Nonspecific amplification inhibitor for use in isothermal nucleic acid amplification, isothermal nucleic acid amplification composition containing same, and isothermal nucleic acid amplification method

Abstract

The present invention is a nonspecific amplification inhibitor for use in isothermal nucleic acid amplification, the nonspecific amplification inhibitor comprising a polymer that contains constituent units derived from a monomer represented by formula (1).　According to the present invention, it is possible to provide a nonspecific amplification inhibitor for isothermal nucleic acid amplification, the nonspecific amplification inhibitor being capable of inhibiting nonspecific amplification and improving detection sensitivity merely by being added to an isothermal nucleic acid amplification composition. (In the formula, X1 represents a (meth)acryloyloxy group or a (meth)acryloyl amide group; L1 represents a C2-4 alkylene group optionally having one hydroxy group, or a C2-4 alkylene oxyalkylene group; and each of R1 to R3 independently represents a C1-3 alkyl group.)

Description

Non-specific amplification inhibitor for use in isothermal nucleic acid amplification, composition for isothermal nucleic acid amplification containing the same, and isothermal nucleic acid amplification method

[0001] The present invention relates to a non-specific amplification inhibitor used in an isothermal nucleic acid amplification method, a composition for isothermal nucleic acid amplification containing the same, and an isothermal nucleic acid amplification method.

[0002] Isothermal nucleic acid amplification is a method of amplifying target nucleic acids at a constant temperature. While PCR (Polymerase Chain Reaction) requires heating and cooling to proceed, isothermal nucleic acid amplification proceeds at a constant temperature, enabling much faster testing compared to PCR. LAMP, one of the isothermal nucleic acid amplification methods, is a technique for performing an amplification reaction using four types of primers containing six regions. When the four types of primers, a strand-displacing DNA polymerase, and template DNA are mixed and reacted isothermally, several reaction steps are performed, and an amplified DNA product with a structure in which complementary sequences are alternately repeated accumulates (see, for example, Patent Document 1). LAMP is known as a highly sensitive measurement method because it can detect even a few copies of nucleic acid.

[0003] However, the use of multiple primers often leads to problems such as the formation of primer dimers and false positives due to nonspecific amplification. Methods for suppressing nonspecific amplification include changing the primer design (see, for example, Patent Document 2) and adding additives (betaine, DMSO, urea, etc.).

[0004] WO00 / 28082 Publication No. JP 2023-113880 A

[0005] However, although the design change of the primer described in Patent Document 2 suppresses nonspecific amplification, it takes time and effort to examine the conditions, and even if a method of adding an additive is used, the addition of betaine, DMSO, etc. is not sufficiently effective. The present invention has been made in consideration of the above problems, and aims to provide a nonspecific amplification inhibitor for isothermal nucleic acid amplification that can suppress nonspecific amplification and improve detection sensitivity simply by adding it to a composition for isothermal nucleic acid amplification in an isothermal nucleic acid amplification method such as the LAMP method, as well as an isothermal nucleic acid amplification composition and an isothermal nucleic acid amplification method containing the same.

[0006] As a result of intensive research, the present inventors have found that a polymer containing a structural unit derived from a monomer represented by the following formula (1) can be used as a non-specific amplification inhibitor for isothermal nucleic acid amplification, which can achieve the above-mentioned object. The present invention is as follows [1] to [6].

[0007] [1] A non-specific amplification inhibitor for use in an isothermal nucleic acid amplification method, comprising a polymer containing a structural unit derived from a monomer represented by formula (1): (In the formula, X １ represents a (meth)acryloyloxy group or a (meth)acryloylamide group, L １ represents an alkylene group having 2 to 4 carbon atoms, which may have one hydroxy group, or an alkyleneoxyalkylene group having 2 to 4 carbon atoms; R １ ~R ３ each independently represents an alkyl group having 1 to 3 carbon atoms. [2] The nonspecific amplification inhibitor according to [1] above, wherein the polymer is a copolymer further comprising a structural unit derived from a monomer represented by formula (2): (In the formula, R ４ represents a hydrogen atom or a methyl group, R ５ represents a group having a phosphate group or an alkyl group having 1 to 20 carbon atoms.) [3] The nonspecific amplification inhibitor according to [1] or [2] above, wherein the polymer is a copolymer further comprising a structural unit derived from a monomer represented by formula (3): (In the formula, R ６ represents a hydrogen atom or a methyl group, and R ７ represents an alkyl group having 3 to 6 carbon atoms and having two or more hydroxy groups.) [4] A composition for isothermal nucleic acid amplification comprising a nonspecific amplification inhibitor used in the isothermal nucleic acid amplification method according to any one of [1] to [3] above, and a polymerase. [5] The composition for isothermal nucleic acid amplification according to [4] above, further comprising a primer. [6] An isothermal nucleic acid amplification method comprising mixing the composition for isothermal nucleic acid amplification according to [4] or [5] above, with a sample containing nucleic acid to be amplified, to prepare a reaction solution for nucleic acid amplification.

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

[0009] 1 shows the amplification curve results when a positive control was added in Examples 1-1, 2, and 3 and Comparative Example 1-1. FIG. 2 shows the amplification curve results when dd Water was added in Examples 1-1, 2, and 3 and Comparative Example 1-1.

[0010] The present invention will be described in detail below.

[0011] [Nonspecific Amplification Inhibitor] The present invention relates to a nonspecific amplification inhibitor that is a polymer containing a structural unit derived from a monomer represented by formula (1) (hereinafter, sometimes referred to as the "polymer of the present invention"). Furthermore, the polymer of the present invention may be a copolymer containing, in addition to the structural unit derived from the monomer represented by formula (1), a structural unit derived from a monomer represented by formula (2) and / or a structural unit derived from a monomer represented by formula (3), as described below. The nonspecific amplification inhibitor can be used as a nonspecific amplification inhibitor in an isothermal nucleic acid amplification composition. In this specification, "isothermal nucleic acid amplification composition" refers to a composition used in an isothermal nucleic acid amplification method. In this specification, "nonspecific amplification inhibitor in an isothermal nucleic acid amplification composition" refers to an additive that suppresses the amplification of nucleic acids other than those of interest in an isothermal nucleic acid amplification method.

[0012] The polymer of the present invention is a polymer containing a structural unit derived from a monomer represented by the following formula (1). (In the formula, X １ represents a (meth)acryloyloxy group or a (meth)acryloylamide group, L １ represents an alkylene group having 2 to 4 carbon atoms, which may have one hydroxy group, or an alkyleneoxyalkylene group having 2 to 4 carbon atoms; R １ ~R ３ each independently represents an alkyl group having 1 to 3 carbon atoms.

[0013] Hereinafter, the "monomer represented by formula (1)" may be abbreviated as "monomer (1)." Furthermore, the "structural unit derived from monomer (1)" may be abbreviated as "structural unit (1)."

[0014] The groups in formula (1) will be explained below in order. X in formula (1) １ represents a (meth)acryloyloxy group (i.e., CH ２ =CR-CO-O-, where R is a hydrogen atom or a methyl group) or a (meth)acryloylamide group (i.e., CH ２ =CR-CO-NH-, R: hydrogen atom or methyl group). １ is preferably a (meth)acryloyloxy group, more preferably a methacryloyloxy group. １ represents an alkylene group having 2 to 4 carbon atoms which may have one hydroxy group, or an alkyleneoxyalkylene group having 2 to 4 carbon atoms. The alkylene group may be linear or branched. Examples of the alkylene group having 2 to 4 carbon atoms which may have one hydroxy group include, for example, -C ２ H ４ Examples of the alkyleneoxyalkylene group having 2 to 4 carbon atoms include -C ２ H ４ -O-C ２ H ４ From the viewpoint of raw material availability, L １ is preferably -C ２ H ４ -or-C ２ H ４ -O-C ２ H ４ -, more preferably -C ２ H ４ - (i.e., an ethylene group).

[0015] R in formula (1) １ ~R ３ each independently represents an alkyl group having 1 to 3 carbon atoms. The alkyl group may be linear or branched. Examples of the alkyl group having 1 to 3 carbon atoms include a methyl group, an ethyl group, and a propyl group. From the viewpoint of raw material availability, R １ ~R ３ Preferably, both of X are methyl groups. １ is a (meth)acryloyloxy group, and L１ But, -C ２ H ４ -or-C ２ H ４ -O-C ２ H ４ - and R １ ~R ３ is a methyl group. １ is a (meth)acryloyloxy group, and L １ is an ethylene group, and R １ ~R ３ is a methyl group (i.e., 2-(meth)acryloyloxyethyl phosphorylcholine). A more preferred monomer (1) is 2-methacryloyloxyethyl phosphorylcholine. Commercially available products can be used as the monomer (1).

[0016] In this specification, "(meth)acryloyl" basically means "acryloyl or methacryloyl." For example, in a case where a plurality of the above-mentioned 2-(meth)acryloyloxyethyl phosphorylcholines may be present, "2-(meth)acryloyloxyethyl phosphorylcholine" means "2-acryloyloxyethyl phosphorylcholine and / or 2-methacryloyloxyethyl phosphorylcholine." Other terms similar to "(meth)acryloyl" (for example, (meth)acrylic, (meth)acrylate, etc.) are also defined in accordance with the above explanation of "(meth)acryloyl."

[0017] The weight-average molecular weight of the polymer of the present invention is not particularly limited, but is preferably 10,000 to 500,000, more preferably 10,000 to 250,000, and even more preferably 10,000 to 200,000. The weight-average molecular weight can be determined in terms of polyethylene glycol by gel filtration chromatography using, for example, an EcoSEC system (manufactured by Tosoh Corporation).

[0018] As described below, the polymer of the present invention may be a copolymer having the structural unit (1) and a structural unit other than the structural unit (1). In this case, the amount of the structural unit (1) (i.e., the amount of the monomer (1) used in the polymerization) is preferably 5 to 95 moles, more preferably 10 to 90 moles, and even more preferably 30 to 85 moles, relative to a total of 100 moles of all structural units in the polymer (i.e., a total of 100 moles of the monomers used in the polymerization). The copolymer may be a random copolymer, a block copolymer, or a copolymer containing both a random portion and a block portion.

[0019] The polymer of the present invention is preferably a copolymer that further contains, in addition to the structural unit (1), a structural unit derived from a monomer represented by the following formula (2): (In the formula, R ４ represents a hydrogen atom or a methyl group, R ５ represents a group having a phosphate group or an alkyl group having 1 to 20 carbon atoms.

[0020] Hereinafter, the "monomer represented by formula (2)" may be abbreviated as "monomer (2)." Furthermore, the "structural unit derived from monomer (2)" may be abbreviated as "structural unit (2)."

[0021] The groups in formula (2) will be explained in order below. ４ represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of the polymer, R ４ is preferably a methyl group. ５ represents a group having a phosphate group or an alkyl group having 1 to 20 carbon atoms. The alkyl group may be linear or branched. ５ is preferably a group having a phosphate group or an alkyl group having 2 to 20 carbon atoms, more preferably a group having a phosphate group or an alkyl group having 3 to 19 carbon atoms, and even more preferably a group having a phosphate group or a linear alkyl group having 4 to 18 carbon atoms.

[0022] Specific examples of the monomer (2) include 2-methacryloyloxyethyl acid phosphate, 10-methacryloyloxydecyl dihydrogen phosphate, methacryloyloxypolypropylene glycol phosphate, 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, and stearyl (meth)acrylate. The monomer (2) may be used alone or in combination of two or more. Commercially available products can be used as the monomer (2).

[0023] Among the specific examples of the monomer (2), (i) 2-methacryloyloxyethyl acid phosphate, methacryloyloxypolypropylene glycol phosphate, 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, and stearyl (meth)acrylate are preferred; (ii) 2-methacryloyloxyethyl acid phosphate, methacryloyloxypolypropylene glycol phosphate, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, 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; (iii) methacryloyloxypolypropylene glycol phosphate, butyl (meth)acrylate, and stearyl (meth)acrylate are even more preferred; and (iv) methacryloyloxypolypropylene glycol phosphate, butyl methacrylate, and stearyl methacrylate are particularly preferred.

[0024] One embodiment of the polymer of the present invention is a copolymer composed of structural unit (1) and structural unit (2) (hereinafter sometimes abbreviated as "copolymer (1-2)"). In copolymer (1-2), relative to a total of 100 moles of structural unit (1) and structural unit (2) (i.e., a total of 100 moles of monomer (1) and monomer (2) used in the polymerization), the amount of structural unit (1) (i.e., monomer (1) used in the polymerization) is preferably 5 to 95 moles, more preferably 10 to 95 moles, even more preferably 15 to 95 moles, and particularly preferably 20 to 90 moles, and the amount of structural unit (2) (i.e., monomer (2) used in the polymerization) is preferably 5 to 95 moles, more preferably 5 to 90 moles, even more preferably 5 to 85 moles, and particularly preferably 10 to 80 moles. The copolymer may be a random copolymer or a block copolymer, or may be a copolymer containing both random and block portions.

[0025] The weight-average molecular weight of the copolymer (1-2) is not particularly limited, but is preferably 10,000 to 2,000,000, more preferably 20,000 to 1,500,000, and even more preferably 30,000 to 1,000,000. The weight-average molecular weight can be determined in terms of polyethylene glycol by gel filtration chromatography using, for example, an EcoSEC system (manufactured by Tosoh Corporation).

[0026] The copolymer of the present invention preferably further contains, in addition to the structural unit (1), a structural unit derived from a monomer represented by the following formula (3). (In the formula, R ６ represents a hydrogen atom or a methyl group, R ７ represents an alkyl group having 3 to 6 carbon atoms and having two or more hydroxy groups.

[0027] Hereinafter, the "monomer represented by formula (3)" may be abbreviated as "monomer (3)." Furthermore, the "structural unit derived from monomer (3)" may be abbreviated as "structural unit (3)."

[0028] The groups in formula (3) will be explained below in order. ６ represents a hydrogen atom or a methyl group. From the viewpoint of storage stability of the polymer, R６ is preferably a methyl group. ７ represents an alkyl group having 3 to 6 carbon atoms and having two or more hydroxy groups. ７ The number of hydroxy 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.

[0029] Specific examples of monomer (3) include glycerin mono(meth)acrylate, threitol mono(meth)acrylate, erythritol mono(meth)acrylate, xylitol mono(meth)acrylate, arabitol mono(meth)acrylate, mannitol mono(meth)acrylate, galactitol mono(meth)acrylate, and sorbitol mono(meth)acrylate. 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. Monomer (3) may be a commercially available product or may be produced by a known method. For example, monomer (3) can be produced by an esterification reaction between (meth)acrylic acid or a derivative thereof (e.g., an acid chloride) and a polyhydric alcohol having three or more hydroxy groups. Esterification reactions are well known, and those skilled in the art can perform the reaction by appropriately setting the conditions.

[0030] One embodiment of the polymer of the present invention is a copolymer composed of structural unit (1) and structural unit (3) (hereinafter sometimes abbreviated as "copolymer (1-3)"). In copolymer (1-3), 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 in the polymerization), the amount of structural unit (1) (i.e., monomer (1) used in the polymerization) is preferably 25 to 95 moles, more preferably 30 to 90 moles, and even more preferably 35 to 85 moles, and the amount of structural unit (3) (i.e., monomer (3) used in the polymerization) is preferably 5 to 75 moles, more preferably 10 to 70 moles, and even more preferably 15 to 65 moles. The copolymer may be a random copolymer or a block copolymer, or may be a copolymer containing both random and block portions.

[0031] The weight average molecular weight of the copolymer (1-3) is not particularly limited, but is preferably 100,000 to 1,000,000, more preferably 100,000 to 800,000, and even more preferably 100,000 to 500,000.

[0032] One embodiment of the polymer of the present invention is a copolymer composed of structural units (1), (2), and (3) (hereinafter sometimes abbreviated as "copolymer (1-2-3)"). In copolymer (1-2-3), 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 in the polymerization), the amount of structural unit (1) (i.e., monomer (1) used in the 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 in the 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 in the polymerization) is preferably 10 to 60 moles, more preferably 10 to 50 moles, and even more preferably 10 to 40 moles.

[0033] The weight average molecular weight of the copolymer (1-2-3) is not particularly limited, but is preferably 10,000 to 500,000, more preferably 10,000 to 100,000, and even more preferably 10,000 to 50,000.

[0034] The copolymer of the present invention may contain other structural units derived from other monomers different from the monomers (1) to (3), as long as the effects of the present invention are not impaired. The other monomers may be used alone or in combination of two or more. The other monomers are not particularly limited, but examples thereof include potassium 3-sulfopropyl methacrylate.

[0035] In one embodiment of the present invention, the polymer of the present invention is preferably at least one selected from the group consisting of homopolymer (1), which is a homopolymer of monomer (1), copolymer (1-2), copolymer (1-3), and copolymer (1-2-3), more preferably at least one selected from the group consisting of homopolymer (1), copolymer (1-2), and copolymer (1-2-3), and even more preferably at least one selected from the group consisting of copolymer (1-2) and copolymer (1-2-3). In this embodiment, examples of monomers (1) to (3) for forming the structural units (1) to (3) include those described above.

[0036] Each monomer used in preparing the polymer of the present invention may be a commercially available product or may be produced by a known method. The polymer of the present invention can be produced by a known method (for example, the method described in International Publication No. 2018 / 216628). The nonspecific amplification inhibitor of the present invention (i.e., the polymer of the present invention) can be easily used as a nonspecific amplification inhibitor by being contained in a reaction composition of a desired isothermal nucleic acid amplification method (i.e., a composition for isothermal nucleic acid amplification described below) before the start of the reaction.

[0037] [Isothermal nucleic acid amplification composition] The present invention provides an isothermal nucleic acid amplification composition (hereinafter sometimes referred to as "the composition of the present invention") containing the non-specific amplification inhibitor of the present invention. The non-specific amplification inhibitor of the present invention has been described above.

[0038] Examples of isothermal nucleic acid amplification methods in which the compositions of the present invention can be used include loop-mediated isothermal amplification (LAMP), transcription-mediated amplification (TMA), isothermal and chimeric primer-initiated amplification of nucleicacids (ICAN), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). The isothermal nucleic acid amplification method is preferably loop-mediated isothermal amplification (LAMP). That is, the compositions of the present invention are more preferably used in LAMP. In this specification, "loop-mediated isothermal amplification (LAMP)" includes not only typical LAMP methods using DNA as the target nucleic acid, but also reverse transcription-loop-mediated isothermal amplification (RT-LAMP) methods using RNA as the target nucleic acid. Furthermore, in this specification, the "LAMP method" includes not only typical reactions in which DNA is the target nucleic acid, but also reactions in which RNA is the target nucleic acid. The amount of the nonspecific amplification inhibitor in the composition of the present invention is preferably 0.001 to 5 w / v %, more preferably 0.01 to 1 w / v %, and even more preferably 0.01 to 0.1 w / v %.

[0039] [Other Components] The composition of the present invention may contain components other than the polymer of the present invention (hereinafter, these may be referred to as "other components"). As the other components, known components used in isothermal nucleic acid amplification methods can be used. Examples of the other components include reaction reagents such as polymerase, primer, buffer, substrate, fluorescent dye, salt, surfactant, protein, and nucleic acid. Each of the other components may be used alone or in combination of two or more.

[0040] The buffer is not particularly limited, but examples include a buffer prepared by mixing a base such as tris(hydroxymethyl)aminomethane, tricine, or bicine with an acid such as sulfuric acid, hydrochloric acid, acetic acid, or phosphoric acid to adjust the pH to about 6 to 9, and more preferably about 7 to 8. The buffer desirably contains a magnesium salt and / or a manganese salt as appropriate. The buffer may further contain a water-soluble organic solvent such as dimethyl sulfoxide, dimethylformamide, formamide, or glycerin. The buffer may further contain a surfactant such as polyoxysorbitan fatty acid ester or polyoxyethylene alkylphenyl ether. The buffer may further contain a protein such as bovine serum albumin. The buffer may further contain a water-soluble polymer such as polyethylene glycol.

[0041] The substrate is not particularly limited, but examples thereof include a mixture (dNTPs) of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and deoxycytidine triphosphate (dCTP). Here, it is also possible to replace part and / or all of the dTTP with deoxyuridine triphosphate (dUTP). The primer is not particularly limited, but examples thereof include oligonucleotides of approximately 15 to 30 bases designed and prepared by known methods. A four-type primer set may be used, or a six-type primer set including a loop primer may be used.

[0042] Examples of polymerases include DNA polymerases, and known DNA polymerases can be used. The DNA polymerase may be one or more selected from DNA-dependent DNA polymerases, RNA-dependent DNA polymerases (reverse transcriptases), or enzymes that combine both functions, depending on the purpose of nucleic acid amplification. In addition to the aforementioned primers, any DNA and / or RNA may be used as an exogenous control gene, for example. Here, the nucleic acid may be synthesized in vitro or prepared by known methods from cells, microorganisms, viruses, etc. Here, the cells, microorganisms, viruses, etc. may be collected from nature, the environment, humans, animals, plants, or may be isolated and cultured. The reaction reagent may further contain an oil such as mineral oil. The reaction reagent may also contain a solid phase carrier such as silica beads or magnetic beads.

[0043] The composition of the present invention can contain a sample to be tested. Examples of such samples include viruses, bacteria, cells, body fluids, tissues, etc. to be tested, suspensions thereof, nucleic acid extracts prepared therefrom, nucleic acid standard solutions, blank test samples, etc. The sample can be appropriately selected depending on the type of test. It is also preferred to premix a master mix (also called a primer mix, premix, etc.) by selecting multiple components. It is also preferred to combine each component of the isothermal nucleic acid amplification composition with components such as a reaction vessel and a sample collection tool to prepare a test kit. The test kit is preferably an in vitro diagnostic drug.

[0044] [Testing Method] The present invention further provides a testing method using the nonspecific amplification inhibitor of the present invention. Examples of the testing method include an isothermal nucleic acid amplification method comprising mixing the above-described isothermal nucleic acid amplification composition with a sample containing the nucleic acid to be amplified to prepare a reaction solution for isothermal nucleic acid amplification. A preferred form of the testing method includes a method in which a test target nucleic acid is amplified using a composition of the present invention containing the nonspecific amplification inhibitor of the present invention, and the presence or concentration of the test target nucleic acid is determined based on the amount of the amplification reaction product. Examples of such testing include medical testing, veterinary testing, forensic testing, pharmaceutical testing, food testing, environmental testing, and plant disease testing. The presence or concentration of the test target nucleic acid can be determined based on the amount of the amplification reaction product using a known nucleic acid detection method. For example, when using the LAMP method, a method can be used to determine the presence or concentration of the target nucleic acid by measuring the magnesium pyrrolate content of the amplification reaction product using a turbidimeter. Another method is to irradiate the amplification reaction product with black light and determine the presence or absence of fluorescence from the target nucleic acid. Yet another method involves adding an intercalating agent such as FL Green Intercalation Mix to a composition for isothermal nucleic acid amplification and monitoring the optical properties of the intercalating agent.

[0045] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these.

[0046] Synthesis Example 1 8.4 g of 2-methacryloyloxyethyl phosphorylcholine (hereinafter referred to as "MPC") as monomer (1), 2.1 g of butyl methacrylate (hereinafter referred to as "BMA") as monomer (2), and 4.5 g of glycerin monomethacrylate (hereinafter 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, and 42.5 g of purified water and 42.5 g of ethanol were added to dissolve the monomers (1) to (3). To the resulting solution, 0.15 g of Perloyl SA (manufactured by NOF Corporation, hereinafter referred to as "PRSA") was added. The atmosphere in the reaction vessel was thoroughly replaced with nitrogen, and the mixture was heated at 70°C for 6 hours with stirring to carry out polymerization. The resulting reaction solution was ice-cooled and added dropwise to acetone to precipitate a polymer. The precipitate was filtered off, washed with acetone, and then vacuum dried to obtain a white powdery copolymer (hereinafter referred to as "Polymer 1"). The weight-average molecular weight of Polymer 1 was 22,000 in terms of polyethylene glycol, as measured by gel permeation chromatography (hereinafter referred to as "GPC") under the conditions described below.

[0047] Synthesis Example 2 6.0 g of MPC as monomer (1) and 3.3 g of stearyl methacrylate (hereinafter referred to as "SMA") as monomer (2) (monomer (1) / monomer (2) = 80 / 20 (molar ratio)) were weighed into a glass flask for polymerization, 85.0 g of ethanol was added to dissolve the monomers (1) and (2), and 0.06 g of azobisisobutyronitrile was added as a polymerization initiator to the resulting solution. The subsequent procedures were the same as in Synthesis Example 1, and a copolymer (hereinafter referred to as "Polymer 2") was obtained. The weight-average molecular weight of Polymer 2 was 43,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0048] Synthesis Example 3 6.5 g of MPC as monomer (1) and 23.5 g of methacryloxypolypropyleneglycolphosphate (manufactured by Unichemical Corporation, hereinafter referred to as "Phosmer PP") as monomer (2) (monomer (1) / monomer (2)=30 / 70 (molar ratio)) were weighed into a glass flask for polymerization, and a mixed solvent of 135.0 g of purified water and 135.0 g of ethanol was added to dissolve the monomers (1) and (2), and 0.78 g of dimethyl 2,2'-azobis(2-methylpropionate) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "V-601") was added to the resulting solution. After thoroughly replacing the atmosphere in the reaction vessel with nitrogen, the mixture was heated at 70°C for 6 hours under stirring to carry out polymerization. The mixture was then separated and purified using a dialysis membrane to obtain a solution of a copolymer (referred to herein as "Polymer 3") with a solid content of approximately 1.9% by mass. The weight-average molecular weight of Polymer 3 was 80,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0049] <GPC Measurement> GPC measurement of polymers 1 to 3 obtained in Synthesis Examples 1 to 3 was carried out under the following conditions: GPC system: EcoSEC system (manufactured by Tosoh Corporation) Column: Shodex OHpak SB-802.5HQ (manufactured by Showa Denko K.K.) and SB-806HQ (manufactured by Showa Denko K.K.) connected in series Developing solvent: 20 mM sodium phosphate buffer (pH 7.4) Detector: differential refractive index detector Molecular weight standard: EasiVial PEG / PEO (manufactured by Agilent Technologies) Flow rate: 0.5 mL / min Column temperature: 40°C Sample: The obtained polymer was diluted with the developing solvent to a final concentration of 0.1 wt % Injection volume: 100 μL

[0050] The monomers used in Synthesis Examples 1 to 3, their molar ratios, and the weight average molecular weights of the resulting polymers are summarized in Table 1. MPC: 2-methacryloyloxyethyl phosphorylcholine BMA: butyl methacrylate GLM: glycerin monomethacrylate SMA: stearyl methacrylate

[0051] Examples 1-1, 1-2, and 1-3 are examples and comparative examples of the LAMP method in which Candidatus Phytoplasma japonicum (Ca. P. japonicum) is the detection target. Example 1-1 is an example in which polymer 1 was used, Example 1-2 is an example in which polymer 2 was used, and Example 1-3 is an example in which polymer 3 was used. In each of the examples and comparative examples, a composition containing a positive control (a composition containing a target nucleic acid) and a composition containing dd Water (a composition not containing a target nucleic acid) were prepared and evaluated, as described below.

[0052] [Preparation of Solution A] Solution A was prepared by mixing the components shown in Table 2. Solution A was prepared using Nippon Gene's DNA Amplification Reagent Set for LAMP Method - Animal Species and Plant Testing Exclusive B -, Fast Plasma Primer Set 1 - Ca. P. japonicum -, and FL Green Intercalation Mix. Two types of samples were prepared: Solution A-1 containing Fast Plasma Positive Control DNA 1 - Ca. P. japonicum -, and Solution A-2 containing dd Water.

[0053]

[0054] [Preparation of Composition for Nucleic Acid Amplification] The compositions for nucleic acid amplification used in Examples 1-1 to 1-3 were prepared by adding Polymers 1 to 3 obtained in Synthesis Examples 1 to 3, respectively, to the obtained Solution A. 2 μL of an aqueous solution of the polymer diluted to 0.5 w / v % was added to Solution A per well to prepare a composition for nucleic acid amplification (polymer concentration in each composition: 0.04 w / v %).

[0055] [Nucleic Acid Amplification Reaction] The above-mentioned composition for nucleic acid amplification was dispensed into a 96-well plate (manufactured by BIORAD), the plate was sealed, and then the 96-well plate was set in a CFX Opus 96 (manufactured by BIORAD), and nucleic acid amplification was performed by the LAMP method under the conditions shown in Table 3.

[0056]

[0057] Comparative Example 1-1 was carried out in the same manner as in Examples 1-1, 1-2, and 1-3, except that in the preparation of a composition for nucleic acid amplification, 2 μL of distilled water, nuclease-free (manufactured by Nippon Gene Co., Ltd.) was used instead of 2 μL of the aqueous polymer solution.

[0058] The amplification curve results obtained when the positive control was added in Examples 1-1, 1-2, and 1-3 and Comparative Example 1-1 are shown in Figure 1, and the amplification curve results obtained when dd Water was added are shown in Figure 2. As shown in Figure 1, when the positive control was added, no significant differences in the amplification curves or Ct values ​​were observed between Examples 1-1, 1-2, and 1-3 and the Comparative Example. Therefore, it was confirmed that the nonspecific amplification inhibitor of the present invention does not inhibit specific amplification. With the addition of dd Water, nucleic acid amplification did not proceed because no target nucleic acid was added, and no amplification curve was formed. As shown in Figure 2, in Examples 1-1, 1-2, and 1-3, no amplification curve was detected, or an amplification curve was detected after 45 cycles. On the other hand, in Comparative Example 1-1, an amplification curve was detected around 25 cycles. A low cycle number indicates a high level of nonspecific amplification by primers. Therefore, it was confirmed that the nonspecific amplification inhibitor of the present invention inhibits nonspecific amplification by primers. From the above results, it can be seen that the nonspecific amplification inhibitor of the present invention suppresses nonspecific amplification without inhibiting specific amplification.

Claims

1. A non-specific amplification inhibitor for use in an isothermal nucleic acid amplification method, which comprises a polymer containing a structural unit derived from a monomer represented by formula (1). (In the formula, X １ represents a (meth)acryloyloxy group or a (meth)acryloylamide group, L １ represents an alkylene group having 2 to 4 carbon atoms, which may have one hydroxy group, or an alkyleneoxyalkylene group having 2 to 4 carbon atoms; R １ ~R ３ each independently represents an alkyl group having 1 to 3 carbon atoms.

2. The nonspecific amplification inhibitor according to claim 1, wherein the polymer is a copolymer further comprising a structural unit derived from a monomer represented by formula (2). (In the formula, R ４ represents a hydrogen atom or a methyl group, R ５ represents a group having a phosphate group or an alkyl group having 1 to 20 carbon atoms.

3. The nonspecific amplification inhibitor according to claim 1 or 2, wherein the polymer is a copolymer further comprising a structural unit derived from a monomer represented by formula (3). (In the formula, R ６ represents a hydrogen atom or a methyl group, and R ７ represents an alkyl group having 3 to 6 carbon atoms and having two or more hydroxy groups.

4. A composition for isothermal nucleic acid amplification, comprising a nonspecific amplification inhibitor used in the isothermal nucleic acid amplification method according to claim 1 and a polymerase.

5. The composition for isothermal nucleic acid amplification according to claim 4, further comprising a primer.

6. An isothermal nucleic acid amplification method comprising mixing the composition for isothermal nucleic acid amplification according to claim 4 or 5 with a sample containing nucleic acid to be amplified to prepare a reaction solution for nucleic acid amplification.

Images