WO2023054340A1 - 核酸増幅促進剤およびそれを用いる検査方法 - Google Patents

核酸増幅促進剤およびそれを用いる検査方法 Download PDF

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WO2023054340A1
WO2023054340A1 PCT/JP2022/035891 JP2022035891W WO2023054340A1 WO 2023054340 A1 WO2023054340 A1 WO 2023054340A1 JP 2022035891 W JP2022035891 W JP 2022035891W WO 2023054340 A1 WO2023054340 A1 WO 2023054340A1
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meth
acrylate
nucleic acid
polymer
acid amplification
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English (en)
French (fr)
Japanese (ja)
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裕貴 鈴木
将 松田
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NOF Corp
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NOF Corp
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Priority to CN202280066455.6A priority Critical patent/CN118202067A/zh
Priority to JP2023551515A priority patent/JPWO2023054340A1/ja
Priority to KR1020247014351A priority patent/KR20240099236A/ko
Priority to EP22876206.8A priority patent/EP4410994A4/en
Priority to US18/697,245 priority patent/US20250197922A1/en
Publication of WO2023054340A1 publication Critical patent/WO2023054340A1/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
    • 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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • 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
    • 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

Definitions

  • the present invention relates to a nucleic acid amplification accelerator and a test method using the same.
  • 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 gene cloning and genetic testing.
  • a representative method of nucleic acid amplification is the polymerase chain reaction (PCR) method.
  • PCR polymerase chain reaction
  • a typical PCR method was originally a method of amplifying DNA, but it can also be applied to the amplification of RNA. sometimes).
  • complementary DNA hereinafter sometimes abbreviated as "cDNA”
  • cDNA complementary DNA
  • the RT-PCR method is further divided into a 2-step method in which a reverse transcription reaction and a PCR method are performed in separate vessels, and a 1-step method in which these are performed as a series of reactions in the same vessel.
  • 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).
  • RT-qPCR method reverse transcription-quantitative PCR method
  • 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, but many of them are based on the endpoint PCR method.
  • nucleic acid amplification method when the amplification product itself is the target product, when the purpose is to qualitatively or quantitatively measure the initial nucleic acid amount, or when the purpose is to determine the base sequence of the nucleic acid. In either case, increasing the concentration of the amplification product is often a challenge. Therefore, various techniques for promoting nucleic acid amplification in PCR have been investigated.
  • nucleic acid amplification it is widely practiced to try to promote nucleic acid amplification by adding salts (eg, potassium chloride, ammonium sulfate, etc.), betaine, polyhydric alcohols, polyethylene glycol, etc. to the reaction solution of the PCR method.
  • salts eg, potassium chloride, ammonium sulfate, etc.
  • betaine e.g., betaine, polyhydric alcohols, polyethylene glycol, etc.
  • 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 DNA replication promoting factor
  • An object of the present invention is to provide a nucleic acid amplification promoter that is superior in mass productivity and storage stability to additives composed of proteins.
  • a nucleic acid amplification accelerator comprising a polymer containing a structural unit derived from a monomer containing a phosphorylcholine group.
  • Copolymers containing structural units derived from C8 alkyl (meth)acrylate, and (a-5) structural units derived from 2-(meth)acryloyloxyethylphosphorylcholine and structural units derived from polyethylene glycol (meth)acrylate A copolymer containing
  • C 2 -C 20 alkyl (meth)acrylates are 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, Lauryl (meth)acrylate, Tridecyl (meth)acrylate, Myristyl (meth)acrylate, Pentadecyl (meth)acrylate, Cetyl (meth)acrylate, margaryl (meth)acrylate, stearyl (meth)acrylate, nonadecyl (meth)acrylate or arachidyl (meth)acrylate, preferably propyl (meth)acrylate, butyl (meth)acrylate,
  • C 2 -C 8 alkyl (meth)acrylates are ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, ) acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, heptyl (meth)acrylate or octyl (meth)acrylate, preferably propyl (meth)acrylate, butyl (meth)acrylate or pentyl (meth)acrylate , more preferably propyl methacrylate, butyl methacrylate, or pentyl methacrylate, and still more preferably butyl methacrylate.
  • C 2 to C 8 alkyl (meth)acrylates having two or more hydroxy groups as substituents are 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 or sorbitol mono(meth)acrylate, preferably glycerol mono(meth)acrylate or xylitol mono(meth)acrylate.
  • nucleic acid amplification accelerator according to any one of the above [2] to [6], which is an acrylate, more preferably glycerin mono(meth)acrylate, and still more preferably glycerin monomethacrylate.
  • Polyethylene glycol (meth)acrylate is polyethylene glycol mono(meth)acrylate, methoxypolyethyleneglycol (meth)acrylate or ethoxypolyethyleneglycol (meth)acrylate, preferably methoxypolyethyleneglycol (meth)acrylate or ethoxypolyethylene
  • the nucleic acid according to any one of [2] to [7], which is glycol (meth)acrylate, more preferably methoxypolyethyleneglycol methacrylate or ethoxypolyethyleneglycol methacrylate, and still more preferably methoxypolyethyleneglycol methacrylate.
  • Amplification facilitator is glycol mono(meth)acrylate, methoxypolyethyleneglycol (meth)acrylate or ethoxypolyethyleneglycol (meth)acrylate, preferably methoxypolyethyleneglycol (meth)acrylate or ethoxypolyethylene
  • Amplification facilitator is glycol mono(meth)acrylate, meth
  • the nucleic acid amplification accelerator of the present invention can increase the amount of nucleic acid amplification products obtained in the nucleic acid amplification method. Therefore, for example, the nucleic acid amplification accelerator of the present invention is used for improving the yield of the target nucleic acid using the nucleic acid amplification method, improving the performance of the test by the nucleic acid amplification method, improving the performance of base sequencing using the nucleic acid amplification method, etc. can be used.
  • the nucleic acid amplification accelerator of the present invention is a synthetic polymer, it is superior in mass productivity and storage stability to additives made 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”.
  • C x to C y (x, y: integers) means “having x to y carbon atoms”.
  • the nucleic acid amplification promoter of the present invention comprises a polymer containing structural units derived from a phosphorylcholine group-containing monomer (hereinafter sometimes referred to as "the polymer of the present invention").
  • the nucleic acid amplification accelerator means an additive for increasing the amount of amplification product in the nucleic acid amplification method.
  • Nucleic acid amplification methods that can use the nucleic acid amplification promoter of the present invention 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 (ICAN) 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 nucleic acid amplification accelerator of the present invention is preferably used in PCR method.
  • an appropriate method may be selected depending on the type of template nucleic acid, that is, the nucleic acid to be amplified. For example, if the template nucleic acid is double-stranded DNA, a typical PCR method is preferably selected, and For example, if the template nucleic acid is single-stranded RNA, the RT-PCR method is preferably selected.
  • the qPCR method is preferably selected for medical testing. That is, the qPCR method is preferably selected for DNA-targeted tests such as bacterial tests, fungal tests, and DNA virus tests, and the RT-qPCR method is preferably selected for RNA-targeted tests such as RNA virus tests. .
  • the nucleic acid amplification accelerator of the present invention may be used alone or in combination of two or more.
  • the nucleic acid amplification promoter of the present invention may be used in combination with other additives as long as the effects of the present invention are not impaired.
  • the polymer of the present invention has the following formula:
  • phosphorylcholine group-containing unit derived from a monomer containing a phosphorylcholine group represented by
  • Preferred examples of the monomer containing a phosphorylcholine group include 2-(meth)acryloyloxyethylphosphorylcholine, 2-(meth)acrylamidoethylphosphorylcholine, alkylphosphorylcholine allyl ether, and alkylphosphorylcholine vinyl ether. From the viewpoint of storage stability and raw material availability, 2-methacryloyloxyethylphosphorylcholine and 2-methacrylamidoethylphosphorylcholine are more preferred, and 2-methacryloyloxyethylphosphorylcholine is even more preferred.
  • the proportion of phosphorylcholine group-containing units is preferably 30 to 100 mol%, more preferably 70 to 100 mol%, and even more preferably 80 to 100 mol%.
  • the weight average molecular weight of the polymer of the present invention is not particularly limited, it is preferably 5,000 to 2,000,000, more preferably 10,000 to 1,500,000.
  • 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 preferably comprises one or more polymers selected from group a below: [Group a] (a-1) a polymer containing only structural units derived from 2-(meth)acryloyloxyethylphosphorylcholine (that is, a polymer consisting of the structural units) (hereinafter abbreviated as “polymer (a-1)” there is), (a-2) a copolymer containing structural units derived from 2-(meth)acryloyloxyethylphosphorylcholine and structural units derived from (meth)acrylic acid (hereinafter abbreviated as "polymer (a-2)” there is), (a-3) a copolymer containing structural units derived from 2-(meth)acryloyloxyethylphosphorylcholine and structural units derived from C 2 to C 20 alkyl (meth)acrylate (hereinafter “polymer (a-3) ”), (a-4) Structural units derived from 2-(meth)acryl
  • polymer (a-5) A copolymer containing structural units derived from phosphorylcholine and structural units derived from polyethylene glycol (meth)acrylate (hereinafter sometimes abbreviated as "polymer (a-5)”).
  • the copolymer may be a random copolymer, an alternating copolymer, a block copolymer, a graft polymer, or a copolymer having two or more of these structures.
  • random copolymers are preferred from the viewpoint of polymer manufacturability.
  • the polymer (a-1) may be a homopolymer of (meth)acryloyloxyethylphosphorylcholine or a copolymer of acryloyloxyethylphosphorylcholine and methacryloyloxyethylphosphorylcholine.
  • the polymer (a-1) is preferably a homopolymer of 2-(meth)acryloyloxyethylphosphorylcholine, more preferably a homopolymer of 2-methacryloyloxyethylphosphorylcholine.
  • the weight average molecular weight of the polymer (a-1) is not particularly limited, it is preferably 20,000 to 2,000,000, more preferably 100,000 to 1,500,000, still more preferably 500,000 to 1. , 500,000.
  • Only one type of (meth)acryloyloxyethylphosphorylcholine (that is, acryloyloxyethylphosphorylcholine or methacryloyloxyethylphosphorylcholine) may be used for forming the polymer (a-2), or two types (that is, acryloyl oxyethylphosphorylcholine and methacryloyloxyethylphosphorylcholine) may also be used.
  • the (meth)acryloyloxyethylphosphorylcholine for forming the polymer (a-2) methacryloyloxyethylphosphorylcholine is preferable from the viewpoint of the storage stability of the polymer and availability of raw materials.
  • MAc (meth)acrylic acid
  • acrylic acid or methacrylic acid may be used for forming the polymer (a-2)
  • Two may be used (ie acrylic acid and methacrylic acid).
  • MAc methacrylic acid is preferable from the viewpoint of the storage stability of the polymer.
  • the proportion of structural units derived from MAc is preferably 1 to 70 mol%, more preferably 60 to 80 mol%.
  • the rest of the structural units of the polymer (a-2) are preferably structural units derived from (meth)acryloyloxyethylphosphorylcholine.
  • the weight average molecular weight of the polymer (a-2) is not particularly limited, it is preferably 10,000 to 2,000,000, more preferably 100,000 to 1,500,000, still more preferably 500,000 to 1. ,000,000.
  • the polymer (a-2) is Preferably a copolymer of 2-(meth)acryloyloxyethylphosphorylcholine and (meth)acrylic acid, A copolymer of 2-methacryloyloxyethylphosphorylcholine and methacrylic acid is more preferred.
  • (meth)acryloyloxyethylphosphorylcholine Only one type of (meth)acryloyloxyethylphosphorylcholine may be used for forming the polymer (a-3), or two types may be used.
  • the (meth)acryloyloxyethylphosphorylcholine for forming the polymer (a-3) methacryloyloxyethylphosphorylcholine is preferred from the viewpoint of the storage stability of the polymer and availability of raw materials.
  • C 2 -C 20 alkyl (meth)acrylate hereinafter sometimes abbreviated as "C 2 -C 20 AMA"
  • C 2 -C 20 AMA alkyl (meth)acrylate
  • C 2 -C 20 AMA examples include ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, heptyl (meth)acrylate, octyl (meth)acrylate, ) acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, undecyl (meth)acrylate, lauryl (meth)acrylate, tridecyl (meth)acrylate, myristyl (meth)acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate , margaryl (meth)acrylate, stearyl (meth)acrylate, nonadecyl (meth)acrylate, arachidyl (meth)acrylate, and monomers in which the alkyl group portion of these is replaced with the corresponding structural
  • propyl (meth) acrylate, butyl (meth) acrylate, pentyl (meth) acrylate, lauryl (meth) acrylate, myristyl (meth) acrylate, cetyl (meth) acrylate, stearyl (meth) acrylate are preferred, and butyl (Meth)acrylate and stearyl (meth)acrylate are more preferred, and butyl methacrylate and stearyl methacrylate are even more preferred.
  • the proportion of structural units derived from C 2 -C 20 AMA is preferably 5 to 80 mol%, more preferably 10 to 30 mol%.
  • the rest of the structural units of the polymer (a-3) are preferably structural units derived from (meth)acryloyloxyethylphosphorylcholine.
  • the weight average molecular weight of the polymer (a-3) is not particularly limited, it is preferably 5,000 to 2,000,000, more preferably 10,000 to 1,000,000, still more preferably 20,000 to 1. ,000,000.
  • the polymer (a-3) is Preferably 2-(meth)acryloyloxyethylphosphorylcholine, 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, lauryl (meth)acrylate, tridecyl (meth)acrylate, myristyl (meth)acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate ) acrylate, margaryl (meth)acrylate, stearyl (meth)acrylate, nonadecyl (meth)acrylate, or arachidyl (meth)acrylate, and
  • (meth)acryloyloxyethylphosphorylcholine Only one type of (meth)acryloyloxyethylphosphorylcholine may be used for forming the polymer (a-4), or two types may be used.
  • the (meth)acryloyloxyethylphosphorylcholine for forming the polymer (a-4) methacryloyloxyethylphosphorylcholine is preferable from the viewpoint of the storage stability of the polymer and availability of raw materials.
  • C 2 -C 8 alkyl (meth)acrylates for forming polymer (a-4) (specifically, C 2 -C 8 alkyl (meth)acrylates having no hydroxy group, hereinafter “C 2 -C 8 AMA " may be abbreviated) may be used alone, or may be used in combination of two or more.
  • Examples of C 2 -C 8 AMA include ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, heptyl (meth)acrylate, octyl (meth)acrylate, and octyl (meth)acrylate.
  • acrylates and monomers in which the alkyl group portion thereof is substituted with corresponding structural isomers.
  • Propyl (meth)acrylate, butyl (meth)acrylate, and pentyl (meth)acrylate are preferred from the viewpoint of high nucleic acid amplification promoting effect.
  • propyl methacrylate, butyl methacrylate, and pentyl methacrylate are more preferable, and butyl methacrylate is even more preferable.
  • HO-AMA C 2 to C 8 alkyl (meth)acrylate having two or more hydroxy groups as substituents for forming the polymer (a-4) is one may be used alone, or two or more thereof may be used in combination.
  • HO-AMA 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, 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.
  • the proportion of structural units derived from C 2 -C 8 AMA is preferably 10 to 60 mol%, more preferably 20 to 60 mol%, and 30 to 60 mol%. More preferred.
  • the proportion of structural units derived from HO-AMA is preferably 10 to 60 mol%, more preferably 10 to 50 mol%, even more preferably 10 to 40 mol%.
  • the rest of the structural units of the polymer (a-4) are preferably structural units derived from (meth)acryloyloxyethylphosphorylcholine.
  • the “C 2 -C 8 alkyl (meth)acrylate” for forming the polymer (a-4) is the “C 2 -C 20 alkyl (meth)acrylate” for forming the polymer (a-3). subsumed in In the present invention, two or more hydroxy groups are substituted in a copolymer containing structural units derived from 2-(meth)acryloyloxyethylphosphorylcholine and structural units derived from C 2 to C 8 alkyl (meth)acrylates. Copolymers further containing structural units derived from C 2 to C 8 alkyl (meth)acrylates as groups are classified as polymers (a-4), and copolymers not containing such structural units are classified as polymers (a -3).
  • the weight average molecular weight of the polymer (a-4) is not particularly limited, it is preferably 10,000 to 500,000, more preferably 10,000 to 100,000, still more preferably 10,000 to 50,000. .
  • the polymer (a-4) is Preferably 2-(meth)acryloyloxyethylphosphorylcholine and ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, heptyl (meth)acrylate, or Octyl (meth)acrylate, glycerin 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, or a copolymer with sorbitol mono(meth)acrylate, More preferably 2-(meth)acryloyloxyethylphosphorylcholine, propyl (meth)acryl
  • a polymer More preferably, it is a copolymer of 2-methacryloyloxyethylphosphorylcholine, propyl methacrylate, butyl methacrylate, or pentyl methacrylate, and glycerin monomethacrylate, A copolymer of 2-methacryloyloxyethylphosphorylcholine, butyl methacrylate and glycerin monomethacrylate is particularly preferred.
  • (meth)acryloyloxyethylphosphorylcholine Only one type of (meth)acryloyloxyethylphosphorylcholine may be used for forming the polymer (a-5), or two types may be used.
  • the (meth)acryloyloxyethylphosphorylcholine for forming the polymer (a-5) methacryloyloxyethylphosphorylcholine is preferable from the viewpoint of the storage stability of the polymer and availability of raw materials.
  • Polyethylene glycol (meth)acrylate for forming the polymer (a-5) may be used alone or in combination of two or more.
  • PEG-MA includes, for example, polyethylene glycol mono(meth)acrylate, methoxypolyethyleneglycol (meth)acrylate, ethoxypolyethyleneglycol (meth)acrylate and the like.
  • methoxypolyethyleneglycol (meth)acrylate and ethoxypolyethyleneglycol (meth)acrylate are preferred, methoxypolyethyleneglycol methacrylate and ethoxypolyethyleneglycol methacrylate are more preferred, and methoxypolyethyleneglycol methacrylate is even more preferred.
  • the molecular weight or number average molecular weight of the polyethylene glycol chain in the polymer (a-5) is preferably 50 to 10,000, more preferably 50 to 5,000, more preferably 50 to 1, from the viewpoint of raw material availability and nucleic acid amplification promoting effect. ,000 is more preferred, and 100 to 1,000 is particularly preferred.
  • the molecular weight or number average molecular weight of the polyethylene glycol chain in the polymer (a-5) can be calculated from, for example, the molecular weight or number average molecular weight of PEG-MA used to form the polymer (a-5). Also, the molecular weight of PEG-MA can be determined based on the hydroxyl value calculated by the method described in JIS K 1557, for example. Also, the number average molecular weight of PEG-MA can be determined in terms of polyethylene glycol by gel filtration chromatography, for example.
  • the proportion of structural units derived from PEG-MA is preferably 5-40 mol%, more preferably 5-20 mol%.
  • the rest of the structural units of the polymer (a-5) are preferably structural units derived from (meth)acryloyloxyethylphosphorylcholine.
  • the weight average molecular weight of the polymer (a-5) is not particularly limited, it is preferably 100,000 to 1,000,000, more preferably 200,000 to 1,000,000, still more preferably 300,000 to 800. , 000.
  • the polymer (a-5) is Preferably a copolymer of 2-(meth)acryloyloxyethylphosphorylcholine and polyethylene glycol mono(meth)acrylate, methoxypolyethyleneglycol (meth)acrylate or ethoxypolyethyleneglycol (meth)acrylate, More preferably, it is a copolymer of 2-(meth)acryloyloxyethylphosphorylcholine and methoxypolyethyleneglycol (meth)acrylate or ethoxypolyethyleneglycol (meth)acrylate, More preferably, it is a copolymer of 2-methacryloyloxyethylphosphorylcholine and methoxypolyethyleneglycol methacrylate or ethoxypolyethyleneglycol methacrylate, A copolymer of 2-methacryloyloxyethylphosphorylcholine and methoxypolyethylene glycol methacrylate is particularly preferred.
  • the polymer of the present invention may contain structural units derived from monomers other than the above-described monomers (hereinafter referred to as "other monomers") within a range that does not impair the effects of the present invention.
  • Other monomers include, for example, benzyl (meth)acrylate, isobornyl (meth)acrylate and the like.
  • the proportion of structural units derived from other monomers is preferably 20 mol % or less, more preferably 10 mol % or less. More preferably, the polymer of the present invention does not contain structural units derived from other monomers.
  • a commercially available product may be used, or it may be produced by a known method.
  • the polymer of the present invention can be produced by a known method (eg, the method described in WO2018/216628).
  • the nucleic acid amplification promoter of the present invention can be easily used as a nucleic acid amplification promoter by including it in the reaction composition of a desired nucleic acid amplification reaction before starting the reaction.
  • the amount of the nucleic acid amplification promoter of the present invention added to the reaction composition of the nucleic acid amplification reaction is preferably 0.001 to 5 w/v%, more preferably 0.01 to 1 w/v%, and still more preferably 0. .01 to 0.1 w/v%.
  • the present invention further provides a test method using the nucleic acid amplification promoter of the present invention.
  • the nucleic acid of the test object (sample) is amplified by a nucleic acid amplification method using a reaction composition containing the test object (sample) and the nucleic acid amplification accelerator of the present invention, and an amplification reaction product is obtained.
  • a method for determining the presence or absence or concentration of a test target from the amount of is as described above.
  • the reaction composition is preferably a composition (more preferably a mixed solution) containing the nucleic acid amplification accelerator of the present invention, a reaction reagent, and a test object (sample), and is a known nucleic acid amplification method represented by the PCR method. can be prepared according to the method.
  • reaction reagents examples include buffers, substrates, primers, DNA polymerases, fluorescent DNA staining reagents, fluorescent probes, passive references, nucleic acids and the like.
  • the buffer is not particularly limited, 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. is adjusted to about 7 to 8.
  • 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, glycerin and the like.
  • 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 buffer may further contain a water-soluble polymer such as polyethylene glycol.
  • the substrate is not particularly limited. ).
  • part and/or all of dTTP can be replaced with deoxyuridine triphosphate (dUTP).
  • dUTP deoxyuridine triphosphate
  • ddATP dideoxyadenosine triphosphate
  • ddTTP dideoxythymidine triphosphate
  • ddGTP dideoxyguanosine triphosphate
  • ddCTP dideoxycytidine triphosphate
  • a fluorescent it is also preferable to add an appropriate amount of label.
  • the primer is not particularly limited, but may be, for example, an oligonucleotide of about 15 to 30 bases designed and prepared by a known method.
  • the primer may be appropriately fluorescently labeled using fluorescein or the like, or isotope-labeled using a heavy element, or the like. Only one type of primer may be used, two types of primers may be used as one pair, or multiple pairs of primers may be used.
  • a known DNA polymerase can be used as the DNA polymerase.
  • enzymes derived from thermophilic bacteria, thermophilic archaea, hyperthermia bacteria, hyperthermia archaea, and mutant enzymes thereof are preferred.
  • One or more DNA polymerases are appropriately selected from DNA-dependent DNA polymerases, RNA-dependent DNA polymerases (reverse transcriptases), or enzymes having both functions, depending on the purpose of nucleic acid amplification.
  • RNA-dependent DNA polymerases reverse transcriptases
  • enzymes having both functions depending on the purpose of nucleic acid amplification.
  • whether to use a DNA polymerase having nuclease activity or a DNA polymerase having no nuclease activity is appropriately selected.
  • the fluorescent DNA staining reagent is not particularly limited, examples thereof include SYBR TM Green I and the like.
  • examples of the fluorescent probe include, but are not limited to, TaqMan TM probes.
  • the 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.
  • 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.
  • Oil such as mineral oil may be further added to the reaction reagent.
  • a solid phase carrier such as silica beads or magnetic beads may be further added to the reaction reagent.
  • a master mix sometimes called a primer mix, premix, etc.
  • the sample is an object to be inspected.
  • Samples include, for example, viruses, bacteria, cells, body fluids, tissues, etc. to be tested, suspensions thereof, nucleic acid extracts prepared from these, nucleic acid standard solutions, blank test samples, and the like. be done.
  • the sample can be appropriately selected according to the mode of examination.
  • inspections examples include medical inspections, veterinary inspections, forensic inspections, pharmaceutical inspections, food inspections, and environmental inspections.
  • the inspection method of the present invention is preferably used for medical inspection.
  • test kit by combining each component of the reaction composition for the nucleic acid amplification reaction with members such as a reaction container and a sample collection device.
  • Said test kit is preferably an in-vitro diagnostic agent.
  • Determination of the presence or concentration of the test target from the amount of the amplification reaction product can be performed using a known nucleic acid detection method.
  • the detection method includes, for example, a method of purifying an amplification reaction product and measuring the turbidity in the ultraviolet region of the purified nucleic acid solution.
  • the reaction product is developed by gel electrophoresis, the nucleic acid is stained with a nucleic acid staining reagent such as ethidium bromide, and the staining intensity is qualitatively or quantified by visual observation or instrumental analysis. mentioned.
  • Still another method includes, for example, adding an intercalating reagent such as CYBR TM Green I to the reaction composition of the nucleic acid amplification reaction, and monitoring the optical properties of the intercalating reagent.
  • the present invention also provides a method for amplifying nucleic acid in a sample by a nucleic acid amplification method using a reaction composition containing the sample and the nucleic acid amplification accelerator of the present invention (hereinafter referred to as "method of the present invention"). Descriptions of the nucleic acid amplification promoter and the nucleic acid amplification method used in the method of the present invention are as described above.
  • the reaction composition used in the method of the present invention is preferably a composition (more preferably a mixed solution) containing the nucleic acid amplification accelerator of the present invention, a reaction reagent, and a sample. It can be prepared according to the amplification method. Descriptions of the reaction reagents used in the method of the present invention are given above.
  • the present invention also provides the use of the nucleic acid amplification promoter of the present invention for amplifying nucleic acid in a sample in a nucleic acid amplification method (hereinafter referred to as "use of the present invention"). Descriptions of nucleic acid amplification promoters and nucleic acid amplification methods for use in the present invention are provided above.
  • Polymer 1 corresponding to polymer (a-1) was prepared in the following manner. 40.0 g of 2-methacryloyloxyethylphosphorylcholine (hereinafter referred to as “MPC”) was weighed into a glass flask for polymerization, 60.0 g of purified water was added and dissolved, and azobisiso was added to the resulting solution as a polymerization initiator. 0.31 g of butyronitrile (hereinafter referred to as "AIBN”) was added. After the inside of the reaction vessel was sufficiently replaced with nitrogen, polymerization was carried out by heating at 70° C. for 6 hours while stirring.
  • MPC 2-methacryloyloxyethylphosphorylcholine
  • AIBN butyronitrile
  • polymer 1 a white powdery homopolymer (referred to as "polymer 1" in this specification).
  • 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 3-1 a random copolymer (referred to as "Polymer 3-1" in this specification) was obtained.
  • the weight average molecular weight of the polymer 3-1 was 600,000 in terms of polyethylene glycol by GPC measurement under the conditions described later.
  • Polymer 4 a random copolymer (referred to as "Polymer 4" in this specification) was obtained.
  • the weight-average molecular weight of polymer 4 was 22,000 in terms of polyethylene glycol by GPC measurement under the conditions described later.
  • Table 1 shows the monomers used in Synthesis Examples 1 to 5, their molar ratios, and the weight average molecular weights of the obtained polymers.
  • Example 1-1 to 1-7 and Comparative Examples 1-1 to 1-3 Examples and comparative examples are shown where the nucleic acid to be amplified is single-stranded RNA and the nucleic acid amplification method is the RT-PCR method.
  • reaction composition Components of the commercially available RT-PCR kit "SARS-CoV-2 RT-qPCR Detection Kit” (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and PW (manufactured by Nippon Gene Co., Ltd.) were used to perform the reactions shown in Table 2.
  • a composition was prepared. This preparation was carried out on ice, and the nucleic acid amplification accelerator, the reaction reagent, and the sample were added in this order, and the nucleic acid amplification reaction described below was immediately carried out.
  • Each nucleic acid amplification product obtained by the above procedure was quantified by the following procedure.
  • 4 ⁇ L of 6 ⁇ Loading Buffer (manufactured by Nippon Gene Co., Ltd.) was added to 20 ⁇ L of the obtained nucleic acid amplification product to prepare an electrophoresis sample.
  • purified agarose (trade name "Agarose21", manufactured by Nippon Gene Co., Ltd.) was suspended in 0.5x TBE buffer at 3 w/v%, heated and dissolved using a microwave oven, poured into a mold with a comb set, and allowed to cool.
  • An agarose gel for electrophoresis was prepared by compaction.
  • the gel was taken out, immersed in ultrapure water, and gently shaken for 15 minutes to remove excess nucleic acid staining reagent. After that, the gel was set in a gel imager (ChemiDocXRS+, manufactured by Bio-Rad) and photographed under irradiation with UV light for transmission.
  • the staining intensity of the band of the target nucleic acid amplification product (size: 158 bp) was quantified using the image analysis software attached to the apparatus on the left, and the amount of amplified nucleic acid was calculated relative to the measured value of Control 1 as 100. The results (mean values of duplicate tests) are shown in Tables 4 and 5.
  • the nucleic acid amplification enhancing agents of the present invention ie, Polymers 1-5)
  • the amount of nucleic acid amplification product was reduced by about 2.6 to It can be seen that it increases by 6.1 times.
  • the amount of nucleic acid amplification product is about 0.7 to 1.3 times higher than that of Control 1. From these results, it can be said that the nucleic acid amplification promoter of the present invention can favorably promote the RNA amplification reaction.
  • Example 2 As a more generalized example, an example in which the nucleic acid to be amplified is double-stranded DNA and the nucleic acid amplification method is a typical PCR method will be shown.
  • Polymer 1 used in Example 2 was used by including it in the reaction composition described later at the final concentration shown in Table 8.
  • Control 2 is a control run using PW instead of Polymer 1.
  • reaction composition The reaction compositions shown in Table 6 were prepared. This preparation was carried out on ice, and the nucleic acid amplification accelerator, the reaction reagent, and the sample were added in this order, and the nucleic acid amplification reaction described below was immediately carried out.
  • Each nucleic acid amplification product obtained by the above procedure was quantified by the following procedure.
  • 4 ⁇ L of 6 ⁇ Loading Buffer (manufactured by Nippon Gene Co., Ltd.) was added to 20 ⁇ L of the obtained nucleic acid amplification product to prepare an electrophoresis sample.
  • purified agarose (trade name "Agarose S", manufactured by Nippon Gene Co., Ltd.) is suspended in 1x TAE buffer at 1 w/v%, heated and dissolved using a microwave oven, poured into a mold with a comb set, allowed to cool and hardened.
  • an agarose gel for electrophoresis was prepared.
  • the gel was taken out, immersed in ultrapure water, and gently shaken for 15 minutes to remove excess nucleic acid staining reagent. After that, the gel was set in a gel imager (FAS-Digi Compact, manufactured by Nippon Genetics Co., Ltd.) and photographed under blue green LED light irradiation. A 16-bit TIFF image is output from the imaging device, and image analysis software (ImageJ, National Institutes of Health) is used to quantify the staining intensity of the band of the target nucleic acid amplification product (size: 6.4 kbp), and the amount of amplified nucleic acid is determined. , the average of the measured values of Control 2 was set to 100, and shown as relative values. Table 8 shows the results of the triplicate test, together with the p-value determined by Student's t-test (two-tailed test).
  • the use of the nucleic acid amplification promoter of the present invention significantly increased the amount of nucleic acid amplification products compared to Control 2 at the 5% level. I understand. From these results, it can be said that the nucleic acid amplification promoter of the present invention can favorably promote the DNA amplification reaction.
  • the nucleic acid amplification promoter of the present invention can favorably promote the nucleic acid amplification reaction regardless of whether the target of amplification is RNA or DNA.
  • the nucleic acid amplification accelerator of the present invention By using the nucleic acid amplification accelerator of the present invention, it is possible to increase the amount of nucleic acid amplification products obtained in the nucleic acid amplification method (especially the PCR method). Therefore, the nucleic acid amplification accelerator of the present invention is used for improving the yield of the target nucleic acid using the nucleic acid amplification method, improving the performance of testing by the nucleic acid amplification method, improving the performance of base sequencing using the nucleic acid amplification method, etc. It can be preferably used.

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WO2024203576A1 (ja) * 2023-03-30 2024-10-03 日油株式会社 核酸増幅用組成物の凍結融解保護剤およびそれを用いた核酸増幅法
WO2024225350A1 (ja) * 2023-04-28 2024-10-31 日油株式会社 １本鎖核酸吸着抑制剤、核酸溶液、および核酸増幅法
WO2025205410A1 (ja) * 2024-03-25 2025-10-02 日油株式会社 核酸増幅促進剤、これを含む核酸増幅用組成物及び核酸増幅法
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