Nucleic acid amplification sensitizers, nucleic acid amplification compositions, and test kits

Abstract

The present invention provides a sensitizer for nucleic acid amplification, the sensitizer being a polymer containing structural unit derived from a monomer represented by formula (1) (in which the symbols are as defined in the description).

Description

[Technical Field]

[0001] This invention relates to a sensitizer for nucleic acid amplification, a composition for nucleic acid amplification, and a test kit. [Background technology]

[0002] Nucleic acid amplification is a method of amplifying a target nucleic acid by a few copies to tens of thousands of times or more, and it is used in a wide variety of fields for genetic testing, microbiological testing, and viral testing.

[0003] A representative method for nucleic acid amplification is the polymerase chain reaction (PCR) method. In a typical PCR method, nucleic acid amplification is achieved by repeating three steps: (1) denaturation of template DNA (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of primers to single-stranded template DNA, and (3) extension of primers by DNA polymerase. In classical endpoint PCR, the amplification product is detected at the reaction endpoint after a predetermined reaction cycle by visualizing it with a fluorescent compound or by measuring the turbidity of the solution.

[0004] While typical PCR is a method for amplifying DNA, it can also be applied to amplify RNA, and such a PCR method is sometimes abbreviated as reverse transcription polymerase chain reaction (RT-PCR). In RT-PCR, complementary DNA (cDNA) is synthesized from RNA using an enzymatic reaction with reverse transcriptase, and RNA is amplified by performing PCR using this cDNA as a template.

[0005] A method for determining the initial amount of nucleic acid based on the amount of amplification product obtained by PCR is also known, and such a PCR method is called quantitative polymerase chain reaction (hereinafter sometimes abbreviated as "qPCR method"). In a narrow sense, the qPCR method refers to a real-time PCR method that visualizes the amount of DNA amplified in each PCR cycle using a fluorescent DNA staining reagent or fluorescent probe. Among these, the method using a fluorescent probe is known to be a particularly reliable method because it can specifically detect the amplification of the target nucleic acid.

[0006] A method combining RT-PCR and qPCR, known as reverse transcription-quantitative PCR (hereinafter sometimes abbreviated as "RT-qPCR"), is also known. The RT-qPCR method can be further divided into a two-step method, in which cDNA synthesis and qPCR are performed in separate containers, and a one-step method, in which these are performed as a series of reactions in the same container. Of these, the one-step method is superior in terms of ease of operation, minimal contamination from outside the system, and high detection sensitivity (hereinafter sometimes abbreviated as "sensitivity") of the nucleic acid to be measured.

[0007] Furthermore, in recent years, a method called digital PCR (sometimes abbreviated as "dPCR method") has been developed, which applies microfluidic flow path formation technology to distribute the reaction solution into tens to tens of thousands of extremely small compartments, performs PCR simultaneously on all of them, and determines the initial nucleic acid concentration using a statistical model based on the proportion of compartments where amplification occurred. The dPCR method is an advanced version of the endpoint PCR method.

[0008] In addition to determining the presence and / or quantity of target nucleic acids as described above, PCR is also used to determine the base sequence of target nucleic acids (sequencing). There are various sequencing PCR methods, such as the Sanger method, but all of them are based on the endpoint PCR method.

[0009] In the use of nucleic acid amplification methods, sensitivity is often a concern, whether for qualitative, quantitative, or sequencing analysis. Therefore, various techniques to accelerate PCR have been investigated.

[0010] For example, it is widely practiced to enhance sensitivity by adding salts (such as potassium chloride, ammonium sulfate, etc.), betaine, polyhydric alcohols, etc. to the reaction solution of the PCR method. However, there is a limit to the improvement of sensitivity by adding these components, and higher-level improvement of sensitivity has been demanded.

[0011] For example, a method of adding a single-stranded DNA-binding protein (SSB) to destabilize double-stranded DNA is known. Further, Patent Document 1 discloses a mutant PCNA (proliferating cell nuclear antigen) monomer as a highly versatile DNA replication promoting factor (additive) for promoting the DNA elongation reaction. However, additives composed of these proteins have problems such as difficulty in mass production, problems with storage stability, and high manufacturing costs.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0013] An object of the present invention is to provide a sensitizer for nucleic acid amplification that is excellent in mass productivity and storage stability as compared with additives composed of proteins.

Means for Solving the Problems

[0014] As a result of intensive studies by the present inventors, the following formula (1):

[0015]

Chemical Formula

[0016] We have found that polymers containing constituent units derived from monomers represented by [the given formula] can be suitably used as sensitizers for nucleic acid amplification. Based on this finding, the present invention is as follows.

[0017] [1] Formula (1):

[0018] [ka]

[0019] (In the formula, X 1 This represents a (meth)acryloyloxy group or a (meth)acryloylamino group. L 1 This represents a C2-C4 alkylene group that may have one hydroxyl group, or a C2-C4 alkylene oxyalkylene group, and R 1 ~R 3 Each of these independently represents an alkyl group having 1 to 3 carbon atoms. A nucleic acid amplification sensitizer that is a polymer containing constituent units derived from monomers represented by [the formula shown]. [2] The nucleic acid amplification sensitizer according to [1], which is a homopolymer consisting of one type of structural unit derived from the monomer represented by formula (1). [3] Equation (2):

[0020] [ka]

[0021] (In the formula, R 4 represents a hydrogen atom or a methyl group, and R 5 (This represents a hydrogen atom or an alkyl group with 1 to 20 carbon atoms.) The nucleic acid amplification sensitizer according to [1], which is a copolymer further comprising constituent units derived from monomers represented by [1]. [4] R 5 However, the nucleic acid amplification sensitizer described in [3] above is an alkyl group having 12 to 18 carbon atoms. [5] Formula (3):

[0022] [Chemical formula]

[0023] (In the formula, R 6 represents a hydrogen atom or a methyl group, and R 7 represents an alkyl group having 3 to carbon atoms and having two or more hydroxy groups.) The sensitizer for nucleic acid amplification according to [1], [3], or [4] above, which is a copolymer further containing a structural unit derived from the monomer represented by

[0024] [6] A composition for nucleic acid amplification containing the sensitizer for nucleic acid amplification according to any one of [1] to [5] above. [7] The composition for nucleic acid amplification according to [6] above, which is used in the reverse transcription polymerase chain reaction method. [8] The composition for nucleic acid amplification according to [6] or [7] above, which is used in the quantitative polymerase chain reaction method.

[0025] [9] The composition for nucleic acid amplification according to any one of [6] to [8] above, further containing a primer.

[10] The composition for nucleic acid amplification according to [9] above, wherein the primer is an oligonucleotide having a length of 10 to 40 bases.

[11] The composition for nucleic acid amplification according to [9] or

[10] above, wherein the concentration of the primer is 0.1 to 3.0 μM.

[0026]

[12] A test kit containing the composition for nucleic acid amplification according to any one of [6] to

[11] above.

[13] The test kit according to

[12] above, which is for clinical examination.

[14] The test kit according to

[12] or

[13] above, wherein the test target is a virus.

[0027]

[15] A nucleic acid amplification method comprising using any one of the polymers described in [1] to [5] above as a sensitizer.

[16] A nucleic acid amplification method comprising preparing a nucleic acid amplification reaction solution by mixing a nucleic acid amplification composition according to any one of [6] to

[11] above with a sample containing the nucleic acid to be amplified.

[17] The nucleic acid amplification method according to

[15] or

[16] , wherein the method is a reverse transcription polymerase chain reaction method.

[18] A nucleic acid amplification method according to any one of the above

[15] to

[17] , which is a quantitative polymerase chain reaction method. [Effects of the Invention]

[0028] The nucleic acid amplification sensitizer of the present invention can improve the detection sensitivity of nucleic acids in nucleic acid amplification methods. Furthermore, because the nucleic acid amplification sensitizer of the present invention is a synthetic polymer, it has superior mass production capabilities and storage stability compared to additives made of protein. [Modes for carrying out the invention]

[0029] The present invention will be described in detail below. In this specification, "(meth)acryloyloxy group" basically means "acryloyloxy group or methacryloyloxy group." If multiple (meth)acryloyloxy groups are present, "(meth)acryloyloxy group" means "acryloyloxy group and / or methacryloyloxy group." Other terms similar to "(meth)acryloyloxy group" also have the same meaning as "(meth)acryloyloxy group."

[0030] Furthermore, where stepwise numerical ranges are described in this specification, the lower and upper limits of each numerical range can be combined. For example, if it is described as "preferably 10 to 100, more preferably 20 to 90", the "preferred lower limit: 10" and the "more preferred upper limit: 90" can be combined (i.e., the numerical range "10 to 90" is also within the scope of this specification).

[0031] [Sensitizer for nucleic acid amplification] The nucleic acid amplification sensitizer of the present invention (hereinafter sometimes referred to as "the sensitizer of the present invention") is given by the following formula (1):

[0032] [ka]

[0033] This polymer (hereinafter sometimes referred to as "the polymer of the present invention") contains constituent units derived from a monomer represented by (hereinafter sometimes referred to as "monomer (1)"). Here, the sensitizer for nucleic acid amplification refers to an additive used to improve the detection sensitivity of nucleic acids in nucleic acid amplification methods.

[0034] Examples of nucleic acid amplification methods include Polymerase Chain Reaction (PCR), Loop-mediated isothermal Amplification (LAMP), Transcription-mediated Amplification (TMA), Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids (IICAN), Strand Displacement Amplification (SDA), Ligase Chain Reaction (LCR), and Nucleic Acid Seqence-Based Amplification (NASBA). The nucleic acid amplification method is preferably PCR. That is, the sensitizer of the present invention is preferably used in the polymerase chain reaction method.

[0035] As the PCR method, the reverse transcription polymerase chain reaction method described above is preferred. That is, the sensitizer of the present invention is preferably used in the reverse transcription polymerase chain reaction method.

[0036] As the PCR method, the quantitative polymerase chain reaction method described above is preferred. That is, the nuclear sensitizer of the present invention is preferably used in the quantitative polymerase chain reaction method.

[0037] The sensitizer of the present invention may be used alone or in combination of two or more types. Furthermore, the sensitizer of the present invention may be used in combination with other additives.

[0038] A constituent unit derived from monomer (1) (hereinafter sometimes abbreviated as "constituent unit (1)") refers to a constituent unit having a structure formed by the reaction of the carbon-carbon double bond of the (meth)acryloyl group contained in monomer (1). Constituent units derived from other monomers have the same meaning as constituent units derived from monomer (1).

[0039] Monomer (1) may be used alone or in combination of two or more. That is, the sensitizer of the present invention may be a homopolymer consisting of one type of constituent unit (1), or a copolymer containing two or more types of constituent units (1). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions. When the polymer of the present invention does not contain any constituent units other than constituent unit (1), the sensitizer of the present invention is preferably a homopolymer consisting of one type of constituent unit (1), more preferably a homopolymer consisting of constituent units derived from 2-(meth)acryloyloxyethyl phosphorylcholine, and even more preferably a homopolymer consisting of constituent units derived from 2-methacryloyloxyethyl phosphorylcholine.

[0040] The following explains the bases in equation (1) in order. X in equation (1) 1 This represents a (meth)acryloyloxy group (i.e., CH2=CR-CO-O-, R: hydrogen atom or methyl group) or a (meth)acryloylamino group (i.e., CH2=CR-CO-NH-, R: hydrogen atom or methyl group). From the standpoint of raw material availability, X 1 The group is preferably a (meth)acryloyloxy group, and more preferably a methacryloyloxy group.

[0041] L in equation (1) 1This represents a C2-C4 alkylene group that may have one hydroxyl group, or a C2-C4 alkylene oxyalkylene group. The alkylene group may be linear or branched. An example of a C2-C4 alkylene group that may have one hydroxyl group is -C2H4-. An example of a C2-C4 alkylene oxyalkylene group is -C2H4-O-C2H4-. From the viewpoint of raw material availability, L 1 The group is preferably -C2H4- or -C2H4-O-C2H4-, and more preferably -C2H4- (i.e., an ethylene group).

[0042] R in equation (1) 1 ~R 3 Each of these independently represents an alkyl group having 1 to 3 carbon atoms. The alkyl group may be linear or branched. Examples of alkyl groups having 1 to 3 carbon atoms include methyl, ethyl, and propyl groups. From the viewpoint of raw material availability, R 1 ~R 3 Preferably, both are methyl groups.

[0043] A preferred monomer (1) is X 1 However, it is a (meth)acryloyloxy group, L 1 However, it is -C2H4- or -C2H4-O-C2H4-, and R 1 ~R 3 The monomer is a methyl group. A more preferred monomer (1) is X 1 However, it is a (meth)acryloyloxy group, L 1 is an ethylene group, and R 1 ~R 3 The monomer is a methyl group (i.e., 2-(meth)acryloyloxyethyl phosphorylcholine). A more preferred monomer is 2-methacryloyloxyethyl phosphorylcholine. Monomer (1) can be a commercially available product.

[0044] The polymer of the present invention comprises, in addition to the constituent unit (1), formula (2):

[0045] [ka]

[0046] The polymer may further contain constituent units (hereinafter sometimes abbreviated as "constituent units (2)") derived from the monomer represented by (hereinafter sometimes abbreviated as "monomer (2)"). Monomer (2) may be used alone or two or more types may be used in combination. That is, the polymer of the present invention may be a copolymer containing one or more constituent units (1) and one or more constituent units (2). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.

[0047] The following explains the groups in equation (2) in order. 4 R represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of polymers, 4 The group is preferably a methyl group.

[0048] R in equation (2) 5 The group represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms. The alkyl group may be linear or branched. Examples of alkyl groups having 1 to 20 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl groups.

[0049] From the perspective of sensitization effect, R 5Preferably, is an alkyl group having 1 to 20 carbon atoms, more preferably an alkyl group having 2 to 20 carbon atoms, even more preferably an alkyl group having 12 to 18 carbon atoms, and particularly preferably a linear alkyl group having 12 to 18 carbon atoms. Furthermore, if the polymer of the present invention further contains a constituent unit derived from the monomer represented by formula (3) described later, R 5 The alkyl group is preferably a C3-C6 alkyl group.

[0050] Specific examples of 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, pentadecyl (meth)acrylate, cetyl (meth)acrylate, heptadecyl (meth)acrylate, stearyl (meth)acrylate, etc. Monomer (2) can be a commercially available product.

[0051] Among the above specific examples of monomer (2), (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, tetradecyl (meth)acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate, heptadecyl (meth)acrylate, and stearyl (meth)acrylate are more preferred, butyl (meth)acrylate and stearyl (meth)acrylate are even more preferred, and butyl methacrylate and stearyl methacrylate are particularly preferred.

[0052] When the polymer of the present invention contains constituent unit (1) and constituent unit (2), from the viewpoint of sensitization effect, the amount of constituent unit (1) (i.e., monomer (1) used in polymerization) is preferably 30 to 99 moles, more preferably 30 to 90 moles, even more preferably 50 to 90 moles, and particularly preferably 75 to 90 moles, with respect to a total of 100 moles of constituent unit (1) and constituent unit (2) (i.e., a total of 100 moles of monomer (1) and monomer (2) used in polymerization), and the amount of constituent unit (2) (i.e., monomer (2) used in polymerization) is preferably 1 to 70 moles, more preferably 10 to 70 moles, even more preferably 10 to 50 moles, and particularly preferably 10 to 25 moles.

[0053] The polymer of the present invention comprises, in addition to the constituent unit (1), formula (3):

[0054] [ka]

[0055] The polymer may further contain constituent units (hereinafter sometimes abbreviated as "constituent units (3)") derived from the monomer represented by (hereinafter sometimes abbreviated as "monomer (3)"). Monomer (3) may be used alone or two or more types may be used in combination. That is, the polymer of the present invention may be a copolymer containing one or more constituent units (1) and one or more constituent units (3), or a copolymer containing one or more constituent units (1), one or more constituent units (2), and one or more constituent units (3). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.

[0056] The following explains the groups in equation (3) in order. 6 R represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of polymers, 6 The group is preferably a methyl group.

[0057] R in equation (3) 7 R represents an alkyl group with 3 to 6 carbon atoms having two or more hydroxyl groups. 7 The number of hydroxyl groups in the alkyl group is preferably 2 to 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.

[0058] Specific examples of monomer (3) include glycerin mono(meth)acrylate, treitol 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, etc. Among these, 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.

[0059] Monomer (3) may be a commercially available product or may be manufactured by known methods. For example, monomer (3) can be manufactured by the esterification reaction of (meth)acrylic acid or a derivative thereof (e.g., an acid chloride) with a polyhydric alcohol having three or more hydroxyl groups. Esterification reactions are well known and can be carried out by those skilled in the art by setting the conditions appropriately.

[0060] When the polymer of the present invention contains constituent unit (1) and constituent unit (3), from the viewpoint of sensitization effect, the amount of constituent unit (1) (i.e., monomer (1) used in polymerization) is preferably 30 to 80 moles, more preferably 30 to 70 moles, and even more preferably 30 to 60 moles, and the amount of constituent unit (3) (i.e., monomer (3) used in polymerization) is preferably 20 to 70 moles, more preferably 30 to 70 moles, and even more preferably 40 to 70 moles, relative to a total of 100 moles of constituent unit (1) and constituent unit (3) (i.e., a total of 100 moles of monomer (1) and monomer (3) used in polymerization).

[0061] When the polymer of the present invention contains constituent units (1), (2), and (3), from the viewpoint of sensitizing effect, the amount of constituent unit (1) (i.e., monomer (1) used in polymerization) is preferably 30 to 80 moles, more preferably 30 to 70 moles, and even more preferably 30 to 60 moles, relative to a total of 100 moles of constituent units (1), (2), and (3) (i.e., a total of 100 moles of monomers (1), (2), and (3) used in polymerization), and the amount of constituent unit (2) (i.e., monomer (2) used in polymerization) is preferably 10 to 60 moles, more preferably 20 to 60 moles, and even more preferably 30 to 60 moles, and the amount of constituent unit (3) (i.e., monomer (3) used in polymerization) is preferably 10 to 60 moles, more preferably 10 to 50 moles, and even more preferably 10 to 40 moles.

[0062] The polymer of the present invention may contain other constituent units derived from monomers other than monomers (1) to (3) described above, to the extent that the effects of the present invention are not impaired. Only one other monomer may be used, or two or more may be used in combination. The other monomers are not particularly limited, but examples include benzyl (meth)acrylate and isobornyl (meth)acrylate. The amount of other constituent units in the polymer of the present invention is preferably 20 mol% or less relative to the total constituent units. It is more preferable that the polymer of the present invention does not contain other constituent units.

[0063] The polymer of the present invention is preferably at least one selected from the group consisting of a homopolymer consisting of one type of structural unit (1), a copolymer consisting of structural unit (1) and structural unit (2), and a copolymer consisting of structural unit (1), structural unit (2), and structural unit (3), and more preferably a homopolymer consisting of one type of structural unit (1), a copolymer consisting of structural unit (1) and structural unit (2), or a copolymer consisting of structural unit (1), structural unit (2), and structural unit (3). In this specification, "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), "copolymer consisting of structural unit (1) and structural unit (2)" means a copolymer in which all of its structural units (repeating units) consist of structural unit (1) and structural unit (2), and "copolymer consisting of structural unit (1), structural unit (2), and structural unit (3)" means a copolymer in which all of its structural units (repeating units) consist of structural unit (1), structural unit (2), and structural unit (3). Other similar expressions have the same meaning.

[0064] The weight-average molecular weight of the polymer of the present invention is not particularly limited, but is preferably 10,000 to 1,000,000. This weight-average molecular weight can be determined, for example, by gel filtration chromatography using an EcoSEC system (manufactured by Tosoh Corporation) in terms of polyethylene glycol.

[0065] The polymer of the present invention can be produced by known methods (for example, the method described in International Publication No. 2018 / 216628).

[0066] The amount of the sensitizer of the present invention (i.e., the polymer of the present invention) used is determined by its concentration in the nucleic acid amplification composition described later. 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 / v%, and even more preferably 0.01 to 0.5 w / v%, from the viewpoint of sensitizing effect and suppression of viscosity increase of the composition. When two or more sensitizers are used, the above concentration means the sum of the concentrations of the two or more sensitizers. Similarly, when two or more of the other components described below are used, the above concentration means the sum of the concentrations of the two or more components.

[0067] [Suitable examples of the sensitizer (polymer of the present invention) of the present invention] Suitable examples of the sensitizer (polymer of the present invention) of the present invention include the following sensitizers (I) (polymer (I)) to sensitizers (IV) (polymer (IV)).

[0068] <Inventive sensitizer (I) (Inventive polymer (I))> The sensitizer (I) of the present invention (polymer (I) of the present invention) is at least one selected from the group consisting of the following homopolymers (I-1), copolymers (I-2), and copolymers (I-3), and is preferably the following homopolymer (I-1), copolymer (I-2), or copolymer (I-3): A homopolymer (I-1) consisting of a constituent unit (1) derived from 2-(meth)acryloyloxyethyl phosphorylcholine; A constituent unit (1) derived from 2-(meth)acryloyloxyethyl phosphorylcholine, (2) Constituent units derived from (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, or stearyl (meth)acrylate and Copolymer (I-2) comprising, and wherein, for a total of 100 moles of constituent units (1) and (2), the amount of constituent unit (1) is 30 to 99 moles and the amount of constituent unit (2) is 1 to 70 moles; and A constituent unit (1) derived from 2-(meth)acryloyloxyethyl phosphorylcholine, A constituent unit (2) derived from (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, or stearyl (meth)acrylate, Constituent units (3) derived from glycerin mono(meth)acrylate or xylitol mono(meth)acrylate and A copolymer (I-3) comprising, and wherein, for a total of 100 moles of constituent units (1), (2), and (3), the amount of constituent unit (1) is 30 to 80 moles, the amount of constituent unit (2) is 10 to 60 moles, and the amount of constituent unit (3) is 10 to 60 moles.

[0069] In the sensitizer (I) of the present invention (polymer (I) of the present invention), it is preferable that the amount of constituent unit (1) in copolymer (I-2) is 30 to 90 moles and the amount of constituent unit (2) is 10 to 70 moles, and in copolymer (I-3) the amount of constituent unit (1) is 30 to 70 moles, the amount of constituent unit (2) is 20 to 60 moles and the amount of constituent unit (3) is 10 to 50 moles. The standard amount of the constituent units in copolymer (I-2) is a total of 100 moles of constituent unit (1) and constituent unit (2), and the standard amount of the constituent units in copolymer (I-3) is a total of 100 moles of constituent unit (1), constituent unit (2), and constituent unit (3).

[0070] In the sensitizer (I) of the present invention (polymer (I) of the present invention), it is more preferable that the amount of constituent unit (1) in copolymer (I-2) is 50 to 90 moles and the amount of constituent unit (2) is 10 to 50 moles, and that the amount of constituent unit (1) in copolymer (I-3) is 30 to 60 moles, the amount of constituent unit (2) is 30 to 60 moles and the amount of constituent unit (3) is 10 to 40 moles. The standard amount of the constituent units in copolymer (I-2) is a total of 100 moles of constituent unit (1) and constituent unit (2), and the standard amount of the constituent units in copolymer (I-3) is a total of 100 moles of constituent unit (1), constituent unit (2), and constituent unit (3).

[0071] In the sensitizer (I) of the present invention (polymer (I) of the present invention), it is more preferable that the amount of constituent unit (1) in copolymer (I-2) is 75 to 90 moles and the amount of constituent unit (2) is 10 to 25 moles, and that the amount of constituent unit (1) in copolymer (I-3) is 30 to 60 moles, the amount of constituent unit (2) is 30 to 60 moles and the amount of constituent unit (3) is 10 to 40 moles. The standard for the amount of constituent units in copolymer (I-2) is a total of 100 moles of constituent unit (1) and constituent unit (2), and the standard for the amount of constituent units in copolymer (I-3) is a total of 100 moles of constituent unit (1), constituent unit (2), and constituent unit (3).

[0072] <Inventive invention sensitizer (II) (Inventive invention polymer (II))> The sensitizer (II) of the present invention (polymer (II) of the present invention) is at least one selected from the group consisting of the following homopolymers (II-1), copolymers (II-2), and copolymers (II-3), and is preferably the following homopolymer (II-1), copolymer (II-2), or copolymer (II-3): A homopolymer (II-1) consisting of a constituent unit (1) derived from 2-methacryloyloxyethyl phosphorylcholine; A constituent unit (1) derived from 2-methacryloyloxyethyl phosphorylcholine, Constituent units (2) derived from butyl (meth)acrylate, dodecyl (meth)acrylate, tridecyl (meth)acrylate, tetradecyl (meth)acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate, heptadecyl (meth)acrylate, or stearyl (meth)acrylate and Copolymer (II-2) comprising, and wherein, for a total of 100 moles of constituent units (1) and (2), the amount of constituent unit (1) is 30 to 90 moles and the amount of constituent unit (2) is 10 to 70 moles; and A constituent unit (1) derived from 2-methacryloyloxyethyl phosphorylcholine, A constituent unit (2) derived from butyl (meth)acrylate, dodecyl (meth)acrylate, tridecyl (meth)acrylate, tetradecyl (meth)acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate, heptadecyl (meth)acrylate, or stearyl (meth)acrylate, Constituent units (3) derived from glycerin mono(meth)acrylate and A copolymer (II-3) comprising, and wherein, for a total of 100 moles of constituent units (1), (2), and (3), the amount of constituent unit (1) is 30 to 70 moles, the amount of constituent unit (2) is 20 to 60 moles, and the amount of constituent unit (3) is 10 to 50 moles.

[0073] In the sensitizer (II) of the present invention (polymer (II) of the present invention), it is preferable that the amount of constituent unit (1) in copolymer (II-2) is 50 to 90 moles and the amount of constituent unit (2) is 10 to 50 moles, and in copolymer (II-3) the amount of constituent unit (1) is 30 to 60 moles, the amount of constituent unit (2) is 30 to 60 moles and the amount of constituent unit (3) is 10 to 40 moles. The standard amount of the constituent units in copolymer (II-2) is a total of 100 moles of constituent unit (1) and constituent unit (2), and the standard amount of the constituent units in copolymer (II-3) is a total of 100 moles of constituent unit (1), constituent unit (2), and constituent unit (3).

[0074] In the sensitizer (II) of the present invention (polymer (II) of the present invention), it is more preferable that the amount of constituent unit (1) in copolymer (II-2) is 75 to 90 moles and the amount of constituent unit (2) is 10 to 25 moles, and that the amount of constituent unit (1) in copolymer (II-3) is 30 to 60 moles, the amount of constituent unit (2) is 30 to 60 moles and the amount of constituent unit (3) is 10 to 40 moles. The standard amount of the constituent units in copolymer (II-2) is a total of 100 moles of constituent unit (1) and constituent unit (2), and the standard amount of the constituent units in copolymer (II-3) is a total of 100 moles of constituent unit (1), constituent unit (2), and constituent unit (3).

[0075] <Inventive invention sensitizer (III) (Inventive invention polymer (III))> The sensitizer (III) of the present invention (polymer (III) of the present invention) is at least one selected from the group consisting of the following homopolymers (III-1), copolymers (III-2), and copolymers (III-3), and is preferably the following homopolymer (III-1), copolymer (III-2), or copolymer (III-3): A homopolymer (III-1) consisting of a constituent unit (1) derived from 2-methacryloyloxyethyl phosphorylcholine; A constituent unit (1) derived from 2-methacryloyloxyethyl phosphorylcholine, Constituent units (2) derived from butyl (meth)acrylate or stearyl (meth)acrylate and A copolymer (III-2) comprising, and wherein, for a total of 100 moles of constituent units (1) and (2), the amount of constituent unit (1) is 50 to 90 moles and the amount of constituent unit (2) is 10 to 50 moles; and A constituent unit (1) derived from 2-methacryloyloxyethyl phosphorylcholine, Constituent units (2) derived from butyl (meth)acrylate or stearyl (meth)acrylate and Constituent units (3) derived from glycerol monomethacrylate and A copolymer (III-3) comprising, and wherein, for a total of 100 moles of constituent units (1), (2), and (3), the amount of constituent unit (1) is 30 to 60 moles, the amount of constituent unit (2) is 30 to 60 moles, and the amount of constituent unit (3) is 10 to 40 moles.

[0076] In the sensitizer (III) of the present invention (polymer (III) of the present invention), it is preferable that the amount of constituent unit (1) in copolymer (III-2) is 75 to 90 moles, and the amount of constituent unit (2) is 10 to 25 moles. The standard amount of the aforementioned constituent units in copolymer (III-2) is 100 moles in total for constituent unit (1) and constituent unit (2).

[0077] <Inventive invention sensitizer (IV) (Inventive invention polymer (IV))> The sensitizer (IV) of the present invention (polymer (IV) of the present invention) is at least one selected from the group consisting of the following homopolymers (IV-1), copolymers (IV-2), and copolymers (IV-3), and is preferably the following homopolymer (IV-1), copolymer (IV-2), or copolymer (IV-3): A homopolymer (IV-1) consisting of a constituent unit (1) derived from 2-methacryloyloxyethyl phosphorylcholine; A constituent unit (1) derived from 2-methacryloyloxyethyl phosphorylcholine, Constituent units (2) derived from butyl methacrylate or stearyl methacrylate and Copolymer (IV-2) comprising, and wherein, for a total of 100 moles of constituent units (1) and (2), the amount of constituent unit (1) is 75 to 90 moles and the amount of constituent unit (2) is 10 to 25 moles; and A constituent unit (1) derived from 2-methacryloyloxyethyl phosphorylcholine, A constituent unit (2) derived from butyl methacrylate or stearyl methacrylate, Constituent units (3) derived from glycerol monomethacrylate and A copolymer (IV-3) comprising, and wherein, for a total of 100 moles of constituent units (1), (2), and (3), the amount of constituent unit (1) is 30 to 60 moles, the amount of constituent unit (2) is 30 to 60 moles, and the amount of constituent unit (3) is 10 to 40 moles.

[0078] [Compositions 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"). In the composition of the present invention, the sensitizer of the present invention may be used alone or in combination of two or more types. Here, the composition for nucleic acid amplification means a composition used in a nucleic acid amplification method. The nucleic acid amplification method is described above. The nucleic acid amplification method is preferably the PCR method. That is, the composition of the present invention is preferably used in the polymerase chain reaction method.

[0079] As the PCR method, the reverse transcription polymerase chain reaction method described above is preferred. That is, the composition of the present invention is preferably used in the reverse transcription polymerase chain reaction method.

[0080] As the PCR method, the quantitative polymerase chain reaction method described above is preferred. That is, the composition of the present invention is preferably used in the quantitative polymerase chain reaction method.

[0081] The composition of the present invention can be prepared by dissolving the sensitizer of the present invention, together with other components used for nucleic acid amplification as needed, in a solvent such as water. That is, the composition of the present invention is preferably a composition comprising the sensitizer of the present invention and water (and other components as needed). The concentration of the sensitizer of the present invention in the composition of the present invention is described above.

[0082] Other components used for nucleic acid amplification include known components used in known nucleic acid amplification methods such as PCR. Examples of such components include buffers, substrates, primers, DNA polymerase, fluorescent DNA staining reagents, fluorescent probes, passive references, and nucleic acids. Each of these other components may be used individually or in combination of two or more.

[0083] The buffer is not particularly limited, but examples include a mixture of a base such as tris(hydroxymethyl)aminomethane, tricine, or bicine with an acid such as sulfuric acid, hydrochloric acid, acetic acid, or phosphoric acid, with the pH adjusted to 6-9, more preferably 7-8. The buffer is also preferably to contain magnesium salts and / or manganese salts as appropriate. Furthermore, the buffer may further contain salts such as potassium chloride or ammonium sulfate. The buffer may also further contain water-soluble organic solvents such as dimethyl sulfoxide, dimethylformamide, formamide, or glycerin. The buffer may further contain surfactants such as polyoxysorbitan fatty acid esters or polyoxyethylene alkylphenyl ethers. The buffer may also further contain proteins such as bovine serum albumin.

[0084] The substrate is not particularly limited, but examples include a mixture of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dCTP) (dNTPs). Here, it is also possible to substitute some and / or all of dTTP with deoxyuridine triphosphate (dUTP). Furthermore, 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 versions thereof.

[0085] Examples of primers include oligonucleotides with a length of 10 to 40 bases. The length of the oligonucleotide is preferably 15 to 30 bases, more preferably 15 to 25 bases. The oligonucleotide can be designed and prepared by known methods. The oligonucleotide may have a fluorescent group formed from fluorothane or the like.

[0086] The primer may be used alone, or two primers may be used as a pair, or multiple primers may be used to amplify multiple regions simultaneously. The primer concentration in the composition of the present invention is preferably 0.1 to 25 μM, more preferably 0.1 to 15 μM, and even more preferably 0.5 to 10 μM.

[0087] As the DNA polymerase, any known DNA polymerase can be used. From the viewpoint of heat resistance, enzymes derived from thermophilic bacteria, thermophilic archaea, hyperthermia bacteria, hyperthermia archaea, and their mutant enzymes are preferred. The DNA polymerase can be appropriately selected from DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, or an enzyme possessing both functions, depending on the purpose of nucleic acid amplification. Furthermore, whether to use a DNA polymerase with nuclease activity or a DNA polymerase without nuclease activity can be appropriately selected.

[0088] The fluorescent DNA staining reagent is not particularly limited, but for example, SYBR TM Green I is one example. The fluorescent probe is not particularly limited, but for example, TaqMan TM A probe is mentioned. A passive reference should be appropriately selected depending on the purpose of nucleic acid amplification. An example of a passive reference is ROX. TM Examples include dyes, etc.

[0089] In addition to the primers and fluorescent probes mentioned above, any DNA and / or RNA may be used as nucleic acids, for example, as exogenous control genes. Here, the nucleic acids may be synthesized in vitro, or prepared from cells, microorganisms, viruses, etc., by known methods. Here, the cells, microorganisms, viruses, etc., may be collected from nature or the environment, from humans or animals and plants, or they may be isolated and cultured.

[0090] The composition of the present invention may further contain an oil such as mineral oil, or a solid support such as glass beads or magnetic beads.

[0091] Alternatively, you may use kit-type products that combine multiple of the above-mentioned components, or master mix (sometimes called primer mix, premix, etc.) type products that contain these components pre-mixed.

[0092] [Test kit] The composition of the present invention can be combined with necessary components to form a test kit. The present invention provides a test kit containing the composition of the present invention. The components are not particularly limited, but examples include a sample collection device, a sample collection container, a sample pretreatment reagent, a calibration standard, a test instrument, consumables, a measurement cassette, an instruction manual, etc. The components are appropriately selected depending on the type of test. The components may be included in the test kit. Commercially available products may be used as the components. Furthermore, components conforming to specified standards may be used.

[0093] The test kit of the present invention can be used, for example, for genetic testing, microbiological testing, and viral testing, and is preferably used for viral testing. That is, the target of testing for the test reagent test kit of the present invention is preferably a virus.

[0094] Examples of tests in which the test kit of the present invention is used include tests conducted in fields such as medicine, veterinary medicine, forensic medicine, pharmaceutical analysis, food analysis, and environmental surveys. Among these, tests conducted in the fields of medicine and veterinary medicine are preferred, and tests conducted in the medical field are more preferred. The test kit of the present invention is preferably for clinical testing and more preferably for in vitro diagnostics.

[0095] [Nucleic acid amplification method] The present invention also provides (i) a nucleic acid amplification method comprising using the polymer of the present invention as a sensitizer, and (ii) a nucleic acid amplification method comprising preparing a reaction solution for nucleic acid amplification by mixing the composition of the present invention with a sample containing the nucleic acid to be amplified. The description of the polymer of the present invention and the composition of the present invention in the nucleic acid amplification method of the present invention is as described above. The description of the nucleic acid amplification method is also as described above unless otherwise specified.

[0096] The sample used in the nucleic acid amplification method of the present invention contains the nucleic acid to be amplified. Preferably, the sample is a sample solution containing water and the nucleic acid to be amplified. The sample may contain one type of nucleic acid or two or more types of nucleic acids. The concentration of 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 / μL, preferably 1-10 10 copies / μL, more preferably 1 to 10 5 The values ​​are copies / μL, particularly preferably 1 to 500 copies / μL, and most preferably 1 to 100 copies / μL.

[0097] In the diffusion amplification method of the present invention, the amount of sample used is preferably a trace amount of ~1 μL, more preferably 0.01 to 1 μL, and even more preferably 0.2 to 0.4 μL, per 1 μL of the composition of the present invention.

[0098] The nucleic acid amplification method of the present invention is preferably a reverse transcription polymerase chain reaction method. Furthermore, the nucleic acid amplification method of the present invention is preferably a quantitative polymerase chain reaction method. [Examples]

[0099] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0100] [Synthesis of polymers (sensitizers for nucleic acid amplification)] [Synthesis Example 1] 40.0 g of 2-methacryloyloxyethyl phosphorylcholine (hereinafter sometimes abbreviated as "MPC") as monomer (1) was weighed into a polymerization glass flask, and 60.0 g of purified water was added to dissolve monomer (1). To the resulting solution, 0.31 g of azobisisobutyronitrile (hereinafter referred to as "AIBN") was added as a polymerization initiator. After thoroughly purging the reaction vessel with nitrogen, polymerization was carried out by heating at 70°C for 6 hours under stirring. The resulting reaction solution was cooled with ice, and the polymer was precipitated by adding it dropwise to diethyl ether. The precipitate was filtered off, washed with diethyl ether, and then vacuum dried to obtain a white powdery homopolymer (hereinafter referred to as "polymer 1"). The weight-average molecular weight of polymer 1 was 1,030,000 in terms of polyethylene glycol, as measured by gel filtration chromatography (hereinafter sometimes abbreviated as "GPC") under the conditions described later.

[0101] [Synthesis Example 2] 6.0 g of MPC as monomer (1) and 4.0 g of methacrylic acid (hereinafter sometimes abbreviated as "MA") as monomer (2) (monomer (1) / monomer (2) = 30 / 70 (molar ratio)) were weighed into a polymerization glass flask, and 90.0 g of purified water was added to dissolve monomers (1) and (2). 0.78 g of AIBN was added to the resulting solution. A random copolymer (hereinafter referred to as "polymer 2") was obtained in the same manner as in Synthesis Example 1. The weight-average molecular weight of polymer 2 was 680,000 in terms of polyethylene glycol, as measured by GPC under the conditions described later.

[0102] [Synthesis Example 3] 19.4 g of MPC as monomer (1) and 2.2 g of butyl methacrylate (hereinafter sometimes abbreviated as "BMA") as monomer (2) (monomer (1) / monomer (2) = 80 / 20 (molar ratio)) were weighed into a polymerization glass flask. 39.3 g of purified water and 39.3 g of ethanol were added to dissolve monomers (1) and (2), and 0.02 g of AIBN was added to the resulting solution. After thoroughly purging the flask with nitrogen, polymerization was carried out by heating at 60°C for 5 hours under stirring. A random copolymer (hereinafter referred to as "polymer 3") was obtained in the same manner as in Synthesis Example 1. The weight-average molecular weight of polymer 3 was 600,000 in terms of polyethylene glycol, as measured by GPC under the conditions described later.

[0103] [Synthesis Example 4] 11.7 g of MPC as monomer (1) and 3.3 g of stearyl methacrylate (hereinafter sometimes abbreviated as "SMA") as monomer (2) (monomer (1) / monomer (2) = 80 / 20 (molar ratio)) were weighed into a polymerization glass flask, 85 g of ethanol was added to dissolve monomers (1) and (2), and 0.06 g of AIBN was added to the resulting solution. After thoroughly purging the flask with nitrogen, polymerization was carried out by heating at 60°C for 6 hours under stirring. A random copolymer (hereinafter referred to as "polymer 4") was obtained in the same manner as in Synthesis Example 1. The weight-average molecular weight of polymer 4 was 43,000 in terms of polyethylene glycol, as measured by GPC under the conditions described later.

[0104] [Synthesis Example 5] 8.4 g of MPC as monomer (1), 2.1 g of BMA as monomer (2), and 4.5 g of glycerin monomethacrylate (hereinafter sometimes abbreviated as "GLM") as monomer (3) (monomer (1) / monomer (2) / monomer (3) = 40 / 40 / 20 (molar ratio)) were weighed into a polymerization glass flask, and 42.5 g of purified water and 42.5 g of ethanol were added to dissolve monomers (1), (2), and (3). 0.15 g of AIBN was added to the resulting solution. A random copolymer (hereinafter referred to as "polymer 5") was obtained in the same manner as in Synthesis Example 3. The weight-average molecular weight of polymer 5 was 22,000 in terms of polyethylene glycol, as measured by GPC under the conditions described later.

[0105] [GPC measurement] GPC measurements of polymers 1-5 obtained in synthesis examples 1-5 were performed under the following conditions. GPC System: EcoSEC System (manufactured by Tosoh Corporation) Columns: 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: 0.5mL / min Column temperature: 40℃ Sample: Dilute the obtained polymer with the developing solvent to a final concentration of 0.1% by weight. Injection volume: 100μL

[0106] Table 1 summarizes the monomers and their molar ratios used in Synthesis Examples 1-5, as well as the weight-average molecular weight of the resulting polymers.

[0107] [Table 1]

[0108] [Examples 1-5 and Comparative Example 1] [Sample solution] The Positive Control RNA, N set No.2 (N2) included with the SARS-CoV-2 RT-qPCR Detection Kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted to 10 copies / μL with nuclease-free purified water (hereinafter sometimes referred to as "PW (nuclease-free)") according to the instructions in the kit's package insert to prepare the sample solution. PW (nuclease-free) was used as a negative control instead of the above sample solution.

[0109] [Reaction solution] Using the components of the aforementioned kit and 50x ROX Passive Reference (manufactured by Nippon Gene Co., Ltd.), solutions with the common composition shown in Table 2 were prepared. A predetermined amount of the polymers obtained in Synthesis Examples 1-5 was added to these solutions, and a predetermined amount of PW (nuclease-free) was added to prepare a nucleic acid amplification composition. 15 μL of the obtained nucleic acid amplification composition was then placed on a PCR plate (MicroAmp). TM The sample solution was dispensed into a Fast Optical 96-well Reaction Plate with Barcode (0.1 mL, AppliedBioscience), and then 5 μL of the sample solution was spiked in to prepare a total of 20 μL of reaction solution. The composition of the obtained reaction solution is shown in Table 2 below.

[0110] In the above procedure, 2 μL of an aqueous polymer solution with a concentration 10 times that of the final concentration of the polymer in the reaction solution was added. For example, under the condition that "the final concentration of polymer 1 in the reaction solution is 0.1 w / v%", 2 μL of a 1 w / v% aqueous solution of polymer 1 and 13 μL of a solution of the common components and PW (nuclease-free) were mixed to prepare 15 μL of a nucleic acid amplification composition, and 5 μL of the sample solution was added to this to prepare 20 μL of a reaction solution.

[0111] [Table 2]

[0112] [reaction] The PCR plate containing the reaction solution is placed in StepOnePlus TM The samples were placed in a Real-Time PCR System (AppliedBioscience) and RT-qPCR was performed using the temperature program shown in Table 3. Fluorescence intensity was read in step #5 of Table 3.

[0113] [Table 3]

[0114] [result] Under the conditions shown in Table 4-1, perform the RT-qPCR method according to the procedure described above, and measure the endpoint fluorescence intensity and the following formula: Endpoint enhancement rate (%) = 100 × (F - F0) / F0 (In the formula, F represents the endpoint fluorescence intensity when the polymer is added, and F0 represents the endpoint fluorescence intensity when the polymer is not added (Comparative Example 1).) The endpoint enhancement rate was then calculated. Here, endpoint fluorescence intensity refers to the fluorescence intensity at the final cycle (60th cycle) of the PCR method, and fluorescence intensity refers to the ΔRn value. The results are shown in Table 4-1.

[0115] [Table 4-1]

[0116] [Comparative Examples 2-5] In Comparative Examples 2-5, the sensitizers of the present invention used in Examples 1-5 (i.e., polymers 1-5) were replaced with the additives shown in Table 4-2 (i.e., bovine serum albumin (hereinafter referred to as "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.)) and the endpoint enhancement rate (%) was calculated. Specifically, RT-qPCR was performed under the conditions shown in Table 4-2 according to the procedure in [Examples 1-5 and Comparative Example 1], and the endpoint fluorescence intensity and the following formula were used: Endpoint enhancement rate (%) = 100 × (F - F0) / F0 (In the formula, F represents the endpoint fluorescence intensity when the additive is added, and F0 represents the endpoint fluorescence intensity when the additive is not added (Comparative Example 1).) The endpoint enhancement rate was calculated. The results are shown in Table 4-2.

[0117] [Table 4-2]

[0118] As shown in Table 4-1, the endpoint enhancement rates for Examples 1-5 using the sensitizer of the present invention (i.e., polymers 1-5) ranged from 372% to 583%. On the other hand, as shown in Table 4-2, the endpoint enhancement rates for 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) ranged from 76% to 157%. These results indicate that the detection sensitivity of nucleic acids can be improved by using the sensitizer of the present invention.

[0119] [Examples 6-10 and Comparative Example 6] [sample] Positive Control RNA, N set No.2 (N2), included with the SARS-CoV-2 RT-qPCR Detection Kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), was diluted with PW (nuclease-free) according to the kit's instructions to prepare sample solutions with nucleic acid concentrations of 160, 40, 10, 2.5, or 0.0625 copies / μL. PW (nuclease-free) was also used as a negative control in place of the sample solutions.

[0120] [Reaction solution] Using the components of the aforementioned kit, 50x ROX Passive Reference (manufactured by Nippon Gene Co., Ltd.), and the polymers obtained in Synthesis Examples 1-5, 15 μL of nucleic acid amplification composition was prepared in the same manner as in Examples 1-5 and Comparative Example 1. 5 μL of the sample solution was spiked into this nucleic acid amplification composition to prepare a total of 20 μL of reaction solution. The final concentrations of the polymer in the reaction solution are as shown in Table 5 below.

[0121] [reaction] RT-qPCR was performed in the same manner as in Examples 1-5 and Comparative Example 1.

[0122] [result] RT-qPCR was performed according to the procedure described above under each condition shown in Table 5. For each condition, a reaction was performed with N=2. Samples showing amplification in both conditions were classified as "strongly detected" (indicated as "PP" in Table 5), samples showing amplification in one condition were classified as "weakly detected" (indicated as "P" in Table 5), and samples showing no amplification in either condition were classified as "not detected" (indicated as "N" in Table 5). The lowest nucleic acid concentration in the sample solution classified as "strongly detected" or "weakly detected" was determined as the respective detection limit. In cases where "weakly detected" was not detected, the detection limit for "weakly detected" was considered equal to the detection limit for "strongly detected." The results are shown in Table 5.

[0123] [Table 5]

[0124] As shown in Table 5, in Examples 6 to 10, which used the sensitizers of the present invention (i.e., polymers 1 to 5), the detection limits for "strong detection" and / or "weak detection" were lowered compared to Comparative Example 6, which did not use these sensitizers. These results indicate that the detection sensitivity of nucleic acids, which are the target of measurement, can be improved by using the sensitizers of the present invention. [Industrial applicability]

[0125] The sensitizer of the present invention can improve the detection sensitivity of nucleic acids to be measured by nucleic acid amplification methods (particularly PCR). The sensitizer of the present invention can be suitably used in nucleic acid amplification methods for genetic testing, microbiological testing, viral testing, and the like.

[0126] This application is based on Japanese Patent Application No. 2021-12400, the contents of which are fully encompassed in the specification of this application.

Claims

1. Formula (1): 【Chemistry 1】 (In the formula, X １ This represents a (meth)acryloyloxy group or a (meth)acryloylamino group. L １ This represents a C2-C4 alkylene group that may have one hydroxyl group, or a C2-C4 alkyleneoxyalkylene group, and R １ ~R ３ Each of these independently represents an alkyl group having 1 to 3 carbon atoms. A nucleic acid amplification sensitizer that is a polymer containing constituent units derived from monomers represented by [the formula shown].

2. The nucleic acid amplification sensitizer according to claim 1, which is a homopolymer consisting of one type of structural unit derived from the monomer represented by formula (1) above.

3. Formula (2): 【Chemistry 2】 (In the formula, R ４ represents a hydrogen atom or a methyl group, and R ５ (This represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.) The nucleic acid amplification sensitizer according to claim 1, which is a copolymer further comprising a constituent unit derived from a monomer represented by

4. R ５ However, the nucleic acid amplification sensitizer according to claim 3, wherein the alkyl group has 12 to 18 carbon atoms.

5. Formula (3): 【Transformation 3】 (In the formula, R ６ represents a hydrogen atom or a methyl group, and R ７ represents an alkyl group having 2 or more hydroxy groups and having 3 to 6 carbon atoms.) A nucleic acid amplification sensitizer according to claim 1, 3, or 4, which is a copolymer further comprising a constituent unit derived from a monomer represented by .

6. A composition for nucleic acid amplification comprising a nucleic acid amplification sensitizer according to any one of claims 1 to 5.

7. A nucleic acid amplification composition according to claim 6, used in a reverse transcription polymerase chain reaction method.

8. A nucleic acid amplification composition according to claim 6 or 7, used in a quantitative polymerase chain reaction method.

9. A test kit comprising the nucleic acid amplification composition according to any one of claims 6 to 8.

10. A test kit according to claim 9, which is for clinical testing.

11. The test kit according to claim 9 or 10, wherein the test subject is a virus.

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