Nucleic acid amplification method

Abstract

The present invention is a nucleic acid amplification method comprising: an extraction step for extracting a nucleic acid from a biological sample; a step for preparing a nucleic acid mixture by mixing the extracted nucleic acid and a polymer containing a constituent unit derived from a monomer containing a phosphorylcholine group; and a step for reacting the nucleic acid mixture to amplify the nucleic acid.　According to the present invention, it is possible to provide a nucleic acid amplification method that suppresses nucleic acid amplification inhibition caused by contaminants, such as amplification inhibitors, included in a specimen or a simple extraction kit.

Description

Nucleic acid amplification method

[0001] The present invention relates to a method for amplifying nucleic acids.

[0002] Nucleic acid amplification is a method for amplifying target genes and is a technology used in molecular biology research, clinical diagnostics, scientific investigations, etc. Polymerase Chain Reaction (PCR) is a known nucleic acid amplification method for amplifying target genes. Because PCR can detect nucleic acids with high sensitivity, it is used for diagnosing infectious diseases, detecting viruses, etc.

[0003] In clinical diagnostic testing using PCR, there are two types of pre-testing processes: one is to use nucleic acids extracted and purified from biological samples such as saliva or nasopharyngeal swabs using a spin column or other method, and the other is to use nucleic acid extracts extracted simply from biological samples using surfactants or other methods. Of these, simple extraction methods that use nucleic acid extracts as samples offer the advantage of labor-saving and time-saving by eliminating the nucleic acid purification step. However, nucleic acid amplification inhibition caused by contaminants such as proteins contained in the sample and surfactants used in simple extraction is often a problem. Therefore, there is a need for a method to suppress nucleic acid amplification inhibition when simple extraction methods are used as sample preparation methods.

[0004] For example, Patent Document 1 describes that adding a specific polypeptide to an RT-PCR (Reverse Transcription PCR) solution can suppress a decrease in fluorescence intensity caused by insoluble substances contained in the reaction solution, thereby overcoming a decrease in detection sensitivity. Furthermore, for example, Patent Document 2 describes a nucleic acid extraction method that suppresses the inhibition of nucleic acid amplification caused by an ionic surfactant that occurs when nucleic acids are extracted from a specimen using an ionic surfactant. Specifically, the inhibition of nucleic acid amplification is suppressed by adding a nonionic surfactant during nucleic acid amplification.

[0005] JP 2021-019558 A JP 2009-136221 A

[0006] However, the method described in Patent Document 1 suppresses amplification inhibition in the presence of amplification inhibitors such as humic acid and tannic acid, but is ineffective in the presence of some PCR inhibitors such as bilirubin and SDS (sodium dodecyl sulfate), leaving room for improvement. Furthermore, the method described in Patent Document 2 adds a nonionic surfactant during nucleic acid amplification to increase the yield of nucleic acid, but this may also increase nonspecific amplification products, leaving room for improvement. In view of the above problems, the present invention aims to provide a nucleic acid amplification method that suppresses nucleic acid amplification inhibition caused by contaminants such as amplification inhibitors contained in samples and simple extraction kits.

[0007] The present inventors have found that adding a polymer containing a constitutional unit derived from a monomer containing a phosphorylcholine group to an extract obtained by extracting nucleic acids from a biological sample and then performing nucleic acid amplification can suppress amplification inhibition caused by contaminants such as amplification inhibitors contained in specimens or simple extraction kits. That is, the present invention provides the following [1] and [2].

[0008] [1] A nucleic acid amplification method comprising the steps of: extracting nucleic acid from a biological sample; mixing the extracted nucleic acid with a polymer containing a constitutional unit derived from a monomer containing a phosphorylcholine group to prepare a nucleic acid mixture; and reacting the nucleic acid mixture to amplify the nucleic acid. [2] The nucleic acid amplification method according to [1] above, wherein the polymer contains one or more polymers selected from the following Group a. [Group a] (a-1) A polymer containing only constitutional units derived from 2-(meth)acryloyloxyethyl phosphorylcholine; (a-2) A polymer containing only constitutional units derived from 2-(meth)acryloyloxyethyl phosphorylcholine and C ４ ~C ２０ (a-3) A copolymer containing a structural unit derived from alkyl (meth)acrylate, a structural unit derived from 2-(meth)acryloyloxyethyl phosphorylcholine, C ４ ~C ２０ C having a structural unit derived from alkyl (meth)acrylate and two or more hydroxy groups as substituents ２ ~C ８(a-4) A copolymer containing a structural unit derived from alkyl (meth)acrylate. (a-5) A copolymer containing a structural unit derived from 2-(meth)acryloyloxyethyl phosphorylcholine and a structural unit derived from polyethylene glycol (meth)acrylate.

[0009] The nucleic acid amplification method of the present invention can suppress inhibition of nucleic acid amplification caused by contaminants such as amplification inhibitors contained in samples or simple extraction kits.

[0010] The present invention will be described in detail below. In this specification, "(meth)acryloyloxy" means "acryloyloxy or methacryloyloxy", "(meth)acrylamide" means "acrylamide or methacrylamide", and "(meth)acrylate" means "acrylate or methacrylate". ａ ~C ｂ The alkyl(meth)acrylate means an alkyl(meth)acrylate having an alkyl group with a to b carbon atoms.

[0011] [Nucleic acid amplification method] The nucleic acid amplification method of the present invention comprises an extraction step of extracting nucleic acid from a biological sample, a step of mixing the extracted nucleic acid with a polymer containing a constituent unit derived from a monomer containing a phosphorylcholine group to prepare a nucleic acid mixture, and a step of reacting the nucleic acid mixture to amplify the nucleic acid.

[0012] <Biological Sample> A biological sample is a sample collected from a living organism, and examples thereof include blood, plasma, serum, cerebrospinal fluid, saliva, sputum, urine, feces, throat swabs, nasopharyngeal swabs, nasal swabs, nasal discharge, and tears. Nucleic acids extracted from biological samples may be any DNA and / or RNA. The nucleic acids 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, or plants, or may be isolated and cultured. The method for extracting nucleic acids from a biological sample is not particularly limited, and any nucleic acid extraction method commonly used in the relevant field may be used.

[0013] <Polymer> After the extraction step of extracting nucleic acids from a biological sample, the extracted nucleic acids are mixed with a polymer containing constitutional units derived from a monomer containing a phosphorylcholine group to prepare a nucleic acid mixture.

[0014] Examples of monomers containing a phosphorylcholine group include 2-(meth)acryloyloxyethyl phosphorylcholine, 2-(meth)acrylamidoethyl phosphorylcholine, alkyl phosphorylcholine allyl ether, alkyl phosphorylcholine vinyl ether, etc. From the viewpoints of polymer stability, availability of raw materials, and reactivity, 2-acrylamidoethyl phosphorylcholine and 2-methacryloyloxyethyl phosphorylcholine are preferred, and 2-methacryloyloxyethyl phosphorylcholine is more preferred.

[0015] Of all the constituent units of a polymer containing constituent units derived from a monomer containing a phosphorylcholine group, the proportion of constituent units derived from a monomer containing a phosphorylcholine group is preferably 20 to 100 mol %, more preferably 30 to 100 mol %. The weight-average molecular weight of a polymer containing constituent units derived from a monomer containing a phosphorylcholine group is not particularly limited, but is preferably 5,000 to 2,000,000, more preferably 10,000 to 1,500,000.

[0016] The polymer containing a structural unit derived from a monomer containing a phosphorylcholine group preferably contains one or more polymers selected from the following [Group a]: [Group a] (a-1) A polymer containing only structural units derived from 2-(meth)acryloyloxyethyl phosphorylcholine (hereinafter, sometimes abbreviated as "polymer (a-1)"); (a-2) A polymer containing structural units derived from 2-(meth)acryloyloxyethyl phosphorylcholine and C ４ ~C ２０ (a-3) A copolymer containing a structural unit derived from 2-(meth)acryloyloxyethylphosphorylcholine, C ４ ~C ２０ C having a structural unit derived from alkyl (meth)acrylate and two or more hydroxy groups as substituents ２ ~C ８ (a-4) A copolymer containing a structural unit derived from 2-(meth)acryloyloxyethyl phosphorylcholine and a structural unit derived from polyethylene glycol (meth)acrylate (hereinafter, sometimes abbreviated as "polymer (a-4)"). (a-5) A copolymer containing a structural unit derived from 2-(meth)acryloyloxyethyl phosphorylcholine and a structural unit derived from a monomer containing a carboxyl group (hereinafter, sometimes abbreviated as "polymer (a-5)").

[0017] That is, the polymer containing a structural unit derived from a monomer containing a phosphorylcholine group preferably contains one or more polymers selected from the group consisting of polymer (a-1), polymer (a-2), polymer (a-3), polymer (a-4), and polymer (a-5). Of these, the polymer containing a structural unit derived from a monomer containing a phosphorylcholine group more preferably contains one or more polymers selected from the group consisting of polymer (a-1), polymer (a-2), and polymer (a-3). When the polymer is a copolymer, the copolymer may be a random copolymer, an alternating copolymer, a block copolymer, a graft copolymer, or a copolymer having two or more of these structures, but a random copolymer is preferred from the viewpoint of polymer production.

[0018] <Polymer (a-1)> The polymer (a-1) may be a homopolymer of 2-(meth)acryloyloxyethyl phosphorylcholine, or a copolymer of 2-acryloyloxyethyl phosphorylcholine and 2-methacryloyloxyethyl phosphorylcholine. The polymer (a-1) is preferably a homopolymer of 2-(meth)acryloyloxyethyl phosphorylcholine, and more preferably a homopolymer of 2-methacryloyloxyethyl phosphorylcholine. The weight-average molecular weight of the polymer (a-1) is not particularly limited, but is preferably 20,000 to 2,000,000, more preferably 100,000 to 1,500,000, and even more preferably 500,000 to 1,500,000.

[0019] <Polymer (a-2)> The polymer (a-2) is a polymer obtained by polymerizing 2-(meth)acryloyloxyethyl phosphorylcholine and C ４ ~C ２０It is formed by polymerizing a monomer mixture containing alkyl (meth)acrylate. Only one type of 2-(meth)acryloyloxyethyl phosphorylcholine may be used for forming polymer (a-2), or two types may be used. From the viewpoints of storage stability of the copolymer and availability of raw materials, 2-methacryloyloxyethyl phosphorylcholine is preferred as the 2-(meth)acryloyloxyethyl phosphorylcholine for forming polymer (a-2). Of all the structural units of polymer (a-2), the proportion of structural units derived from 2-(meth)acryloyloxyethyl phosphorylcholine is preferably 10 to 95 mol%, more preferably 20 to 90 mol%, and even more preferably 25 to 85 mol%.

[0020] C for forming polymer (a-2) ４ ~C ２０ The alkyl (meth)acrylate may be used alone or in combination of two or more kinds. ４ ~C ２０ Examples of the alkyl (meth)acrylate include butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, heptyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, undecyl (meth)acrylate, lauryl (meth)acrylate, tridecyl (meth)acrylate, myristyl (meth)acrylate, pentadecyl (meth)acrylate, cetyl (meth)acrylate, margaryl (meth)acrylate, stearyl (meth)acrylate, nonadecyl (meth)acrylate, arachidyl (meth)acrylate, and monomers in which the alkyl group moiety of these is replaced with the corresponding structural isomer. Among these, butyl (meth)acrylate, hexyl (meth)acrylate, octyl (meth)acrylate, lauryl (meth)acrylate, myristyl (meth)acrylate, cetyl (meth)acrylate, and stearyl (meth)acrylate are preferred, and butyl (meth)acrylate and stearyl (meth)acrylate are more preferred. ４ ~C ２０The proportion of structural units derived from alkyl(meth)acrylate is preferably from 5 to 90 mol %, more preferably from 10 to 80 mol %, and even more preferably from 15 to 75 mol %.

[0021] The polymer (a-2) contains a structural unit derived from 2-(meth)acryloyloxyethyl phosphorylcholine and C ４ ~C ２０ The copolymer may contain other structural units than the structural units derived from alkyl (meth)acrylate. However, the other structural units are not limited to the C hydroxyl groups of the polymer (a-3) described below, which have two or more hydroxy groups as substituents. ２ ~C ８ The polymer (a-2) is a structural unit other than a structural unit derived from alkyl (meth)acrylate. ４ ~C ２０ It is preferable that the polymer is composed only of structural units derived from alkyl (meth)acrylate.

[0022] The weight average molecular weight of the polymer (a-2) is not particularly limited, but is preferably 5,000 to 2,000,000, more preferably 10,000 to 1,000,000, and even more preferably 20,000 to 1,000,000.

[0023] <Polymer (a-3)> The polymer (a-3) is a 2-(meth)acryloyloxyethyl phosphorylcholine, C ４ ~C ２０ Alkyl (meth)acrylate, C having two or more hydroxyl groups as substituents ２ ~C ８It is formed by polymerizing a monomer mixture containing alkyl (meth)acrylate. Only one type of 2-(meth)acryloyloxyethyl phosphorylcholine may be used for forming polymer (a-3), or two types may be used. From the viewpoints of storage stability of the copolymer and availability of raw materials, 2-methacryloyloxyethyl phosphorylcholine is preferred as the 2-(meth)acryloyloxyethyl phosphorylcholine for forming polymer (a-3). Of all the structural units of polymer (a-3), the proportion of structural units derived from 2-(meth)acryloyloxyethyl phosphorylcholine is preferably 20 to 60 mol%, more preferably 30 to 60 mol%, and even more preferably 30 to 50 mol%.

[0024] C for forming polymer (a-3) ４ ~C ２０ The alkyl (meth)acrylate may be used alone or in combination of two or more kinds. ４ ~C 20 Among alkyl (meth)acrylates, C ４ ~C ８ Alkyl (meth)acrylates are preferred. ４ ~C 20 Examples of alkyl (meth)acrylates include butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, heptyl (meth)acrylate, octyl (meth)acrylate, and monomers in which the alkyl group moiety of these is substituted with the corresponding structural isomer. Of these, butyl methacrylate is preferred. Of all the structural units of polymer (a-3), C ４ ~C 20 The proportion of structural units derived from alkyl(meth)acrylate is preferably from 10 to 60 mol %, more preferably from 20 to 60 mol %, and even more preferably from 30 to 60 mol %.

[0025] C having two or more hydroxyl groups as substituents for forming polymer (a-3) ２ ~C ８The alkyl (meth)acrylate may be used alone or in combination of two or more. ２ ~C ８ Examples of alkyl(meth)acrylates 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 is preferred. Among all the structural units of the polymer (a-3), C having two or more hydroxyl groups as substituents is preferred. ２ ~C ８ The proportion of structural units derived from alkyl(meth)acrylate is preferably from 10 to 60 mol %, more preferably from 10 to 50 mol %, and even more preferably from 10 to 40 mol %.

[0026] The polymer (a-3) contains a structural unit derived from 2-(meth)acryloyloxyethyl phosphorylcholine, C ４ ~C ２０ C having a structural unit derived from alkyl (meth)acrylate and two or more hydroxyl groups as substituents ２ ~C ８ The polymer (a-3) may contain other structural units in addition to the structural units derived from alkyl (meth)acrylate. Among these, the polymer (a-3) contains structural units derived from 2-(meth)acryloyloxyethyl phosphorylcholine, C ４ ~C ２０ C having a structural unit derived from alkyl (meth)acrylate and two or more hydroxyl groups as substituents ２ ~C ８ It is preferable that the polymer is composed only of structural units derived from alkyl (meth)acrylate.

[0027] The weight average molecular weight of the polymer (a-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.

[0028] <Polymer (a-4)> Polymer (a-4) is formed by polymerizing a monomer mixture containing 2-(meth)acryloyloxyethyl phosphorylcholine and polyethylene glycol (meth)acrylate. The 2-(meth)acryloyloxyethyl phosphorylcholine used to form polymer (a-4) may be one type or two types. From the viewpoints of the storage stability of the copolymer and availability of raw materials, 2-methacryloyloxyethyl phosphorylcholine is preferred as the 2-(meth)acryloyloxyethyl phosphorylcholine used to form polymer (a-4). Of all the structural units of polymer (a-4), the proportion of structural units derived from 2-(meth)acryloyloxyethyl phosphorylcholine is preferably 50 to 95 mol%, more preferably 70 to 95 mol%, and even more preferably 85 to 95 mol%.

[0029] The polyethylene glycol (meth)acrylate for forming the polymer (a-4) may be used alone or in combination of two or more. The polyethylene glycol (meth)acrylate is a polyethylene glycol [HO—(CH ２ CH ２ O) ｎ -H: n is 2 to 90], at least one of the hydroxyl groups is bonded to an acryloyl group or a methacryloyl group. The polyethylene glycol (meth)acrylate may be either polyethylene glycol mono(meth)acrylate or polyethylene glycol di(meth)acrylate, with polyethylene glycol mono(meth)acrylate being preferred. Of all the structural units of polymer (a-4), the proportion of structural units derived from polyethylene glycol (meth)acrylate is preferably 5 to 50 mol%, more preferably 5 to 30 mol%, and even more preferably 5 to 15 mol%. The weight-average molecular weight of polymer (a-4) is not particularly limited, but is preferably 10,000 to 2,000,000, more preferably 25,000 to 1,500,000, and even more preferably 50,000 to 1,000,000.

[0030] <Polymer (a-5)> Polymer (a-5) is formed by polymerizing a mixture of 2-(meth)acryloyloxyethyl phosphorylcholine and a carboxyl group-containing monomer. The 2-(meth)acryloyloxyethyl phosphorylcholine used to form polymer (a-5) may be used alone or in combination of two or more. From the viewpoints of the storage stability of the copolymer and availability of raw materials, 2-methacryloyloxyethyl phosphorylcholine is preferred as the 2-(meth)acryloyloxyethyl phosphorylcholine used to form polymer (a-5). Of all the constituent units of polymer (a-5), the proportion of constituent units derived from 2-(meth)acryloyloxyethyl phosphorylcholine is preferably 10 to 90 mol%, more preferably 15 to 70 mol%, and even more preferably 20 to 50 mol%. The carboxyl group-containing monomer used to form polymer (a-5) may be used alone or in combination of two or more types. Examples of monomers containing a carboxyl group include compounds containing a carboxyl group and a vinyl group, such as (meth)acrylic acid, crotonic acid, 3-methylcrotonic acid, angelic acid, tiglic acid, fumaric acid, maleic acid, itaconic acid, and citraconic acid. From the viewpoint of ease of polymerization, (meth)acrylic acid is preferred, and methacrylic acid is more preferred. Of all the structural units of polymer (a-5), the proportion of the monomer containing a carboxyl group is preferably 10 to 90 mol%, more preferably 30 to 85 mol%, and even more preferably 50 to 80 mol%. The weight-average molecular weight of polymer (a-5) is not particularly limited, but is preferably 10,000 to 2,000,000, more preferably 25,000 to 1,500,000, and even more preferably 50,000 to 1,000,000.

[0031] Each monomer used in the synthesis of the polymer used in 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 (e.g., the method described in International Publication No. 2018 / 216628). The polymer used in the present invention is mixed with a nucleic acid solution extracted from a biological sample to prepare a nucleic acid mixture, thereby reducing the effects of amplification inhibition by contaminants such as amplification inhibitors contained in the biological sample. The polymer used in the present invention has a toxicity-reducing effect on surfactants used in simple nucleic acid extraction kits, etc., and mixing it with the surfactant before nucleic acid extraction may reduce the nucleic acid extraction efficiency of the surfactant. Therefore, it is desirable to mix it with a nucleic acid solution extracted from a biological sample. The extraction method for nucleic acid from a biological sample is not particularly limited, and nucleic acid extraction methods commonly used in the field can be used.

[0032] <Nucleic Acid Mixture> In the nucleic acid amplification method of the present invention, a step of preparing a nucleic acid mixture is carried out by mixing a solution containing extracted nucleic acid (nucleic acid solution) with a polymer containing a constitutional unit derived from a monomer containing a phosphorylcholine group. In the step of preparing the nucleic acid mixture, the order of mixing the components is not particularly limited, but it is preferable to prepare a master mix by mixing the polymer used in the present invention with reagents necessary for performing the nucleic acid amplification reaction described below, and then mix the master mix with the nucleic acid solution.

[0033] The concentration of the polymer contained in the nucleic acid mixture 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). The nucleic acid mixture is prepared by mixing the polymer with known reagents necessary for carrying out a nucleic acid amplification reaction. Examples of the known reagents include a buffer, a substrate, a primer, a DNA polymerase, a fluorescent DNA staining reagent, a fluorescent probe, a passive reference, and a nucleic acid.

[0034] 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 salt such as potassium chloride or ammonium sulfate. The buffer may further contain a water-soluble organic solvent such as dimethyl sulfoxide, dimethylformamide, formamide, or glycerin. The buffer may further contain a surfactant such as polyoxysorbitan fatty acid ester or polyoxyethylene alkylphenyl ether. The buffer may further contain a protein such as bovine serum albumin. The buffer may further contain a water-soluble polymer such as polyethylene glycol.

[0035] The substrate is not particularly limited, and for example, a mixture (dNTPs) of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and deoxycytidine triphosphate (dCTP) can be mentioned. Here, it is also possible to replace part and / or all of dTTP with deoxyuridine triphosphate (dUTP). In addition, in sequencing PCR and the like, it is also preferable to add an appropriate amount of a mixture of dideoxyadenosine triphosphate (ddATP), dideoxythymidine triphosphate (ddTTP), dideoxyguanosine triphosphate (ddGTP), and dideoxycytidine triphosphate (ddCTP), or fluorescently labeled products thereof.

[0036] The primer is not particularly limited, but examples include oligonucleotides of about 15 to 30 bases designed and prepared by known methods. The primer may be fluorescently labeled with fluorescein or the like, or isotope-labeled with a heavy element, as appropriate. Only one type of primer may be used, or two types of primers may be used as a pair, or multiple pairs of primers may be used.

[0037] Known DNA polymerases can be used as the DNA polymerase. From the viewpoint of heat resistance, enzymes derived from thermophilic bacteria, thermophilic archaea, hyperthermophilic bacteria, and hyperthermophilic archaea, and mutant enzymes thereof are preferred. The DNA polymerase is selected from DNA-dependent DNA polymerase, RNA-dependent DNA polymerase (reverse transcriptase), or an enzyme having both functions, as appropriate, depending on the purpose of nucleic acid amplification. In addition, whether to use a DNA polymerase with nuclease activity or a DNA polymerase without nuclease activity is appropriately selected. The fluorescent DNA staining reagent is not particularly limited, but examples thereof include SYBR ＴＭ Green I and the like.

[0038] The fluorescent probe is not particularly limited, but may be, for example, TaqMan ＴＭ The passive reference may be appropriately selected depending on the purpose of nucleic acid amplification. Examples of the passive reference include ROX. ＴＭ Dye and the like.

[0039] In addition to the aforementioned primers and fluorescent probes, any DNA and / or RNA may be used as the nucleic acid, for example, as an exogenous control gene. 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 or the environment, or from humans, animals, or plants, or may be isolated and cultured. The nucleic acid mixture may further contain an oil such as mineral oil. Furthermore, a solid phase carrier such as silica beads or magnetic beads may also be added.

[0040] Methods for amplifying nucleic acids by reacting the nucleic acid mixture include the Polymerase Chain Reaction (PCR) method, Loop-Mediated Isothermal Amplification (LAMP) method, Transcription-Mediated Amplification (TMA) method, Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids (IICAN) method, Strand Displacement Amplification (SDA) method, and Ligase Chain Reaction (LCR) method. Examples of such methods include the Linear Reaction (LCR) method and the Nucleic Acid Sequence-Based Amplification (NASBA) method, with the PCR method being preferred.

[0041] As the PCR method, an appropriate method may be selected depending on the type of template nucleic acid, i.e., the nucleic acid to be amplified. For example, if the template nucleic acid is double-stranded DNA, a typical PCR method is preferably selected, and if the template nucleic acid is single-stranded nucleic acid, an RT-PCR (Reverse Transcription PCR) method is preferably selected.

[0042] Furthermore, in medical tests, the qPCR (quantitative PCR) method is preferably selected. That is, the qPCR method is preferably selected for tests targeting DNA, such as bacterial tests, fungal tests, and DNA virus tests, while the RT-qPCR method is preferably selected for tests targeting RNA, such as RNA virus tests.

[0043] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these.

[0044] [Synthesis of Polymer] The polymer of the present invention was synthesized as follows.

[0045] Synthesis Example 1 Polymer 1 corresponding to polymer (a-1) was synthesized as follows. 40.0 g of 2-methacryloyloxyethyl phosphorylcholine (hereinafter referred to as "MPC") was weighed into a polymerization recovery flask, and 60.0 g of purified water was added and dissolved. The atmosphere in the reaction vessel was thoroughly purged with nitrogen, and the temperature was raised to 70°C with stirring. 0.31 g of azobisisobutyronitrile (hereinafter referred to as "AIBN") was added as a polymerization initiator, and polymerization was carried out for 6 hours. The resulting reaction solution was ice-cooled and added dropwise to diethyl ether to precipitate a polymer. The precipitate was filtered, washed with diethyl ether, and then vacuum-dried to obtain a white powdery homopolymer (referred to as "polymer (a-1)" in this specification). A 5 wt % aqueous solution was prepared using ultrapure water and used for testing. The weight average molecular weight of Polymer 1 was 1,030,000 in terms of polyethylene glycol, as measured by gel permeation chromatography (hereinafter referred to as "GPC") under the conditions described below.

[0046] Synthesis Example 2 Polymer 2 corresponding to polymer (a-2) was synthesized as follows. 11.7 g of MPC and 3.3 g of stearyl methacrylate (hereinafter referred to as SMA) (MPC / SMA = 80 / 20 (molar ratio)) were weighed into a polymerization flask, and 85.0 g of ethanol was added and dissolved. The atmosphere in the reaction vessel was thoroughly replaced with nitrogen, and the temperature was raised to 60°C with stirring. 0.06 g of AIBN was added as a polymerization initiator, and polymerization was carried out for 6 hours. Thereafter, a random copolymer (referred to herein as "polymer (a-2)") was obtained in the same manner as in Synthesis Example 1. A 5 wt % aqueous solution was prepared using ultrapure water and used for testing. 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.

[0047] Synthesis Example 3 Polymer 3, corresponding to polymer (a-3), was synthesized as follows. 8.4 g of MPC, 2.1 g of butyl methacrylate (hereinafter referred to as BMA), and 4.5 g of glycerin monomethacrylate (hereinafter referred to as GrMA) (MPC / BMA / GrMA = 40 / 40 / 20 (molar ratio)) were weighed into a polymerization recovery flask, and 42.5 g of purified water and 42.5 g of ethanol were added and dissolved. The atmosphere inside the reaction vessel was thoroughly replaced with nitrogen, and the temperature was raised to 70°C with stirring. 0.15 g of AIBN was added as a polymerization initiator, and polymerization was carried out for 6 hours. Thereafter, a random copolymer (referred to herein as "polymer (a-3)") was obtained in the same manner as in Synthesis Example 1. A 5 wt % aqueous solution was prepared using ultrapure water and used for testing. The weight-average molecular weight of polymer 3 was 22,000 in terms of polyethylene glycol, as determined by GPC measurement under the conditions described below.

[0048] [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: Eco SEC 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

[0049] Table 1 shows the monomers used in Synthesis Examples 1 to 3, their molar ratios, and the weight average molecular weights of the resulting polymers.

[0050]

[0051] [Preparation of Clinical Mock Samples] The preparation method of the clinical mock samples used in each Example and Comparative Example is as follows: Samples containing either a nasopharyngeal sample, a nasal sample, or a saliva sample spiked with SARS-CoV-2 virus (clinical mock samples) and samples not containing a clinical sample were prepared according to the following procedures.

[0052] (Nasopharyngeal Sample) A nasopharyngeal swab collected from a healthy subject was suspended in 600 μL of Dulbecco's phosphate-buffered saline (D-PBS). 50 μL of SARS-CoV-2 virus solution was added to 450 μL of the prepared suspension to prepare a nasopharyngeal sample. (Nasal Sample) A nasal swab collected from a healthy subject was suspended in 600 μL of D-PBS. 50 μL of SARS-CoV-2 virus solution was added to 450 μL of the prepared suspension to prepare a nasal sample. (Saliva Sample) A saliva sample was prepared by adding 50 μL of SARS-CoV-2 virus solution to 450 μL of the serous portion of saliva collected from a healthy subject. (Sample without Clinical Sample) A sample without clinical sample was prepared by adding 50 μL of SARS-CoV-2 virus solution to 450 μL of D-PBS.

[0053] [Preparation of Master Mix] The method for preparing the master mix used in each example and comparative example is as follows. RT-qPCR was performed using the commercially available SARS-CoV-2 RT-qPCR Detection Kit Ver. 2 (Fujifilm Wako Pure Chemical Corporation). The master mix was prepared by mixing the reagents included in the kit in the amounts listed in Tables 2 and 3 below.

[0054]

[0055]

[0056] (Examples 1-1 to 1-3 and Comparative Example 1) Examples and Comparative Examples are shown below in which samples not containing clinical specimens were used as clinical simulants. [Preparation of Simple RNA Extraction Solution] RNA was simply extracted using Solution A and Solution B contained in commercially available SARS-CoV-2 Lysis Buffer Ver. 2 (Fujifilm Wako Pure Chemical Corporation). 30 μL of Solution A was added to 150 μL of the above-mentioned sample not containing a clinical specimen, and the mixture was left to stand at room temperature for 5 minutes or more, followed by incubation at 95°C for 5 minutes. Then, 30 μL of Solution B was added and mixed on ice to prepare a simple RNA extraction solution.

[0057] [Reverse transcription / nucleic acid amplification reaction] 5.0 μL of the above-mentioned simple RNA extraction solution was added to 15.0 μL of master mix, and an RT-qPCR reaction was performed using a QuantStudio6Pro real-time PCR system (Thermo Fisher Scientifics) under the conditions shown in Table 4 below.

[0058] Denaturation and Annealing & Extension were performed for 45 cycles.

[0059] [Determination] The cycle number at which the QuantStudio6Pro real-time PCR system automatically detected the amplification of the SARS-CoV-2 gene was determined as the Ct value. The results are shown in Table 5.

[0060]

[0061] As shown in Table 5, when a polymer was added to the master mix (Examples 1-1 to 1-3), the Ct value was lower than that of Comparative Example 1, which shows that the delay in Ct value caused by impurities that are incorporated into the simple RNA extract when RNA is extracted from a virus using a simple extraction kit is improved.

[0062] (Examples 2-1 to 2-3 and Comparative Examples 2-1 to 2-2) Examples and Comparative Examples are shown below in which nasopharyngeal specimens and samples not containing clinical specimens were used as clinical simulants. The procedures for [preparation of simple RNA extract], [reverse transcription / nucleic acid amplification reaction], and [assessment] were the same as those in Example 1-1, except that nasopharyngeal specimens were used as clinical simulants. The results are shown in Table 6.

[0063]

[0064] As shown in Table 6, when a nasopharyngeal sample was used (Comparative Example 2-1), the Ct value was higher than when a sample not containing a clinical sample was used (Comparative Example 2-2), confirming that the Ct value was delayed due to impurities contained in the nasopharyngeal sample. On the other hand, when polymers 1 to 3 were added to the master mix (Examples 2-1 to 2-3), the Ct value was earlier than in Comparative Example 2-1 and was close to that of Comparative Example 2-2, indicating that the delay in Ct value due to impurities contained in the nasopharyngeal sample was improved.

[0065] (Examples 3-1 to 3-3 and Comparative Example 3-1) Examples and Comparative Examples are shown below in which nasal specimens were used as clinical simulants. The procedures for [preparation of simple RNA extract], [reverse transcription / nucleic acid amplification reaction], and [assessment] were the same as those in Example 1-1, except that nasal specimens were used as clinical simulants. The results are shown in Table 7.

[0066]

[0067] As shown in Table 7, when nasal samples were used and when polymers 1 to 3 were added to the master mix (Examples 3-1 to 3-3), the Ct values ​​were earlier than in Comparative Example 3-1, which indicates that the delay in Ct values ​​due to impurities contained in nasal samples was improved.

[0068] (Examples 4-1 to 4-3 and Comparative Example 4-1) These are examples and comparative examples in which saliva samples were used as clinical simulants. The procedures for [preparation of simple RNA extract], [reverse transcription / nucleic acid amplification reaction], and [assessment] were the same as those in Example 1-1, except that saliva samples were used as clinical simulants. The results are shown in Table 8.

[0069]

[0070] As shown in Table 8, when a saliva sample was used and polymers 1 to 3 were added to the master mix (Examples 4-1 to 4-3), the Ct value was earlier than in Comparative Example 4-1, which indicates that the delay in Ct value due to impurities contained in the saliva sample was improved. From the above results, it can be seen that the nucleic acid amplification method of the present invention can suppress the inhibition of nucleic acid amplification caused by impurities contained in the sample or simple extraction kit.

Claims

1. A nucleic acid amplification method comprising: an extraction step of extracting nucleic acid from a biological sample; a step of mixing the extracted nucleic acid with a polymer containing a constitutional unit derived from a monomer containing a phosphorylcholine group to prepare a nucleic acid mixture; and a step of reacting the nucleic acid mixture to amplify the nucleic acid.

2. The nucleic acid amplification method according to claim 1, wherein the polymer comprises one or more polymers selected from the following Group a: [Group a] (a-1) Polymers containing only structural units derived from 2-(meth)acryloyloxyethyl phosphorylcholine; (a-2) Polymers containing structural units derived from 2-(meth)acryloyloxyethyl phosphorylcholine and C ４ ~C ２０ (a-3) A copolymer containing a structural unit derived from alkyl (meth)acrylate, a structural unit derived from 2-(meth)acryloyloxyethyl phosphorylcholine, C ４ ~C ２０ C having a structural unit derived from alkyl (meth)acrylate and two or more hydroxy groups as substituents ２ ~C ８ (a-4) A copolymer containing a structural unit derived from alkyl (meth)acrylate. (a-5) A copolymer containing a structural unit derived from 2-(meth)acryloyloxyethyl phosphorylcholine and a structural unit derived from polyethylene glycol (meth)acrylate.

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