Sensitizers for nucleic acid amplification, compositions for nucleic acid amplification, and test kits

Abstract

The present invention provides a sensitizer for nucleic acid amplification, which is a polymer comprising a structural unit derived from a monomer represented by the following formula (1): wherein the symbols are as described in the specification.

Description

Technical Field

[0001] This invention relates to sensitizers for nucleic acid amplification, compositions for nucleic acid amplification, and diagnostic kits. Background Technology

[0002] Nucleic acid amplification is a method that amplifies the target nucleic acid from a few copies to tens of thousands of times or more. It is effectively used in a variety of fields for gene testing, microbial testing, and virus testing.

[0003] The representative method for nucleic acid amplification is polymerase chain reaction (PCR). In a typical PCR method, nucleic acid amplification is achieved by repeating the following three steps: (1) denaturation of template DNA (dissociation from double-stranded DNA to single-stranded DNA), (2) primer annealing to single-stranded template DNA, and (3) primer extension by DNA polymerase. In the classic endpoint PCR method, the amplified products are detected at the endpoint of the reaction after a specified reaction cycle by visualization with fluorescent compounds or by measuring the turbidity of the solution.

[0004] Typical PCR is a method for amplifying DNA, but it can also be applied to amplify RNA. This type of PCR is called reverse transcription polymerase chain reaction (hereinafter sometimes simply referred to as "RT-PCR"). In RT-PCR, complementary DNA (hereinafter sometimes simply referred to as "cDNA") is synthesized from RNA using an enzymatic reaction of reverse transcriptase. This cDNA is then used as a template for PCR, thereby amplifying RNA.

[0005] It is also known to determine the initial amount of nucleic acid based on the amount of amplified products obtained in a PCR method; this type of PCR is called quantitative polymerase chain reaction (hereinafter sometimes simply referred to as "qPCR"). In a narrow sense, qPCR refers to real-time PCR, which is a method that visualizes the amount of DNA amplified in each PCR cycle using fluorescent DNA staining reagents or fluorescent probes. Among these, the method using fluorescent probes is known for its high reliability in specifically detecting the amplification of the target nucleic acid.

[0006] Reverse transcription-quantitative PCR (RT-qPCR), which combines RT-PCR and qPCR, is also known. RT-qPCR can be further divided into two-step methods: one where cDNA synthesis and qPCR are performed in separate containers; and a one-step method where they are performed as a series of reactions in the same container. The one-step method is superior in terms of ease of operation, fewer contaminants from outside the system, and high detection sensitivity (sometimes referred to as "sensitivity") of the nucleic acids being measured.

[0007] Furthermore, in recent years, digital PCR (hereinafter sometimes referred to as "dPCR") has been developed. This method utilizes microfluidic circuit formation technology to distribute the reaction solution into tens to tens of thousands of extremely small regions, which are then subjected to PCR simultaneously. The initial nucleic acid concentration is determined using a statistical model based on the proportion of regions with amplification. dPCR is an evolution of endpoint PCR.

[0008] In addition to determining the presence and / or quantity of target nucleic acids as mentioned above, PCR is also used to determine the nucleotide sequence of target nucleic acids (sequencing). Various methods exist within sequencing PCR, such as the Sanger method, but all are based on the fundamental principle of endpoint PCR.

[0009] In this context, sensitivity is often a problem when using nucleic acid amplification methods, particularly in qualitative, quantitative, and sequence determination. Therefore, various studies have been conducted on techniques to facilitate PCR.

[0010] For example, it has been widely studied to improve sensitivity by adding salts (such as potassium chloride, ammonium sulfate, etc.), betaine, polyols, etc., to the reaction solution of PCR. However, there are limits to the ability to improve sensitivity by adding these components, and efforts are being made to improve sensitivity at a higher level.

[0011] For example, methods for destabilizing double-stranded DNA by adding single-stranded DNA-binding proteins (SSBs) are known. Furthermore, Patent Document 1 discloses a mutant PCNA (proliferating nuclear antigen) monomer as a highly versatile DNA replication-promoting factor (additive) for promoting DNA elongation. However, additives composed of these proteins suffer from difficulties in mass production, issues with storage stability, and high manufacturing costs.

[0012] Existing technical documents

[0013] Patent documents

[0014] Patent Document 1: International Publication No. 2007 / 004654. Summary of the Invention

[0015] The problem that the invention aims to solve

[0016] The purpose of this invention is to provide a nucleic acid amplification sensitizer with superior mass production and storage stability compared to protein-based additives.

[0017] Methods for solving problems

[0018] Through repeated and in-depth research, the inventors discovered that polymers containing structural units derived from monomers represented by the following formula (1) are suitable for use as sensitizers for nucleic acid amplification:

[0019] [Chemical Formula 1]

[0020]

[0021] Based on this insight, the present invention is as follows.

[0022] [1] A sensitizer for nucleic acid amplification is a polymer comprising structural units derived from monomers represented by the following formula (1):

[0023] [Chemical Formula 2]

[0024]

[0025] (in the formula,

[0026] X 1 It represents (meth)acryloyloxy or (meth)acryloylamino.

[0027] L 1 This refers to an alkylene group having 2 to 4 carbon atoms or an alkyleneoxyalkylene group having 2 to 4 carbon atoms, and...

[0028] R 1 ~R 3 Each can be independently represented as an alkyl group having 1 to 3 carbon atoms.

[0029] [2] The nucleic acid amplification sensitizer described in [1] above is a homopolymer composed of a structural unit derived from a monomer represented by the above formula (1).

[0030] [3] The sensitizer for nucleic acid amplification according to [1] above is a copolymer further comprising structural units derived from monomers represented by the following formula (2):

[0031] [Chemical Formula 3]

[0032]

[0033] (in the formula,

[0034] R 4 Represents a hydrogen atom or a methyl group, and

[0035] R 5 (representing an alkyl group having 1 to 20 hydrogen atoms or carbon atoms).

[0036] [4] According to the nucleic acid amplification sensitizer described in [3] above, wherein R 5 It is an alkyl group with 12 to 18 carbon atoms.

[0037] [5] The nucleic acid amplification sensitizer according to [1], [3] or [4] above is a copolymer further comprising structural units derived from monomers represented by the following formula (3):

[0038] [Chemical Formula 4]

[0039]

[0040] (in the formula,

[0041] R 6 Represents a hydrogen atom or a methyl group, and

[0042] R 7 (Refers to alkyl groups having 3 to 6 carbon atoms and having 2 or more hydroxyl groups).

[0043] [6] A composition for nucleic acid amplification, comprising any one of the above [1] to [5] sensitizers for nucleic acid amplification.

[0044] [7] The nucleic acid amplification composition according to [6] above is used in reverse transcription polymerase chain reaction.

[0045] [8] The nucleic acid amplification composition according to [6] or [7] above is used in quantitative polymerase chain reaction.

[0046] [9] The nucleic acid amplification composition according to any one of [6] to [8] above further comprises primers.

[0047]

[10] The nucleic acid amplification composition according to [9] above, wherein the primers are oligonucleotides with a length of 10 to 40 bases.

[0048]

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

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

[0049]

[12] Test kit, which contains the nucleic acid amplification composition described in any one of [6] to

[11] above.

[0050]

[13] The test kit described in

[12] above is used for clinical examination.

[0051]

[14] The test kit described in

[12] or

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

[0052]

[15] Nucleic acid amplification method, which includes using the polymer described in any one of [1] to [5] above as a sensitizer.

[0053]

[16] Nucleic acid amplification method, which includes mixing the nucleic acid amplification composition described in any one of [6] to

[11] above with a sample containing the amplified nucleic acid to prepare a nucleic acid amplification reaction solution.

[0054]

[17] The nucleic acid amplification method described in

[15] or

[16] above is the reverse transcription polymerase chain reaction method.

[0055]

[18] The nucleic acid amplification method according to any one of

[15] to

[17] above is a quantitative polymerase chain reaction method.

[0056] Invention Effects

[0057] The nucleic acid amplification sensitizer of the present invention can improve the detection sensitivity of nucleic acids in nucleic acid amplification methods. Furthermore, since the nucleic acid amplification sensitizer of the present invention is a synthetic polymer, it exhibits superior mass production and storage stability compared to protein-based additives. Detailed Implementation

[0058] The present invention will now be described in detail.

[0059] In this specification, "(meth)acryloyloxy" essentially means "acryloyloxy or methacryloyloxy". Where multiple (meth)acryloyloxy groups may be present, "(meth)acryloyloxy" means "acryloyloxy and / or methacryloyloxy". Other terms similar to "(meth)acryloyloxy" have the same meaning.

[0060] Furthermore, in this specification, when a range of numerical values ​​is described in stages, the lower limit and upper limit of each range can be combined. For example, when “preferred 10 to 100, more preferred 20 to 90” is described, “preferred lower limit: 10” and “more preferred upper limit: 90” can be combined (that is, the numerical range of “10 to 90” is also within the scope of this specification).

[0061] [Sensitizer for nucleic acid amplification]

[0062] The nucleic acid amplification sensitizer of the present invention (hereinafter sometimes referred to as "the sensitizer of the present invention") is a polymer (hereinafter sometimes referred to as "the polymer of the present invention") comprising structural units derived from a monomer (hereinafter sometimes simply referred to as "monomer (1)") represented by the following formula (1):

[0063] [Chemical Formula 5]

[0064]

[0065] Here, sensitizers for nucleic acid amplification refer to additives used to improve the detection sensitivity of nucleic acids in nucleic acid amplification methods.

[0066] 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 sequence-based amplification (NASBA). PCR is the preferred nucleic acid amplification method. Specifically, the sensitizer of this invention is preferably used in the polymerase chain reaction method.

[0067] As a 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.

[0068] As a PCR method, the quantitative polymerase chain reaction (PCR) method described above is preferred. That is, the nuclear sensitizer of the present invention is preferably used in the quantitative polymerase chain reaction (PCR) method.

[0069] The sensitizer of the present invention may be used in combination with only one type or in combination with two or more types. In addition, the sensitizer of the present invention may be used in combination with other additives.

[0070] The structural unit derived from monomer (1) (hereinafter sometimes simply referred to as "structural unit (1)") refers to a structural unit that has a structure formed by the reaction of the carbon-carbon double bond of the (meth)acryloyl group contained in monomer (1). Structural units derived from other monomers have the same meaning as structural units derived from monomer (1).

[0071] Monomer (1) may be used in combination with only one type or in combination with two or more types. That is, the sensitizer of the present invention may be a homopolymer composed of one type of structural unit (1) or a copolymer containing two or more types of structural units (1). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions. In the case where the polymer of the present invention does not contain structural units other than structural unit (1), the sensitizer of the present invention is preferably a homopolymer composed of one type of structural unit (1), more preferably a homopolymer composed of structural units derived from 2-(methacryloyloxyethylphosphorylcholine), and even more preferably a homopolymer composed of structural units derived from 2-methacryloyloxyethylphosphorylcholine.

[0072] The groups in formula (1) will be explained in turn below. X in formula (1) 1This indicates (meth)acryloyloxy (i.e., CH2=CR-CO-O-, R: hydrogen atom or methyl) or (meth)acryloylamino (i.e., CH2=CR-CO-NH-, R: hydrogen atom or methyl). From the point of view of raw material availability, X 1 Preferably, it is (meth)acryloyloxy, more preferably methacryloyloxy.

[0073] L in equation (1) 1 This refers to an alkylene group having 2 to 4 carbon atoms or an alkylene oxide having 2 to 4 carbon atoms, which may have one hydroxyl group. The aforementioned alkylene group can be linear or branched. Examples of alkylene groups having 2 to 4 carbon atoms and having one hydroxyl group include -C2H4-. Examples of alkylene oxides having 2 to 4 carbon atoms include -C2H4-O-C2H4-. From the viewpoint of raw material availability, L 1 Preferably -C2H4- or -C2H4-O-C2H4-, more preferably -C2H4- (i.e., ethylene).

[0074] R in equation (1) 1 ~R 3 Each alkyl group independently represents an alkyl group having 1 to 3 carbon atoms. These alkyl groups can be straight-chain or branched. Examples of alkyl groups having 1 to 3 carbon atoms include methyl, ethyl, and propyl. From the viewpoint of raw material availability, R 1 ~R 3 Preferably, all are methyl groups.

[0075] The preferred monomer (1) is X 1 (Meth)acryloyloxy, L 1 It is -C2H4- or -C2H4-O-C2H4- and R 1 ~R 3 The monomer is methyl. A more preferred monomer (1) is X. 1 (Meth)acryloyloxy, L 1 It is ethylene and R 1 ~R 3 The monomer is methyl (i.e., 2-(meth)acryloyloxyethyl phosphorylcholine). A further preferred monomer is 2-methacryloyloxyethyl phosphorylcholine. Monomer (1) can be a commercially available product.

[0076] In addition to structural unit (1), the polymer of the present invention may further include structural units (hereinafter referred to as "structural unit (2)") derived from monomers represented by formula (2) (hereinafter sometimes simply referred to as "monomer (2)"):

[0077] [Chemical Formula 6]

[0078]

[0079] Monomer (2) may be used in combination with one type or in combination with two or more types. That is, the polymer of the present invention may be a copolymer comprising one or more structural units (1) and one or more structural units (2). The copolymer may be a random copolymer, a block copolymer, or a copolymer comprising both random and block portions.

[0080] The groups in formula (2) will be explained in turn below. R in formula (2) 4 Represents a hydrogen atom or a methyl group. From the perspective of polymer storage stability, R 4 Methyl is preferred.

[0081] R in equation (2) 5 This refers to an alkyl group having 1 to 20 hydrogen atoms or carbon atoms. The alkyl group can be straight-chain 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, heptadecanyl, octadecyl, nonadecanyl, and eicosyl.

[0082] From the perspective of sensitization effect, R 5 Preferably, it is an alkyl group having 1 to 20 carbon atoms, more preferably an alkyl group having 2 to 20 carbon atoms, further preferably an alkyl group having 12 to 18 carbon atoms, and particularly preferably a straight-chain alkyl group having 12 to 18 carbon atoms. Furthermore, when the polymer of the present invention further comprises structural units derived from monomers represented by the following formula (3), R 5 Preferably, it is an alkyl group having 3 to 6 carbon atoms.

[0083] Specific examples of monomer (2) include (meth)acrylic acid, (meth)acrylate, (meth)ethyl acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, stearate, etc. Monomer (2) may be commercially available.

[0084] In the above specific examples of monomer (2), (meth)acrylic acid, (meth)acrylate ...

[0085] In the case where the polymer of the present invention comprises structural unit (1) and structural unit (2), from the viewpoint of sensitization effect, 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 for polymerization), the amount of structural unit (1) (i.e., monomer (1) used for polymerization) is preferably 30 to 99 moles, more preferably 30 to 90 moles, further preferably 50 to 90 moles, particularly preferably 75 to 90 moles, and the amount of structural unit (2) (i.e., monomer (2) used for polymerization) is preferably 1 to 70 moles, more preferably 10 to 70 moles, further preferably 10 to 50 moles, particularly preferably 10 to 25 moles.

[0086] In addition to structural unit (1), the polymer of the present invention may further include structural units (hereinafter referred to as "structural units (3)") derived from monomers represented by formula (3) (hereinafter sometimes simply referred to as "monomers (3)"):

[0087] [Chemical Formula 7]

[0088]

[0089] Monomer (3) may be used in combination with one or more types. That is, the polymer of the present invention may be a copolymer containing one or more structural units (1) and one or more structural units (3), or a copolymer containing one or more structural units (1), one or more structural units (2), and one or more structural units (3). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both random and block portions.

[0090] The groups in formula (3) will be explained in turn below. R 6 Represents a hydrogen atom or a methyl group. From the perspective of polymer storage stability, R 6 Methyl is preferred.

[0091] R in equation (3) 7 R indicates an alkyl group having 3 to 6 carbon atoms and having 2 or more hydroxyl groups. 7 The number of hydroxyl groups is preferably 2 to 5. The alkyl group can be straight-chain or branched. Examples of alkyl groups with 3 to 6 carbon atoms include propyl, butyl, pentyl, and hexyl.

[0092] Specific examples of monomer (3) include glyceryl mono(meth)acrylate, threitol mono(meth)acrylate, erythritol mono(meth)acrylate, xylitol mono(meth)acrylate, arabinol mono(meth)acrylate, mannitol mono(meth)acrylate, galactitol mono(meth)acrylate, sorbitol mono(meth)acrylate, etc. Among these, glyceryl mono(meth)acrylate and xylitol mono(meth)acrylate are preferred, glyceryl mono(meth)acrylate is more preferred, and glyceryl mono(meth)acrylate is even more preferred.

[0093] Monomer (3) can be commercially available or manufactured by known methods. For example, monomer (3) can be manufactured by esterification of (meth)acrylic acid or its derivatives (e.g., acyl chlorides) with a polyol having three or more hydroxyl groups. Esterification is well known, and those skilled in the art can appropriately set its conditions.

[0094] In the case where the polymer of the present invention comprises structural unit (1) and structural unit (3), from the viewpoint of sensitization effect, relative to a total of 100 moles of structural unit (1) and structural unit (3) (i.e., a total of 100 moles of monomer (1) and monomer (3) used for polymerization), the amount of structural unit (1) (i.e., monomer (1) used for 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 structural unit (3) (i.e., monomer (3) used for polymerization) is preferably 20 to 70 moles, more preferably 30 to 70 moles, and even more preferably 40 to 70 moles.

[0095] In the case where the polymer of the present invention comprises structural unit (1), structural unit (2) and structural unit (3), from the viewpoint of sensitization effect, relative to a total of 100 moles of structural unit (1), structural unit (2) and structural unit (3) (i.e., a total of 100 moles of monomer (1), monomer (2) and monomer (3) used for polymerization), the amount of structural unit (1) (i.e., monomer (1) used for polymerization) is preferably 30 to 80 moles, more preferably 30 to 70 moles, and even more preferably 30 to 60 moles; the amount of structural unit (2) (i.e., monomer (2) used for 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 structural unit (3) (i.e., monomer (3) used for polymerization) is preferably 10 to 60 moles, more preferably 10 to 50 moles, and even more preferably 10 to 40 moles.

[0096] The polymer of the present invention may contain other structural units derived from monomers other than those described above (1) to (3), without impairing the effects of the present invention. Only one other monomer may be used, or two or more may be used in combination. There are no particular limitations on the other monomers, but examples include benzyl (meth)acrylate and isobornyl (meth)acrylate. The amount of other structural units in the polymer of the present invention is preferably 20 mol% or less relative to all structural units. More preferably, the polymer of the present invention does not contain other structural units.

[0097] The polymer of the present invention is preferably selected from at least one of the following: a homopolymer composed of a single structural unit (1), a copolymer composed of structural unit (1) and structural unit (2), and a copolymer composed of structural unit (1), structural unit (2) and structural unit (3); more preferably, it is a homopolymer composed of a single structural unit (1), a copolymer composed of structural unit (1) and structural unit (2), or a copolymer composed of structural unit (1), structural unit (2) and structural unit (3). It should be noted that in this specification, "homopolymer composed of one structural unit (1)" means a homopolymer in which all its structural units (repeating units) are composed of one structural unit (1), "copolymer composed of structural unit (1) and structural unit (2)" means a copolymer in which all its structural units (repeating units) are composed of structural unit (1) and structural unit (2), and "copolymer composed of structural unit (1), structural unit (2) and structural unit (3)" means a copolymer in which all its structural units (repeating units) are composed of structural unit (1), structural unit (2) and structural unit (3). Other similar expressions have the same meaning.

[0098] 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. It should be noted that this weight-average molecular weight can be determined, for example, by conversion of polyethylene glycol using gel filtration chromatography with an EcoSEC system (manufactured by Tosoh Corporation).

[0099] The polymers of the present invention can be manufactured by known methods (e.g., the method described in International Publication No. 2018 / 216628).

[0100] The amount of sensitizer (i.e., the polymer of the present invention) used is determined by its concentration in the nucleic acid amplification composition described below. From the viewpoint of sensitization effect and suppression of viscosity increase in the above composition, the concentration of the sensitizer of the present invention in the above 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%. It should be noted that when using two or more sensitizers, the above concentration refers to the total concentration of the two or more sensitizers. For the concentrations of other components described below, when using two or more of that component, the above concentration also refers to the total concentration of the two or more of that component.

[0101] [Preferred Examples of the Sensitizer (Polymer of the Invention)]

[0102] As preferred examples of the sensitizers (polymers of the present invention), the following are examples of the sensitizers (I) (polymers (I) of the present invention) to (IV) (polymers (IV) of the present invention).

[0103] <The sensitizer (I) of the present invention (the polymer (I) of the present invention)>

[0104] The sensitizer (I) of the present invention (polymer (I) of the present invention) is at least one selected from the following homopolymers (I-1), copolymers (I-2) and copolymers (I-3), preferably the following homopolymers (I-1), copolymers (I-2) or copolymers (I-3):

[0105] The homopolymer (I-1) is composed of structural units (1) derived from 2-((meth)acryloyloxyethylphosphorylcholine);

[0106] copolymer (I-2), which is composed of

[0107] Derived from the structural unit (1) of 2-((meth)acryloyloxyethylphosphorylcholine), and

[0108] Structural units derived from (meth)acrylic acid, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, or (meth)acrylate, stearyl ester (2)

[0109] The composition, and relative to the total of 100 moles of structural units (1) and (2), is such that the amount of structural unit (1) is 30 to 99 moles and the amount of structural unit (2) is 1 to 70 moles; and

[0110] copolymer (I-3), which is composed of

[0111] Derived from the structural unit (1) of 2-((meth)acryloyloxyethylphosphocholine),

[0112] Structural units (2) derived from (meth)acrylic acid, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, (meth)acrylate, or (meth)acrylate, stearyl ester, and

[0113] Structural units derived from glyceryl mono(meth)acrylate or xylitol mono(meth)acrylate (3)

[0114] The structure consists of 100 moles of structural units (1), (2), and (3), with 30 to 80 moles of structural unit (1), 10 to 60 moles of structural unit (2), and 10 to 60 moles of structural unit (3).

[0115] In the sensitizer (I) of the present invention (polymer (I) of the present invention), it is preferred that the amount of structural unit (1) in copolymer (I-2) is 30 to 90 moles and the amount of structural unit (2) is 10 to 70 moles, and the amount of structural unit (1) in copolymer (I-3) is 30 to 70 moles, the amount of structural unit (2) is 20 to 60 moles and the amount of structural unit (3) is 10 to 50 moles. It should be noted that the basis for the amount of the above-mentioned structural units in copolymer (I-2) is a total of 100 moles of structural units (1) and (2), and the basis for the amount of the above-mentioned structural units in copolymer (I-3) is a total of 100 moles of structural units (1), (2) and (3).

[0116] In the sensitizer (I) of the present invention (polymer (I) of the present invention), it is more preferable that the amount of structural unit (1) in copolymer (I-2) is 50 to 90 moles and the amount of structural unit (2) is 10 to 50 moles, and the amount of structural unit (1) in copolymer (I-3) is 30 to 60 moles, the amount of structural unit (2) is 30 to 60 moles and the amount of structural unit (3) is 10 to 40 moles. It should be noted that the basis for the amount of the above-mentioned structural units in copolymer (I-2) is a total of 100 moles of structural units (1) and (2), and the basis for the amount of the above-mentioned structural units in copolymer (I-3) is a total of 100 moles of structural units (1), (2) and (3).

[0117] In the sensitizer (I) of the present invention (polymer (I) of the present invention), it is further preferred that the amount of structural unit (1) in copolymer (I-2) is 75 to 90 moles and the amount of structural unit (2) is 10 to 25 moles, and the amount of structural unit (1) in copolymer (I-3) is 30 to 60 moles, the amount of structural unit (2) is 30 to 60 moles and the amount of structural unit (3) is 10 to 40 moles. It should be noted that the basis for the amount of the above-mentioned structural units in copolymer (I-2) is a total of 100 moles of structural units (1) and (2), and the basis for the amount of the above-mentioned structural units in copolymer (I-3) is a total of 100 moles of structural units (1), (2) and (3).

[0118] <Sensitizer (II) of the present invention (polymer (II) of the present invention)>

[0119] The sensitizer (II) of the present invention (polymer (II) of the present invention) is at least one selected from the following homopolymers (II-1), copolymers (II-2) and copolymers (II-3), preferably the following homopolymers (II-1), copolymers (II-2) or copolymers (II-3):

[0120] The homopolymer (II-1) is composed of structural units (1) derived from 2-methacryloyloxyethylphosphorylcholine;

[0121] copolymer (II-2), which is composed of

[0122] Structural units derived from 2-methacryloyloxyethylphosphorylcholine (1), and

[0123] Structural units derived from butyl methacrylate, dodecyl methacrylate, tridecyl methacrylate, tetradecyl methacrylate, pentadecyl methacrylate, cetyl methacrylate, heptadecanyl methacrylate, or stearyl methacrylate (2)

[0124] The composition comprises, and relative to the total of 100 moles of structural unit (1) and structural unit (2), the amount of structural unit (1) is 30 to 90 moles and the amount of structural unit (2) is 10 to 70 moles; and

[0125] copolymer (II-3), which is composed of

[0126] The structural unit derived from 2-methacryloyloxyethylphosphorylcholine (1),

[0127] Structural units (2) derived from butyl methacrylate, dodecyl methacrylate, tridecyl methacrylate, tetradecyl methacrylate, pentadecyl methacrylate, cetyl methacrylate, heptadecanyl methacrylate, or stearyl methacrylate, and

[0128] Structural units derived from mono(meth)acrylate (3)

[0129] The composition consists of 100 moles of structural units (1), (2) and (3), with 30 to 70 moles of structural unit (1), 20 to 60 moles of structural unit (2) and 10 to 50 moles of structural unit (3).

[0130] In the sensitizer (II) of the present invention (polymer (II) of the present invention), it is preferred that the amount of structural unit (1) in copolymer (II-2) is 50 to 90 moles and the amount of structural unit (2) is 10 to 50 moles, and the amount of structural unit (1) in copolymer (II-3) is 30 to 60 moles, the amount of structural unit (2) is 30 to 60 moles and the amount of structural unit (3) is 10 to 40 moles. It should be noted that the basis for the amount of the above-mentioned structural units in copolymer (II-2) is a total of 100 moles of structural units (1) and (2), and the basis for the amount of the above-mentioned structural units in copolymer (II-3) is a total of 100 moles of structural units (1), (2) and (3).

[0131] In the sensitizer (II) of the present invention (polymer (II) of the present invention), it is more preferable that the amount of structural unit (1) in copolymer (II-2) is 75 to 90 moles and the amount of structural unit (2) is 10 to 25 moles, and the amount of structural unit (1) in copolymer (II-3) is 30 to 60 moles, the amount of structural unit (2) is 30 to 60 moles and the amount of structural unit (3) is 10 to 40 moles. It should be noted that the basis for the amount of the above-mentioned structural units in copolymer (II-2) is a total of 100 moles of structural units (1) and (2), and the basis for the amount of the above-mentioned structural units in copolymer (II-3) is a total of 100 moles of structural units (1), (2) and (3).

[0132] <Sensitizer (III) of the present invention (polymer (III) of the present invention)>

[0133] The sensitizer (III) of the present invention (polymer (III) of the present invention) is at least one selected from the following homopolymers (III-1), copolymers (III-2) and copolymers (III-3), preferably the following homopolymers (III-1), copolymers (III-2) or copolymers (III-3):

[0134] The homopolymer (III-1) is composed of structural units (1) derived from 2-methacryloyloxyethylphosphorylcholine;

[0135] copolymer (III-2), which is composed of

[0136] Structural units derived from 2-methacryloyloxyethylphosphorylcholine (1), and

[0137] Structural units derived from butyl methacrylate or stearyl methacrylate (2)

[0138] The composition comprises, and relative to the total of 100 moles of structural unit (1) and structural unit (2), the amount of structural unit (1) is 50 to 90 moles and the amount of structural unit (2) is 10 to 50 moles; and

[0139] copolymer (III-3), which is composed of

[0140] The structural unit derived from 2-methacryloyloxyethylphosphorylcholine (1),

[0141] Structural units (2) derived from butyl methacrylate or stearyl methacrylate, and

[0142] Structural units derived from glyceryl monomethacrylate (3)

[0143] The composition is such that, relative to the total of 100 moles of structural units (1), (2) and (3), the amount of structural unit (1) is 30 to 60 moles, the amount of structural unit (2) is 30 to 60 moles and the amount of structural unit (3) is 10 to 40 moles.

[0144] In the sensitizer (III) of the present invention (polymer (III) of the present invention), it is preferred that the amount of structural unit (1) in the copolymer (III-2) is 75 to 90 moles and the amount of structural unit (2) is 10 to 25 moles. It should be noted that the basis for the amount of the above-mentioned structural units in the copolymer (III-2) is a total of 100 moles of structural unit (1) and structural unit (2).

[0145] <The Sensitizer (IV) of the Invention (The Polymer (IV) of the Invention)>

[0146] The sensitizer (IV) of the present invention (the polymer (IV) of the present invention) is at least one selected from the following homopolymers (IV-1), copolymers (IV-2) and copolymers (IV-3), preferably the following homopolymers (IV-1), copolymers (IV-2) or copolymers (IV-3):

[0147] The homopolymer (IV-1) is composed of structural units (1) derived from 2-methacryloyloxyethylphosphorylcholine;

[0148] copolymer (IV-2), which is composed of

[0149] Structural units derived from 2-methacryloyloxyethylphosphorylcholine (1), and

[0150] Structural units derived from butyl methacrylate or stearyl methacrylate (2)

[0151] The composition, and relative to the total of 100 moles of structural unit (1) and structural unit (2), the amount of structural unit (1) is 75-90 moles and the amount of structural unit (2) is 10-25 moles; and

[0152] copolymer (IV-3), which is composed of

[0153] The structural unit derived from 2-methacryloyloxyethylphosphorylcholine (1),

[0154] Structural units derived from butyl methacrylate or stearyl methacrylate (2), and

[0155] Structural units derived from glyceryl monomethacrylate (3)

[0156] The composition is such that, relative to the total of 100 moles of structural units (1), (2) and (3), the amount of structural unit (1) is 30 to 60 moles, the amount of structural unit (2) is 30 to 60 moles and the amount of structural unit (3) is 10 to 40 moles.

[0157] Composition for nucleic acid amplification

[0158] This invention also provides nucleic acid amplification compositions comprising the sensitizers of this invention (hereinafter sometimes referred to as "compositions of this invention"). In the compositions of this invention, only one sensitizer may be used, or two or more may be used in combination. Here, "compositions for nucleic acid amplification" means compositions used in nucleic acid amplification methods. The nucleic acid amplification method is described above. The nucleic acid amplification method is preferably PCR. That is, the compositions of this invention are preferably used in polymerase chain reaction (PCR).

[0159] As a PCR method, the reverse transcription polymerase chain reaction method described above is preferred. That is, the composition of the present invention is preferably used for reverse transcription polymerase chain reaction.

[0160] As a PCR method, the quantitative polymerase chain reaction (PCR) method described above is preferred. That is, the composition of the present invention is preferably used for quantitative polymerase chain reaction.

[0161] The compositions of the present invention can be prepared by dissolving the sensitizer of the present invention, along with other components for nucleic acid amplification as needed, in a solvent such as water. That is, the compositions of the present invention are preferably compositions containing the sensitizer of the present invention and water (and other components as needed). The concentration of the sensitizer of the present invention in the compositions of the present invention is explained as described above.

[0162] Other components used for nucleic acid amplification may include those known to be used in known nucleic acid amplification methods, such as PCR. Examples of such components include, for instance, buffers, substrates, primers, DNA polymerase, fluorescent DNA staining reagents, fluorescent probes, passive references, and nucleic acids. Only one of these components may be used, or two or more may be used in combination.

[0163] The buffer solution is not particularly limited, but examples include buffer solutions that are prepared by mixing bases such as tris(hydroxymethyl)aminomethane, tricine, and bicine with acids such as sulfuric acid, hydrochloric acid, acetic acid, and phosphoric acid to adjust the pH to around 6-9, more preferably 7-8. It is also desirable for the buffer solution to contain magnesium and / or manganese salts. Furthermore, the buffer solution may further contain salts such as potassium chloride and ammonium sulfate. Additionally, the buffer solution may further contain water-soluble organic solvents such as dimethyl sulfoxide, dimethylformamide, formamide, and glycerol. Furthermore, the buffer solution may further contain surfactants such as polyoxyethylene sorbitan fatty acid esters and polyoxyethylene alkylphenyl ethers. Furthermore, the buffer solution may further contain proteins such as bovine serum albumin.

[0164] The substrate is not particularly limited, but examples include mixtures of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dCTP) (dNTPs). Alternatively, some or all of the dTTP may be replaced with deoxyuridine triphosphate (dUTP). Furthermore, in sequencing PCR and the like, it is preferable to add appropriate amounts of mixtures of dideoxyadenosine triphosphate (ddATP), dideoxythymidine triphosphate (ddTTP), dideoxyguanosine triphosphate (ddGTP), and dideoxythymidine triphosphate (ddCTP), or their fluorescently labeled forms.

[0165] 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 modulated using known methods. The oligonucleotide may have a fluorescent group formed from fluorescein or the like.

[0166] A single primer may be used, 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 0.1–25 μM, more preferably 0.1–15 μM, and even more preferably 0.5–10 μM.

[0167] Known DNA polymerases can be used. From the viewpoint of thermostability, enzymes derived from thermophilic bacteria, thermophilic archaea, hyperthermophilic bacteria, hyperthermophilic archaea, and their mutant forms are preferred. The DNA polymerase is appropriately selected from DNA-dependent DNA polymerases, RNA-dependent DNA polymerases, or enzymes possessing both functions, depending on the purpose of nucleic acid amplification. Furthermore, it is appropriate to choose whether to use a DNA polymerase with nuclease activity or a DNA polymerase without nuclease activity.

[0168] As a fluorescent DNA staining reagent, there are no particular limitations, but examples include SYBR. TM Green I et al.

[0169] As a fluorescent probe, there are no particular limitations, but for example, TaqMan can be cited. TM Probe.

[0170] An inert reference can be appropriately selected based on the purpose of nucleic acid amplification. Examples of inert references include ROX. TM Dyes, etc.

[0171] In addition to the primers and fluorescent probes mentioned above, any DNA and / or RNA can be used as an exogenous control gene for nucleic acids. Here, the nucleic acid can be synthesized in vitro or modulated from cells, microorganisms, viruses, etc., using known methods. These cells, microorganisms, viruses, etc., can be collected from humans or animals and plants in nature or the environment, and can also be isolated / cultured.

[0172] In the composition of the present invention, mineral oil or other oils, glass beads, magnetic beads or other solid carriers may be further added.

[0173] In addition, kit-type products that combine multiple of the above-mentioned components, and master mix-type products (sometimes also called primer mix, premix, etc.) that further premix these components can be used.

[0174] [Test Kit]

[0175] The compositions of the present invention can be combined with necessary constituent components to form a test kit. The present invention provides a test kit comprising the compositions of the present invention. There are no particular limitations on the aforementioned constituent components, but examples include sample collection equipment, sample collection containers, sample pretreatment reagents, calibration standards, testing equipment, consumables, test kits, and instruction manuals. The aforementioned constituent components are appropriately selected according to the testing protocol. The aforementioned constituent components can be packaged together with the test kit. Furthermore, commercially available products can be used as the aforementioned constituent components. Additionally, specified standard components can be used as the aforementioned constituent components.

[0176] The test kit of the present invention can be used for, for example, gene testing, microbial testing, and virus testing, and is preferably used for virus testing. That is, the test target of the test kit of the present invention is preferably a virus.

[0177] Examples of tests using the test kit of the present invention include those performed in the fields of medicine, veterinary medicine, forensic medicine, pharmaceutical analysis, food analysis, and environmental investigation. Among these, tests performed in the fields of medicine and veterinary medicine are preferred, and tests performed in the medical field are more preferred. The test kit of the present invention is preferably used for clinical examinations, and more preferably for in vitro diagnostics.

[0178] [Nucleic Acid Amplification Method]

[0179] 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 mixing the composition of the present invention with a sample containing the amplified nucleic acid to prepare a reaction solution for nucleic acid amplification. 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. Furthermore, unless otherwise specified, the description of the nucleic acid amplification method is also as described above.

[0180] The nucleic acid amplification method of the present invention uses a sample containing the amplified nucleic acid. The sample is preferably a sample solution containing water and the amplified nucleic acid. 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 can be appropriately determined according to the purpose of nucleic acid amplification, but where the concentration can be adjusted, it is preferably 1 to 10. 20 Copy / μL, more preferably 1 to 10 10 copies / μL, more preferably 1 to 10 5 The concentration is 1 to 500 copies / μL, particularly preferably 1 to 100 copies / μL.

[0181] In the computational amplification method of the present invention, the amount of sample used is preferably trace to 1 μL, more preferably 0.01 to 1 μL, and even more preferably 0.2 to 0.4 μL, relative to the amount of the composition of the present invention (1 μL).

[0182] The nucleic acid amplification method of the present invention is preferably the reverse transcription polymerase chain reaction (RT-PCR) method. Alternatively, the nucleic acid amplification method of the present invention is preferably the quantitative polymerase chain reaction (QPCR) method.

[0183] Example

[0184] The present invention will be specifically described below through examples, but the present invention is not limited to these examples.

[0185] Synthesis of polymers (sensitizers for nucleic acid amplification)

[0186] [Synthesis example 1]

[0187] 40.0 g of 2-methacryloyloxyethyl phosphorylcholine (hereinafter sometimes referred to as "MPC") as monomer (1) was weighed into a glass flask for polymerization. 60.0 g of purified water was added to dissolve monomer (1), and 0.31 g of azobisisobutyronitrile (hereinafter referred to as "AIBN") as polymerization initiator was added to the resulting solution. After the reaction vessel was fully purged with nitrogen, the mixture was heated at 70 °C for 6 hours with stirring to carry out polymerization. The resulting reaction solution was cooled with ice and added dropwise to diethyl ether to precipitate the polymer. The precipitate was filtered out, washed with diethyl ether, and dried under vacuum to obtain a white powdery homopolymer (hereinafter referred to as "polymer 1"). The weight-average molecular weight of polymer 1 was determined by gel filtration chromatography (hereinafter sometimes referred to as "GPC") under the following conditions and converted to polyethylene glycol as 1,030,000.

[0188] [Synthesis example 2]

[0189] 6.0 g of MPC as monomer (1) and 4.0 g of methacrylic acid (hereinafter sometimes simply referred to as "MA") as monomer (2) (monomer (1) / monomer (2) = 30 / 70 (molar ratio)) were weighed into a glass flask for polymerization. 90.0 g of purified water was added to dissolve monomer (1) and monomer (2), and 0.78 g of AIBN was added to the resulting solution. The process was then repeated as in Synthesis Example 1 to obtain a random copolymer (hereinafter referred to as "polymer 2"). The weight-average molecular weight of polymer 2 was determined by GPC under the following conditions and converted to polyethylene glycol as 680,000.

[0190] [Synthesis example 3]

[0191] 19.4 g of MPC as monomer (1) and 2.2 g of butyl methacrylate (hereinafter sometimes referred to as "BMA") as monomer (2) (monomer (1) / monomer (2) = 80 / 20 (molar ratio)) were weighed into a glass flask for polymerization. 39.3 g of purified water and 39.3 g of ethanol were added to dissolve monomer (1) and monomer (2). 0.02 g of AIBN was added to the resulting solution. After the flask was completely purged with nitrogen, the mixture was heated at 60 °C for 5 hours with stirring to carry out polymerization. The same procedure as in Synthesis Example 1 was then followed to obtain a random copolymer (hereinafter referred to as "polymer 3"). The weight-average molecular weight of polymer 3 was determined by GPC under the following conditions and converted to polyethylene glycol as 600,000.

[0192] [Synthesis Example 4]

[0193] 11.7 g of MPC as monomer (1) and 3.3 g of stearyl methacrylate (hereinafter sometimes referred to as "SMA") as monomer (2) (monomer (1) / monomer (2) = 80 / 20 (molar ratio)) were weighed into a glass flask for polymerization. 85 g of ethanol was added to dissolve monomer (1) and monomer (2), and 0.06 g of AIBN was added to the resulting solution. After the flask was completely purged with nitrogen, the mixture was heated at 60 °C for 6 hours with stirring to carry out polymerization. The same procedure as in Synthesis Example 1 was then followed to obtain a random copolymer (hereinafter referred to as "polymer 4"). The weight-average molecular weight of polymer 4 was determined by GPC under the following conditions and converted to polyethylene glycol as 43,000.

[0194] [Synthesis example 5]

[0195] 8.4 g of MPC as monomer (1), 2.1 g of BMA as monomer (2), and 4.5 g of glyceryl monomethacrylate (hereinafter sometimes referred to as "GLM") as monomer (3) (monomer (1) / monomer (2) / monomer (3) = 40 / 40 / 20 (molar ratio)) were weighed into a glass flask for polymerization. 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. The process was then repeated as in Synthesis Example 3 to obtain a random copolymer (hereinafter referred to as "polymer 5"). The weight-average molecular weight of polymer 5 was determined by GPC under the following conditions and converted to polyethylene glycol as 22,000.

[0196] [GPC Measurement]

[0197] The GPC determination of polymers 1 to 5 obtained in Synthetic Examples 1 to 5 was carried out under the following conditions.

[0198] GPC System: EcoSEC System (manufactured by Tosoh Corporation);

[0199] Column: Connect Shodex OHpak SB-802.5HQ (manufactured by Showa Denko Co., Ltd.) and SB-806HQ (manufactured by Showa Denko Co., Ltd.) in series;

[0200] Development solvent: 20 mM sodium phosphate buffer (pH 7.4);

[0201] Detector: Differential refractive index detector;

[0202] Molecular weight standard: EasiVial PEG / PEO (manufactured by Agilent Technologies);

[0203] Flow rate: 0.5 mL / min;

[0204] Column temperature: 40℃;

[0205] Sample: The obtained polymer was diluted with an expanding solvent to a final concentration of 0.1% by weight;

[0206] Injection volume: 100μL.

[0207] The monomers used in Synthetic Examples 1 to 5, their molar ratios, and the weight-average molecular weights of the resulting polymers are summarized in Table 1 below.

[0208] [Table 1]

[0209] Polymer 1 MPC - - 100 / 0 / 0 1,030,000 Polymer 2 MPC MA - 30 / 70 / 0 680,000 Polymer 3 MPC BMA - 80 / 20 / 0 600,000 Polymer 4 MPC SMA - 80 / 20 / 0 43,000 Polymer 5 MPC BNM GLM 40 / 40 / 20 22,000

[0210] The molar ratio of monomers = monomer(1) / monomer(2) / monomer(3);

[0211] MPC: 2-Methacryloxyethylphosphocholine;

[0212] MA: Methacrylic acid;

[0213] BMA: Butyl methacrylate;

[0214] SMA: Stearyl methacrylate;

[0215] GLM: Glyceryl monomethacrylate.

[0216] [Examples 1-5 and Comparative Example 1]

[0217] [Sample Solution]

[0218] The positive control RNA, N set No. 2 (N2), provided with the SARS-CoV-2 RT-qPCR detection kit (manufactured by Fujifilm and Koko 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 kit's instructions. PW (nuclease-free) was used instead of the above sample solution as a negative control.

[0219] [Reaction Solution]

[0220] Using the components of the above-described kit and a 50×ROX inert reference (manufactured by Nippon Gene Co., Ltd.), a solution with the general composition shown in Table 2 was prepared. A specified amount of the polymer obtained in Synthetic Examples 1-5 was added, along with a specified amount of PW (without nuclease), to prepare the nucleic acid amplification composition. 15 μL of the obtained nucleic acid amplification composition was dispensed into a PCR plate (a barcoded MicroAmp). TM A rapid optical 96-well reaction plate (0.1 mL, manufactured by Applied Bioscience) was used, and 5 μL of the above sample solution was added to prepare a total of 20 μL of reaction solution. The composition of the resulting reaction solution is described in Table 2 below.

[0221] It should be noted that in the above operation, 2 μL of a polymer aqueous solution with a concentration 10 times that of the final polymer concentration 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 common components and PW (without nuclease) were mixed to prepare 15 μL of nucleic acid amplification composition, and 5 μL of sample solution was added to prepare 20 μL of reaction solution.

[0222] [Table 2]

[0223]

[0224] *Components of the SARS-CoV-2 RT-qPCR detection kit (manufactured by Fujifilm and Koichi Pure Chemical Industries, Ltd.);

[0225] Manufactured by Nippon Gene Co., Ltd.

[0226] [reaction]

[0227] Set the PCR plate containing the above reaction solution onto the StepOnePlus plate. TM The real-time PCR system (manufactured by Applied Bioscience) was used for RT-qPCR according to the temperature program shown in Table 3. It should be noted that fluorescence intensity readings were performed according to step #5 in Table 3.

[0228] [Table 3]

[0229]

[0230] [result]

[0231] Under the conditions shown in Table 4-1, RT-qPCR was performed according to the above steps, and the endpoint enhancement rate was calculated from the endpoint fluorescence intensity and the following formula:

[0232] Endpoint enhancement rate (%) = 100 × (F - F0) / F0

[0233] (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).

[0234] Here, endpoint fluorescence intensity refers to the fluorescence intensity in the final cycle (cycle 60) of the PCR method, and fluorescence intensity is defined as the ΔRn value. The results are shown in Table 4-1.

[0235] [Table 4-1]

[0236]

[0237] Nucleic acid concentration in sample solution: 10 copies / μL

[0238] [Comparative Examples 2-5]

[0239] In Comparative Examples 2-5, the sensitizers of the present invention (i.e., polymers 1-5) used in Examples 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, under the conditions shown in Table 4-2, RT-qPCR was performed according to the steps described above [Examples 1-5 and Comparative Example 1], and the endpoint enhancement rate was calculated from the endpoint fluorescence intensity and the following formula:

[0240] Endpoint enhancement rate (%) = 100 × (F - F0) / F0

[0241] (In the formula, F represents the endpoint fluorescence intensity with the additive added, and F0 represents the endpoint fluorescence intensity without the additive added (Comparative Example 1).

[0242] The results are shown in Table 4-2.

[0243] [Table 4-2]

[0244]

[0245] As shown in Table 4-1, the endpoint enhancement rates of Examples 1-5, which used the sensitizers of the present invention (i.e., polymers 1-5), were 372-583%. On the other hand, as shown in Table 4-2, the endpoint enhancement rates of Comparative Examples 2-5, which used additives other than the sensitizers of the present invention (i.e., BSA, DMSO, PEG 6000, or T4 gene 32 protein), were 76-157%. These results indicate that the detection sensitivity of nucleic acids as the analyte can be improved by using the sensitizers of the present invention.

[0246] [Examples 6-10 and Comparative Example 6]

[0247] [sample]

[0248] The positive control RNA, N set No. 2 (N2), provided with the SARS-CoV-2 RT-qPCR detection kit (manufactured by Fujifilm and Koko 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. Additionally, PW (nuclease-free) was used as a negative control instead of the above sample solutions.

[0249] [Reaction Solution]

[0250] Using the components of the above-described kit and a 50×ROX inert 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 above-described sample solution was incorporated into this nucleic acid amplification composition to prepare a total of 20 μL of reaction solution. It should be noted that the final concentration of the polymer in the reaction solution is shown in Table 5 below.

[0251] [reaction]

[0252] The same procedure as in Examples 1-5 and Comparative Example 1 was followed for RT-qPCR.

[0253] [result]

[0254] RT-qPCR was performed under the conditions shown in Table 5, following the steps described above. Reactions were performed with N=2 under each condition. Amplification was detected in both reactions (denoted as "PP" in Table 5), amplification in one reaction (denoted as "P" in Table 5), and no amplification in either reaction (denoted as "N" in Table 5). Furthermore, the lowest nucleic acid concentration in the sample solution classified as "strong detection" and "weak detection" was determined as their respective limits of detection (LODs). It should be noted that in cases where a "weak detection" criterion did not exist, the LOD of "weak detection" was equal to the LOD of "strong detection." The results are shown in Table 5.

[0255]

[0256] As shown in Table 5, in Examples 6-10, which used the sensitizers of the present invention (i.e., polymers 1-5), 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 as the analyte can be improved by using the sensitizers of the present invention.

[0257] Industry availability

[0258] Using the sensitizer of the present invention can improve the detection sensitivity of nucleic acids as the analyte in nucleic acid amplification methods (especially PCR methods). The sensitizer of the present invention is applicable to nucleic acid amplification methods used for gene testing, microbial testing, virus testing, etc.

[0259] This application is based on Japanese Patent Application No. 2021-12400, the entire contents of which are contained in this application specification.

Claims

1. A sensitizer for nucleic acid amplification, which is a polymer comprising structural units derived from monomers represented by the following formula (1): [Chemical Formula 1] In the formula, X 1 It represents (meth)acryloyloxy or (meth)acryloylamino. L 1 This refers to an alkylene group having one hydroxyl group or without a hydroxyl group, having 2 to 4 carbon atoms, or an alkylene oxide having 2 to 4 carbon atoms, and... R 1 ~R 3 Each can be used independently to represent an alkyl group having 1 to 3 carbon atoms.

2. The sensitizer for nucleic acid amplification according to claim 1 is a homopolymer composed of a structural unit derived from a monomer represented by the above formula (1).

3. The sensitizer for nucleic acid amplification according to claim 1, further comprising a copolymer of structural units derived from a monomer represented by the following formula (2): [Chemical Formula 2] In the formula, R 4 Represents a hydrogen atom or a methyl group, and R 5 It refers to an alkyl group having 1 to 20 hydrogen or carbon atoms.

4. The sensitizer for nucleic acid amplification according to claim 3, wherein R 5 It is an alkyl group with 12 to 18 carbon atoms.

5. The sensitizer for nucleic acid amplification according to claim 1, 3 or 4, further comprising a copolymer of structural units derived from a monomer represented by the following formula (3): [Chemical Formula 3] In the formula, R 6 Represents a hydrogen atom or a methyl group, and R 7 It refers to an alkyl group having 3 to 6 carbon atoms and having 2 or more hydroxyl groups.

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

7. The composition for nucleic acid amplification according to claim 6, used in reverse transcription polymerase chain reaction.

8. The nucleic acid amplification composition according to claim 6 or 7, used in quantitative polymerase chain reaction (PCR).

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

10. The test kit according to claim 9, used for clinical examination.

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

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