience), and RT-qPCR was performed according to the temperature program shown in Table 3. The reading of the fluorescence intensity was performed in step #5 in Table 3.
• Comparative Examples 2 to 5 the sensitizers of the present invention (that is, polymers 1 to 5) used in Examples 1 to 5 were added to each additive shown in Table 4-2 (that is, bovine serum albumin (hereinafter "BSA”, manufactured by Sigma-Aldrich), dimethyl sulfoxide (hereinafter referred to as "DMSO”), polyethylene glycol 6000 (hereinafter referred to as "PEG 6000”), or T4 Gene 32 Protein (manufactured by Nippon Gene Co., Ltd.)).
• BSA bovine serum albumin
• DMSO dimethyl sulfoxide
• PEG 6000 polyethylene glycol 6000
• T4 Gene 32 Protein manufactured by Nippon Gene Co., Ltd.
• the endpoint enhancement rates of Examples 1-5 using the sensitizers of the present invention were 372-583%.
• the endpoints of Comparative Examples 2-5 using additives other than the sensitizer of the present invention i.e., BSA, DMSO, PEG 6000 or T4 Gene 32 Protein
• the enhancement rate was 76-157%.
• Example 6 to 10 and Comparative Example 6 [sample] Positive Control RNA, N set No.2 (N2) attached to the SARS-CoV-2 RT-qPCR Detection Kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was subjected to PW (nuclease-free) according to the package insert of the kit. Sample solutions were prepared by diluting and having nucleic acid concentrations of 160, 40, 10, 2.5 or 0.0625 copies/ ⁇ L. In addition, PW (nuclease-free) was used as a negative control in place of the sample solution.
• reaction solution Using the components of the kit, 50x ROX Passive Reference (manufactured by Nippon Gene Co., Ltd.), and the polymers obtained in Synthesis Examples 1 to 5, nucleic acid amplification was carried out in the same manner as in Examples 1 to 5 and Comparative Example 1. 15 ⁇ L of composition was prepared. This composition for nucleic acid amplification was spiked with 5 ⁇ L of the sample solution to prepare a reaction solution of 20 ⁇ L in total. The final concentration of the polymer in the reaction solution is as shown in Table 5 below.
• the sensitizer of the present invention it is possible to improve the detection sensitivity of the nucleic acid to be measured in the nucleic acid amplification method (especially the PCR method).
• the sensitizer of the present invention can be suitably used in nucleic acid amplification methods for genetic testing, microbial testing, virus testing and the like.

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