Nucleic acid amplification promoter, nucleic acid amplification composition containing same, and nucleic acid amplification method

Abstract

A nucleic acid amplification promoter according to the present invention comprises a copolymer containing a constituent unit derived from a monomer represented by formula (1) and a constituent unit derived from a monomer represented by formula (2).　The present invention can provide a nucleic acid amplification promoter capable of promoting nucleic acid amplification and improving detection sensitivity when added to a nucleic acid amplification composition. (X1 represents a (meth)acryloyloxy group or a (meth)acryloyl amide group; L1 represents an alkylene group having 2 to 4 carbon atoms and optionally having one hydroxy group, or an alkylene oxyalkylene group having 2 to 4 carbon atoms; and R1 to R3 each independently represent an alkyl group having 1 to 3 carbon atoms. R4 represents a hydrogen atom or a methyl group, and R5 is a group having any one of a sulfo group, a phosphate group, a sulfate group, and a phosphonic acid group.)

Description

Nucleic acid amplification promoter, composition for nucleic acid amplification containing the same, and nucleic acid amplification method

[0001] The present invention relates to a nucleic acid amplification promoter, a composition for nucleic acid amplification containing the same, and a method for amplifying nucleic acid.

[0002] Nucleic acid amplification is known as a method for synthesizing a new nucleic acid complementary to a nucleic acid using the nucleic acid as a template. A typical method for nucleic acid amplification is the polymerase chain reaction (PCR). In a typical PCR method, nucleic acid amplification is performed by repeating three steps: (1) thermal denaturation, (2) primer annealing, and (3) primer extension by DNA polymerase. While the usual PCR method is a technique for amplifying DNA, there are also other methods, such as reverse transcription PCR, which combines reverse transcription and PCR reactions to enable RNA detection, and multiplex PCR, which amplifies multiple genomic regions at once.

[0003] These PCR methods enable the specific detection of RNA derived from RNA viruses in throat swabs, saliva samples, blood samples, environmental samples, etc., and of biologically derived RNA in biological samples, etc., and are utilized in research into gene expression, diagnosis of infectious agents or genetic diseases, cDNA generation, etc. However, PCR often suffers from problems such as reduced reverse transcription efficiency and DNA synthesis efficiency, and reduced detection sensitivity due to nonspecific gene amplification. Possible methods for improving detection sensitivity include modifying the primer design, adjusting the magnesium ion concentration, and changing the amount and type of enzyme (see Patent Documents 1 and 2). Other attempts to promote nucleic acid amplification have also been made by adding betaine, DMSO, bovine serum albumin, etc. to the PCR reaction solution.

[0004] JP 8-322597 A JP 2017-108736 A

[0005] However, although these methods improve detection sensitivity, it takes time and effort to examine the conditions (such as changing the primer design or adjusting the magnesium ion concentration), and the addition of betaine, DMSO, etc. is not sufficiently effective. The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a nucleic acid amplification promoter for nucleic acid amplification that can promote nucleic acid amplification and improve detection sensitivity simply by adding it to a composition for nucleic acid amplification in a nucleic acid amplification method such as PCR, a composition for nucleic acid amplification containing the same, and a nucleic acid amplification method.

[0006] As a result of intensive research, the present inventors have found that a copolymer containing a constituent unit derived from a monomer represented by the following formula (1) and a constituent unit derived from a monomer represented by the following formula (2) can be used as a nucleic acid amplification promoter in a composition for nucleic acid amplification that can achieve the above-mentioned object. The explanation based on this finding is as follows.

[0007] [1] A nucleic acid amplification promoter comprising a copolymer containing a structural unit derived from a monomer represented by formula (1) and a structural unit derived from a monomer represented by formula (2). (X １ represents a (meth)acryloyloxy group or a (meth)acryloylamide group, L 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, and R １ ~R ３ R each independently represents an alkyl group having 1 to 3 carbon atoms. ４ represents a hydrogen atom or a methyl group, and R ５ is a group having any one of a sulfonic acid group, a phosphate group, a sulfate group, and a phosphonic acid group.) [2] A composition for nucleic acid amplification, comprising the nucleic acid amplification promoter according to [1] above and a polymerase. [3] The composition for nucleic acid amplification according to [2] above, which is used in a quantitative polymerase chain reaction. [4] The composition for nucleic acid amplification according to [3] above, further comprising a primer. [5] A method for nucleic acid amplification, comprising mixing the composition for nucleic acid amplification according to any one of [2] to [4] above with a sample containing nucleic acid to be amplified to prepare a reaction solution for nucleic acid amplification.

[0008] The nucleic acid amplification promoter of the present invention can promote nucleic acid amplification and improve detection sensitivity by adding it to a composition for nucleic acid amplification.

[0009] The present invention will be described in detail below. In the present specification, when stepwise numerical ranges are described, the lower limit and upper limit of each numerical range can be combined. For example, when "preferably 10 to 100, more preferably 20 to 90" is described, "preferably lower limit: 10" and "more preferably upper limit: 90" can be combined (i.e., the numerical range "10 to 90" is also within the scope of the present specification).

[0010] [Nucleic Acid Amplification Promoter] The present invention relates to a nucleic acid amplification promoter that is a copolymer (hereinafter sometimes referred to as the "copolymer of the present invention") containing a constitutional unit derived from a monomer represented by formula (1) and a constitutional unit derived from a monomer represented by formula (2). The nucleic acid amplification promoter can be used as a nucleic acid amplification promoter in a composition for nucleic acid amplification. In this specification, "composition for nucleic acid amplification" refers to a composition used in a nucleic acid amplification method. In this specification, "nucleic acid amplification promoter in a composition for nucleic acid amplification" refers to an additive for increasing the amount of nucleic acid product in a nucleic acid amplification method.

[0011] The copolymer of the present invention is a copolymer containing a structural unit derived from a monomer represented by the following formula (1). (In the formula, X １ represents a (meth)acryloyloxy group or a (meth)acryloylamide group, L 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, and R １ ~R ３ each independently represents an alkyl group having 1 to 3 carbon atoms.) Hereinafter, the "monomer represented by formula (1)" may be abbreviated as "monomer (1)." Furthermore, the "structural unit derived from monomer (1)" may be abbreviated as "structural unit (1)."

[0012] The groups in formula (1) will be explained below in order. X in formula (1) １represents a (meth)acryloyloxy group (i.e., CH ２ =CR-CO-O-, where R is a hydrogen atom or a methyl group) or a (meth)acryloylamide group (i.e., CH ２ =CR-CO-NH-, R: hydrogen atom or methyl group). １ is preferably a (meth)acryloyloxy group, more preferably a methacryloyloxy group.

[0013] L in formula (1) 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, for example, -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 From the viewpoint of raw material availability, L １ is preferably -C ２ H ４ -or-C ２ H ４ -O-C ２ H ４ -, more preferably -C ２ H ４ - (i.e., an ethylene group).

[0014] R in formula (1) １ ~R ３ each independently represents 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 a methyl group, an ethyl group, and a propyl group. From the viewpoint of raw material availability, R １ ~R ３ Preferably, both of X are methyl groups. １ is a (meth)acryloyloxy group, and L １ But, -C ２ H ４-or-C ２ H ４ -O-C ２ H ４ - and R １ ~R ３ is a methyl group. １ is a (meth)acryloyloxy group, and L １ is an ethylene group, and R １ ~R ３ is a methyl group (i.e., 2-(meth)acryloyloxyethyl phosphorylcholine). A more preferred monomer (1) is 2-methacryloyloxyethyl phosphorylcholine. Commercially available products can be used as monomer (1). In this specification, "2-(meth)acryloyloxyethyl phosphorylcholine" basically means "2-acryloyloxyethyl phosphorylcholine or 2-methacryloyloxyethyl phosphorylcholine." In cases where multiple 2-(meth)acryloyloxyethyl phosphorylcholines may be present, "2-(meth)acryloyloxyethyl phosphorylcholine" means "2-acryloyloxyethyl phosphorylcholine and / or 2-methacryloyloxyethyl phosphorylcholine." Other terms similar to "2-(meth)acryloyloxyethyl phosphorylcholine" also have the same meaning as "2-(meth)acryloyloxyethyl phosphorylcholine."

[0015] The copolymer of the present invention further contains, in addition to the structural unit (1), a structural unit derived from a monomer represented by the following formula (2). (In the formula, R ４ represents a hydrogen atom or a methyl group, and R ５ is a group having a sulfonic acid group, a phosphoric acid group, a sulfate group, or a phosphonic acid group.

[0016] Hereinafter, the "monomer represented by formula (2)" may be abbreviated as "monomer (2)." Furthermore, the "structural unit derived from monomer (2)" may be abbreviated as "structural unit (2)."

[0017] The groups in formula (2) will be explained in order below. ４represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of the polymer, R ４ is preferably a methyl group. ５ is a group having any one of a sulfonic acid group, a phosphoric acid group, a sulfate group, and a phosphonic acid group. The sulfonic acid group, the phosphoric acid group, the sulfate group, and the phosphonic acid group may be in the form of a metal salt, such as an alkali metal salt. ５ is preferably an organic group having 1 to 20 carbon atoms and having any one of a sulfonic acid group, a phosphoric acid group, a sulfate group, and a phosphonic acid group, and the organic group may contain a hydrocarbon group, an oxygen atom, a nitrogen atom, etc. in addition to the sulfonic acid group, a phosphoric acid group, a sulfate group, and a phosphonic acid group. ５ Preferably, R has a repeating unit of an alkylene group having 1 to 4 carbon atoms or an alkylene oxide having 2 to 3 carbon atoms in addition to a sulfonic acid group, a phosphoric acid group, a sulfate group, or a phosphonic acid group. ５ is preferably a group having a sulfonic acid group or a phosphoric acid group among sulfonic acid groups, phosphoric acid groups, sulfate groups and phosphonic acid groups.

[0018] The copolymer of the present invention contains a structural unit derived from the monomer represented by formula (2), and therefore has any one of a sulfonic acid group, a phosphate group, a sulfate group, and a phosphonic acid group. It is believed that the copolymer of the present invention has such a structure, thereby promoting nucleic acid amplification reactions. While the reason for this is unclear, it is presumed that the sulfonic acid group, the phosphate group, the sulfate group, and the phosphonic acid group improve the reaction specificity of primers and probes, thereby promoting nucleic acid amplification reactions.

[0019] Specific examples of monomer (2) include potassium 3-sulfopropyl methacrylate, 2-sulfoethyl methacrylate, 2-acrylamido-2-methylpropanesulfonic acid, 4-[(3-methacrylamidopropyl)dimethylammonio]butane-1-sulfonic acid, 2-methacryloxyethyl acid phosphate, 10-methacryloyloxydecyl dihydrogen phosphate, methacryloxypolypropylene glycol phosphate, etc. Monomer (2) may be used alone or in combination of two or more types. Commercially available products can be used as monomer (2).

[0020] Among the specific examples of the monomer (2), (i) potassium 3-sulfopropyl methacrylate, 2-sulfoethyl methacrylate, 4-[(3-methacrylamidopropyl)dimethylammonio]butane-1-sulfonic acid, 2-methacryloxyethyl acid phosphate, and methacryloxypolypropylene glycol phosphate are preferred, (ii) potassium 3-sulfopropyl methacrylate, 2-sulfoethyl methacrylate, 2-methacryloxyethyl acid phosphate, and methacryloxypolypropylene glycol phosphate are more preferred, and (iii) potassium 3-sulfopropyl methacrylate and methacryloxypolypropylene glycol phosphate are even more preferred.

[0021] The copolymer of the present invention is a copolymer containing the structural unit (1) and the structural unit (2). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both a random portion and a block portion.

[0022] In the copolymer of the present invention, relative to a total of 100 moles of structural unit (1) and structural unit (2) (i.e., a total of 100 moles of monomer (1) and monomer (2) used in the polymerization), the amount of structural unit (1) (i.e., the amount of monomer (1) used in the polymerization) is preferably 1 to 95 moles, more preferably 10 to 95 moles, even more preferably 15 to 95 moles, and particularly preferably 20 to 90 moles, and the amount of structural unit (2) (i.e., the amount of monomer (2) used in the polymerization) is preferably 5 to 99 moles, more preferably 5 to 90 moles, even more preferably 5 to 85 moles, and particularly preferably 10 to 80 moles.

[0023] The weight-average molecular weight of the copolymer of the present invention is not particularly limited, but is preferably 1,000 to 1,000,000, more preferably 5,000 to 500,000, and even more preferably 10,000 to 200,000. The weight-average molecular weight can be determined in terms of polyethylene glycol by gel filtration chromatography using, for example, an EcoSEC system (manufactured by Tosoh Corporation). The copolymer of the present invention may contain other structural units derived from other monomers different from monomer (1) and monomer (2), as long as the effects of the present invention are not impaired. These other monomers may be used alone or in combination of two or more. Examples of such other monomers include, but are not limited to, glycerol methacrylate and isobornyl (meth)acrylate. However, it is preferable that the copolymer of the present invention does not contain other structural units derived from other monomers. That is, the copolymer of the present invention is preferably a copolymer consisting only of structural units (1) and (2). Each monomer used in preparing the copolymer of the present invention may be a commercially available product or may be produced by a known method. The copolymer of the present invention can be produced by a known method (for example, the method described in WO 2018 / 216628 ). The nucleic acid amplification promoter of the present invention (i.e., the copolymer of the present invention) can be easily used as a nucleic acid amplification promoter by being contained in a reaction composition for a desired nucleic acid amplification reaction (i.e., a composition for nucleic acid amplification described below) before the start of the reaction.

[0024] [Nucleic Acid Amplification Composition] The present invention provides a composition for nucleic acid amplification (hereinafter sometimes referred to as "the composition of the present invention") containing the nucleic acid amplification promoter of the present invention. The nucleic acid amplification promoter of the present invention is as described above. Examples of nucleic acid amplification methods in which the composition of the present invention can be used include the polymerase chain reaction (PCR) method, loop-mediated isothermal amplification (LAMP) method, transcription-mediated amplification (TMA) method, isothermal and chimeric primer-initiated amplification of nucleicacids (ICAN) method, strand displacement amplification (SDA) method, ligase chain reaction (LCR) method, and nucleic acid sequence-based amplification (NASBA) method.

[0025] The nucleic acid amplification method is preferably polymerase chain reaction (PCR), more preferably quantitative polymerase chain reaction. That is, the composition of the present invention is more preferably used in quantitative polymerase chain reaction. In this specification, "polymerase chain reaction (PCR)" includes not only typical PCR methods using DNA as the target nucleic acid, but also reverse transcription polymerase chain reaction (RT-PCR) methods using RNA as the target nucleic acid and multiplex PCR methods that amplify two or more gene sequences in the same reaction. In this specification, "quantitative polymerase chain reaction" includes not only typical quantitative polymerase chain reaction methods using DNA as the target nucleic acid, but also quantitative reverse transcription polymerase chain reaction methods using RNA as the target nucleic acid.

[0026] The amount of the nucleic acid amplification promoter in the composition of the present invention is preferably 0.001 to 5 w / v %, more preferably 0.01 to 1 w / v %, and even more preferably 0.01 to 0.1 w / v %.

[0027] [Other Components] The composition of the present invention may contain components other than the copolymer of the present invention (hereinafter, sometimes referred to as "other components"). As the other components, known components used in nucleic acid amplification methods, typified by PCR, can be used. Examples of the other components include reaction reagents such as polymerase, primer, buffer, substrate, fluorescent probe, passive reference, salt, surfactant, protein, and nucleic acid. Each of the other components may be used alone or in combination of two or more.

[0028] The buffer is not particularly limited, but examples include a buffer prepared by mixing a base such as tris(hydroxymethyl)aminomethane, tricine, or bicine with an acid such as sulfuric acid, hydrochloric acid, acetic acid, or phosphoric acid to adjust the pH to about 6 to 9, and more preferably about 7 to 8. The buffer desirably contains an appropriate amount of magnesium salt and / or manganese salt. The buffer may further contain a salt such as potassium chloride or ammonium sulfate. The buffer may further contain a water-soluble organic solvent such as dimethyl sulfoxide, dimethylformamide, formamide, or glycerin. The buffer may further contain a surfactant such as polyoxysorbitan fatty acid ester or polyoxyethylene alkylphenyl ether. 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.

[0029] The substrate is not particularly limited, and examples thereof include a mixture (dNTPs) of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dCTP). Here, it is also possible to replace part and / or all of dTTP with deoxyuridine triphosphate (dUTP). In addition, in sequencing PCR and the like, it is also preferable to add an appropriate amount of a mixture of dideoxyadenosine triphosphate (ddATP), dideoxythymidine triphosphate (ddTTP), dideoxyguanosine triphosphate (ddGTP), and dideoxythymidine triphosphate (ddCTP), or fluorescently labeled products thereof.

[0030] The primer is not particularly limited, but examples include oligonucleotides of about 15 to 30 bases designed and prepared by known methods. The primer may be fluorescently labeled with fluorescein or the like, or isotope-labeled with a heavy element, as appropriate. Only one type of primer may be used, or two types of primers may be used as a pair, or multiple pairs of primers may be used.

[0031] Examples of polymerases include DNA polymerases, and known DNA polymerases can be used. From the viewpoint of thermostability, enzymes derived from thermophilic bacteria, thermophilic archaea, hyperthermophilic bacteria, and hyperthermophilic archaea, and mutant enzymes thereof, are preferred. The DNA polymerase is selected from one or more DNA-dependent DNA polymerases, RNA-dependent DNA polymerases (reverse transcriptases), or enzymes that combine both functions, depending on the purpose of nucleic acid amplification. In addition, whether a DNA polymerase with nuclease activity or a DNA polymerase without nuclease activity is used can be selected appropriately.

[0032] The fluorescent probe is not particularly limited, but may be, for example, TaqMan ＴＭ The passive reference may be appropriately selected depending on the purpose of nucleic acid amplification. Examples of the passive reference include ROX. ＴＭ Examples of nucleic acids include DNA and / or RNA, such as fluorescent dyes. In addition to the above-mentioned primers and fluorescent probes, any DNA and / or RNA may be used as an exogenous control gene, for example. The nucleic acid may be synthesized in vitro or may be prepared by a known method from cells, microorganisms, viruses, etc. The cells, microorganisms, viruses, etc. may be collected from nature or the environment, or from humans, animals, or plants, or may be isolated and cultured.

[0033] The reaction reagent may further contain oil such as mineral oil, or a solid phase carrier such as silica beads or magnetic beads.

[0034] The composition of the present invention can contain a sample to be tested. Examples of the sample include a virus, bacterial cells, cells, body fluid, tissue, etc. to be tested, a suspension thereof, a nucleic acid extract prepared therefrom, a nucleic acid standard solution, a blank test sample, etc. The sample can be appropriately selected depending on the mode of the test.

[0035] It is also preferred to select multiple components and mix them in advance to prepare a master mix (sometimes called a primer mix, premix, etc.). It is also preferred to combine each component of the nucleic acid amplification composition with components such as a reaction vessel and a sample collection tool to prepare a test kit. The test kit is preferably an in vitro diagnostic drug.

[0036] [Testing Method] The present invention further provides a testing method using the nucleic acid amplification promoter of the present invention. Examples of the testing method include a nucleic acid amplification method comprising mixing the above-described nucleic acid amplification composition with a sample containing the nucleic acid to be amplified to prepare a reaction solution for nucleic acid amplification. A preferred form of the testing method includes a method in which the nucleic acid to be tested is amplified using a composition of the present invention containing the nucleic acid amplification promoter of the present invention, and the presence or concentration of the test target is determined from the amount of the amplification reaction product. Examples of the testing method include medical testing, veterinary testing, forensic testing, pharmaceutical testing, food testing, environmental testing, etc. The testing method of the present invention is preferably used for medical testing.

[0037] The presence or concentration of the test subject can be determined from the amount of the amplification reaction product using a known nucleic acid detection method. Examples of such detection methods include purifying the amplification reaction product and measuring the turbidity of the purified nucleic acid solution in the ultraviolet region. Another example of a method is to develop the reaction product by gel electrophoresis, stain the nucleic acid with a nucleic acid staining reagent such as ethidium bromide, and qualitatively or quantitatively determine the staining intensity visually or by instrumental analysis. Yet another example of a method is to use CYBR ＴＭAn example of such a method is to add an intercalating agent such as Green I to the reaction composition of the nucleic acid amplification reaction, and monitor the optical properties of the intercalating agent.

[0038] The present invention will be specifically described below with reference to examples, but is not limited to these. [Synthesis of Copolymer (Nucleic Acid Amplification Promoting Agent)] The copolymer of the present invention was prepared as follows.

[0039] Synthesis Example 1 15.3 g of 2-methacryloyloxyethyl phosphorylcholine (hereinafter referred to as "MPC") as monomer (1) and 29.7 g of 3-sulfopropyl methacrylate potassium salt (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter referred to as "SPMA") as monomer (2) (monomer (1) / monomer (2)=20 / 80 (molar ratio)) were weighed into a glass flask for polymerization, and 252.9 g of purified water was added to dissolve monomers (1) and (2). To the resulting solution was added 2.15 g of Perloyl SA (manufactured by NOF Corporation, hereinafter referred to as "PRSA"). The atmosphere in the reaction vessel was thoroughly replaced with nitrogen, and polymerization was carried out by heating at 70°C for 6 hours with stirring. The copolymer was then separated and purified using a dialysis membrane to obtain a solution of a copolymer (referred to herein as "Polymer 1") having a solid content of about 5.0% by mass. The weight-average molecular weight of Polymer 1 was 30,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0040] [Synthesis Example 2] A copolymer having a composition ratio of monomer (1) / monomer (2) = 30 / 70 (molar ratio) was synthesized according to Synthesis Example 1. A solution of the copolymer (referred to as "Polymer 2" in this specification) having a solid content of about 2.4 mass% was obtained. The weight average molecular weight of Polymer 2 was 160,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0041] Synthesis Example 3: A copolymer having a composition ratio of monomer (1) / monomer (2)=50 / 50 (molar ratio) was synthesized according to Synthesis Example 1. A solution of a copolymer (referred to as "Polymer 3" in this specification) having a solid content of about 2.0 mass% was obtained. The weight average molecular weight of Polymer 3 was 150,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0042] Synthesis Example 4: A copolymer having a composition ratio of monomer (1) / monomer (2)=70 / 30 (molar ratio) was synthesized according to Synthesis Example 1. A solution of the copolymer (referred to as "Polymer 4" in this specification) having a solid content of about 2.5% by mass was obtained. The weight average molecular weight of Polymer 4 was 110,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0043] Synthesis Example 5: A copolymer having a composition ratio of monomer (1) / monomer (2)=90 / 10 (molar ratio) was synthesized according to Synthesis Example 1. A solution of the copolymer (referred to as "Polymer 5" in this specification) having a solid content of about 4.8 mass% was obtained. The weight average molecular weight of Polymer 5 was 140,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0044] Synthesis Example 6 6.5 g of MPC as monomer (1) and 23.5 g of methacryloxypolypropyleneglycolphosphate (manufactured by Unichemical Corporation, hereinafter referred to as "Phosmer PP") as monomer (2) (monomer (1) / monomer (2)=30 / 70 (molar ratio)) were weighed into a glass flask for polymerization, and a mixed solvent of 135.0 g of purified water and 135.0 g of ethanol was added to dissolve the monomers (1) and (2), and 0.78 g of dimethyl 2,2'-azobis(2-methylpropionate) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "V-601") was added to the resulting solution. After thoroughly replacing the atmosphere in the reaction vessel with nitrogen, the mixture was heated at 70°C for 6 hours under stirring to carry out polymerization. The mixture was then separated and purified using a dialysis membrane to obtain a solution of a copolymer (referred to as "Polymer 6" in this specification) with a solid content of approximately 1.9% by mass. The weight-average molecular weight of Polymer 6 was 80,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0045] [GPC Measurement] GPC measurement of polymers 1 to 6 obtained in synthesis examples 1 to 6 was carried out under the following conditions: GPC system: EcoSEC system (manufactured by Tosoh Corporation) Column: Shodex OHpak SB-802.5HQ (manufactured by Showa Denko K.K.) and SB-806HQ (manufactured by Showa Denko K.K.) connected in series Developing solvent: 20 mM sodium phosphate buffer (pH 7.4) Detector: differential refractive index detector Molecular weight standard: EasiVial PEG / PEO (manufactured by Agilent Technologies) Flow rate: 0.5 mL / min Column temperature: 40°C Sample: The obtained polymer was diluted with the developing solvent to a final concentration of 0.5 wt% Injection volume: 100 μL

[0046] Table 1 shows the monomers used in Synthesis Examples 1 to 6, their molar ratios, and the weight average molecular weights of the resulting polymers.

[0047]

[0048] [Examples 1-1 to 1-4] Examples and comparative examples are shown below in which the nucleic acid to be amplified is single-stranded RNA derived from SARS-CoV-2 and the nucleic acid amplification method is RT-PCR. [Preparation of Solution A] Solution A was prepared by mixing the components shown in Table 2. This preparation was carried out on ice.

[0049]

[0050] [Preparation of Composition for Nucleic Acid Amplification] Compositions for nucleic acid amplification were prepared by adding the polymers obtained in Synthesis Examples 1 to 4 to the obtained Solution A. 2 μL of an aqueous solution of the polymer diluted to 0.5 w / v % was added to Solution A per well to prepare compositions for nucleic acid amplification (polymer concentration in each composition: 0.05 w / v %).

[0051] [Nucleic Acid Amplification Reaction] The nucleic acid amplification composition described above was dispensed into a PCR plate (manufactured by BIORAD), the plate was sealed, and the PCR plate was set in a PCR thermal cycler (manufactured by BIORAD). The RT-PCR reaction (Table 3) and the endpoint fluorescence intensity were measured. The measurement was performed three times for each nucleic acid amplification composition.

[0052]

[0053] Comparative Example 1 was performed in the same manner as in Examples 1-1 to 1-4, except that in the preparation of a composition for nucleic acid amplification, 2 μL of distilled water, nuclease-free (manufactured by Nippon Gene Co., Ltd.) was used instead of 2 μL of the aqueous polymer solution. The endpoint fluorescence intensities (relative values) obtained in Examples 1-1 to 1-4, where the endpoint fluorescence intensity in Comparative Example 1 was taken as 100%, are shown in Table 4 below.

[0054]

[0055] As shown in Table 4, it can be seen that when the nucleic acid amplification promoter of the present invention (i.e., polymers 1 to 4) is used, the amount of nucleic acid amplification product is increased compared to Comparative Example 1. From these results, it can be said that the nucleic acid amplification promoter of the present invention can preferably promote the RNA amplification reaction.

[0056] [Examples 2-1 to 2-8] Examples and comparative examples are shown in which the nucleic acid to be amplified is single-stranded RNA derived from human metapneumovirus and the nucleic acid amplification method is RT-PCR. [Preparation of Solution B] Solution B was prepared by mixing the components shown in Table 5. This preparation was carried out on ice.

[0057] [Preparation of Compositions for Nucleic Acid Amplification] The polymers obtained in Synthesis Examples 1, 2, 5, and 6 were added to the resulting Solution B to prepare compositions for nucleic acid amplification. In Examples 2-1 to 2-3 and 2-6 to 2-8, 2 μL of an aqueous solution of Polymer 1 or 6 was added to Solution B per well, and the compositions were prepared so that the final concentrations of the polymer in the compositions were 0.01, 0.05, or 0.1 w / v%. In Examples 2-4 and 2-5, 2 μL of an aqueous solution of Polymer 2 or 5 was added to Solution B per well, and the compositions were prepared so that the final concentration of the polymer in the compositions was 0.05 w / v%.

[0058] [Nucleic Acid Amplification Reaction] The nucleic acid amplification composition described above was dispensed into a PCR plate (manufactured by BIORAD), the plate was sealed, and the PCR plate was set in a PCR thermal cycler (manufactured by BIORAD). The RT-PCR reaction (Table 3) and the endpoint fluorescence intensity were measured. The measurement was performed three times for each nucleic acid amplification composition.

[0059]

[0060] Comparative Example 2 was performed in the same manner as in Examples 2-1 to 2-8, except that 2 μL of distilled water, nuclease-free (manufactured by Nippon Gene Co., Ltd.) was used instead of 2 μL of the aqueous polymer solution in [Preparation of composition for nucleic acid amplification]. The endpoint fluorescence intensities (relative values) obtained in Examples 2-1 to 2-8, when the endpoint fluorescence intensity in Comparative Example 2 was set to 100%, are shown in Table 7 below.

[0061]

[0062] As shown in Table 7, when the nucleic acid amplification promoter of the present invention (i.e., polymers 1, 2, 5, and 6) is used, the amount of nucleic acid amplification product is increased compared to Comparative Example 2. It is also clear that a sufficient nucleic acid amplification promoting effect can be obtained even when the nucleic acid amplification promoter is added at a concentration of 0.01 w / v%. From these results, it can be said that the nucleic acid amplification promoter of the present invention can preferably promote the RNA amplification reaction.

[0063] [Examples 3-1 to 3-6] These examples and comparative examples show cases where the nucleic acid to be amplified is double-stranded DNA derived from Norovirus and the nucleic acid amplification method is multiplex qPCR. The GI gene and GII gene of Norovirus were detected simultaneously. [Preparation of Solution C] Solution C was prepared by mixing the components shown in Table 8. This preparation was carried out on ice.

[0064]

[0065] [Preparation of Nucleic Acid Amplification Composition] The polymers obtained in Synthesis Examples 1 to 6 were added to the resulting Solution C to prepare compositions for nucleic acid amplification. 2 μL of a 0.5 w / v% diluted aqueous solution of the polymer was added to Solution C per well to prepare compositions for nucleic acid amplification (polymer concentration in each composition: 0.05 w / v%). [Nucleic Acid Amplification Reaction] The nucleic acid amplification composition described above was dispensed into a PCR plate (manufactured by BIORAD) and sealed. The PCR plate was then placed in a PCR thermal cycler (manufactured by BIORAD) and subjected to qPCR using the temperature program shown in Table 9. The measurement was performed three times for each composition for nucleic acid amplification.

[0066]

[0067] Comparative Example 3 was carried out in the same manner as in Examples 3-1 to 3-6, except that in the preparation of a composition for nucleic acid amplification, 2 μL of distilled water, nuclease-free (manufactured by Nippon Gene Co., Ltd.) was used instead of 2 μL of the aqueous polymer solution. The endpoint fluorescence intensities (relative values) obtained in Examples 3-1 to 3-6, when the endpoint fluorescence intensity in Comparative Example 3 was taken as 100%, are shown in Table 10 below.

[0068]

[0069] As shown in Table 10, when the nucleic acid amplification promoter of the present invention (i.e., polymers 1 to 6) is used, the amount of nucleic acid amplification product is increased compared to Comparative Example 3. From these results, it can be said that the nucleic acid amplification promoter of the present invention can preferably promote DNA amplification reactions. From the above examples, it can be said that the nucleic acid amplification promoter of the present invention can preferably promote nucleic acid amplification reactions whether the target of amplification is RNA or DNA.

[0070] The nucleic acid amplification promoter of the present invention can increase the amount of nucleic acid amplification product obtained in a nucleic acid amplification method. Therefore, the nucleic acid amplification promoter of the present invention can be preferably used to improve the yield of target nucleic acid using a nucleic acid amplification method, improve the performance of tests using a nucleic acid amplification method, improve the performance of base sequencing using a nucleic acid amplification method, etc.

Claims

1. A nucleic acid amplification promoter comprising a copolymer containing a structural unit derived from a monomer represented by formula (1) and a structural unit derived from a monomer represented by formula (2). (X １ represents a (meth)acryloyloxy group or a (meth)acryloylamide group, L 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, and R １ ~R ３ R each independently represents an alkyl group having 1 to 3 carbon atoms. ４ represents a hydrogen atom or a methyl group, and R ５ is a group having a sulfonic acid group, a phosphoric acid group, a sulfate group, or a phosphonic acid group.

2. A composition for nucleic acid amplification comprising the nucleic acid amplification promoter of claim 1 and a polymerase.

3. The nucleic acid amplification composition according to claim 2, which is used in a quantitative polymerase chain reaction.

4. The composition for amplifying nucleic acids according to claim 3, further comprising a primer.

5. A method for amplifying nucleic acid, comprising mixing the composition for amplifying nucleic acid according to any one of claims 2 to 4 with a sample containing nucleic acid to be amplified to prepare a reaction solution for amplifying nucleic acid.

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