Nucleic acid extraction method, nucleic acid amplification method, nucleic acid extraction kit, and PCR test kit

Abstract

This nucleic acid extraction method comprises: producing an antibody-copolymer conjugate by preparing, in the absence of a protease, a mixture including at least one sample selected from the group consisting of cells, extracellular vesicles and virions, a copolymer containing a repeating unit represented by formula 1, and a repeating unit represented by formula 2, and an antibody-linker complex obtained by binding an antibody, which binds to the sample, with a linker represented by formula 3 via an amide bond; and heating the mixture, aggregating the antibody-copolymer conjugate, and extracting a nucleic acid included in the sample. According to the nucleic acid extraction method, a nucleic acid included in the sample can be easily extracted even in the absence of a protease and without the need of concentration with a carrier nucleic acid.

Description

Nucleic acid extraction method, nucleic acid amplification method, nucleic acid extraction kit, and PCR test kit

[0001] The present disclosure relates to a nucleic acid extraction method, a nucleic acid amplification method, a nucleic acid extraction kit, and a PCR test kit.

[0002] Biological diagnostics, testing, and analytical procedures require the detection of nucleic acids contained in cells and / or virus particles contained in biological samples (e.g., swabs, etc.). In general, such tests involve pretreatment, such as separation, concentration, and purification of nucleic acids from target substances (e.g., virus specimens) contained in the sample.

[0003] Nucleic acids in samples can form complex associations with contaminants, and concentration and purification often require digestion of the contaminants with protease and increasing the yield (hereinafter also referred to as "concentration") by using "carrier nucleic acids" such as polyadenylic acid. This tendency is particularly pronounced when the sample concentration is low. Note that "sample" here refers to cells and / or virus particles, etc., contained in a biological sample that contain nucleic acids to be extracted.

[0004] For example, in a test using the polymerase chain reaction (PCR) method (hereinafter also referred to as a "PCR test") performed to isolate and identify SARS-CoV-2 infection, treatment with a protease and / or concentration of a sample and / or nucleic acid using a carrier nucleic acid may be performed as pretreatment for reverse transcription and amplification of viral RNA (ribonucleic acid) (e.g., Patent Document 1).

[0005] Japanese Patent Application Laid-Open No. 2004-215676

[0006] Sample treatment with protease and concentration by adding carrier nucleic acid (hereinafter also referred to as "treatment with protease, etc.") requires expensive reagents, complicated experimental procedures, and a certain amount of time. Therefore, treatment with protease, etc., can become a bottleneck in PCR testing. In addition, the inventory management of the various pretreatment reagents used in treatment with protease, etc., is complicated, and the reagents are expensive, which increases testing costs.

[0007] An object of the present disclosure is to provide a nucleic acid extraction method that can easily extract nucleic acids contained in a sample even in the absence of protease and without concentration using a carrier nucleic acid. In other words, it is to provide a nucleic acid extraction method that can purify and concentrate a sample for nucleic acid extraction in one step. Another object of the present disclosure is to provide a nucleic acid amplification method, a nucleic acid extraction kit, and a PCR test kit.

[0008] One embodiment of the nucleic acid extraction method of the present disclosure is a nucleic acid extraction method comprising: preparing, in the absence of a protease, a mixed solution containing at least one specimen selected from the group consisting of cells, extracellular vesicles, and virions; a copolymer including a repeating unit represented by the following formula 1 and a repeating unit represented by the following formula 2; an antibody that binds to the specimen; and an antibody-linker conjugate obtained by binding, via an amide bond, a linker represented by the following formula 3, to generate an antibody-copolymer conjugate; and heating the mixed solution to aggregate the antibody-copolymer conjugate, thereby extracting nucleic acids contained in the specimen.

[0009] In formula 1, X １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; ２ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; L ２ represents a divalent group, and R １ represents a hydrogen atom, a halogen atom, -OR ５ , -NO ２ , -CN, -S(O) ２ R ５ , an alkyl group having 1 to 24 carbon atoms, an alkenyl group having 2 to 24 carbon atoms, and a (hetero)aryl group having 6 to 24 carbon atoms; １ may be the same or different, and two or more of them may be bonded to each other to form a ring; R ５ is selected from the group consisting of a hydrogen atom, a halogen atom, an alkyl group having 1 to 24 carbon atoms, and a (hetero)aryl group having 6 to 24 carbon atoms, and Z is C(R１ ) ２ ,O,S,and ,NR １ a' is an integer from 0 to 8, a" is an integer from 0 to 8, and the sum of a' and a" is less than 10.

[0010] In formula 3, L ３ represents a divalent hydrocarbon group which may have a hetero atom.

[0011] One embodiment of the nucleic acid extraction kit of the present disclosure is a nucleic acid extraction kit used to extract nucleic acids from at least one specimen selected from the group consisting of cells, extracellular vesicles, and virions in the absence of proteases, the nucleic acid extraction kit comprising: a first agent containing a copolymer including a repeating unit represented by the following formula 1 and a repeating unit represented by the following formula 2; and a second agent containing an antibody that binds to the specimen and an antibody-linker complex obtained by bonding, via an amide bond, an antibody represented by the following formula (3):

[0012] In formula 1, X １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; ２ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; L ２ represents a divalent group, and R １ represents a hydrogen atom, a halogen atom, -OR ５ , -NO ２ , -CN, -S(O) ２ R ５ , an alkyl group having 1 to 24 carbon atoms, an alkenyl group having 2 to 24 carbon atoms, and a (hetero)aryl group having 6 to 24 carbon atoms; １ may be the same or different, and two or more of them may be bonded to each other to form a ring; R ５ is selected from the group consisting of a hydrogen atom, a halogen atom, an alkyl group having 1 to 24 carbon atoms, and a (hetero)aryl group having 6 to 24 carbon atoms, and Z is C(R １ ) ２ ,O,S,and ,NR １a' is an integer from 0 to 8, a" is an integer from 0 to 8, and the sum of a' and a" is less than 10.

[0013] In formula 3, L ３ represents a divalent hydrocarbon group which may have a hetero atom.

[0014] The present disclosure provides a nucleic acid extraction method that can easily extract nucleic acids contained in a sample even in the absence of protease and without concentration using a carrier nucleic acid. In other words, the present disclosure provides a nucleic acid extraction method that can purify and concentrate a sample for nucleic acid extraction in one step. The present disclosure also provides a nucleic acid amplification method, a nucleic acid extraction kit, and a PCR test kit.

[0015] 1 is a flow chart showing the procedure of the nucleic acid extraction method of the present invention. 2 is a quantification result by real-time PCR using nucleic acids extracted by the method of Comparative Example 1 and the method of Reference Example 1. 3 is a quantification result by real-time PCR using nucleic acids extracted by the method of Comparative Example 1 and the method of Example 1.

[0016] A first embodiment of the nucleic acid extraction method of the present disclosure is a nucleic acid extraction method comprising: preparing, in the absence of a protease, a mixture containing at least one analyte selected from the group consisting of cells, extracellular vesicles, and virions; a copolymer including a repeating unit represented by Formula 1 (described below) and a repeating unit represented by Formula 2 (described below); an antibody that binds to the analyte; and an antibody-linker conjugate obtained by binding, via an amide bond, a linker represented by Formula 3 (described below); thereby generating an antibody-copolymer conjugate; and heating the mixture to aggregate the antibody-copolymer conjugate, thereby extracting nucleic acids contained in the analyte.

[0017] The antibody-copolymer conjugate is water-soluble and has a lower critical solution temperature (LCST) due to the copolymer structure. Therefore, its solubility in water decreases upon heating, making it prone to precipitation and aggregation. The analyte (antigen) is specifically recognized and bound by the antibody through antigen-antibody interaction, and when heated in this state, the analyte aggregates and concentrates while bound to the conjugate. According to the nucleic acid extraction method of the first embodiment, target nucleic acids can be easily extracted even from samples with low antigen concentrations and / or without pretreatment using proteases.

[0018] A second embodiment of the nucleic acid extraction method of the present disclosure is the nucleic acid extraction method of the first embodiment, wherein the content of the repeating unit represented by Formula 2 described below is 1.0 to 30.0 mol % when all repeating units of the copolymer are taken as 100 mol %.

[0019] The repeating unit (unit 2) represented by formula 2 has two functions in the copolymer (and antibody-copolymer conjugate). One is to serve as a binding site for the antibody, and the other is to adjust the LCST. Regarding the former, unit 2 has one site (click reaction site) containing a cyclic alkyne (alkynylene group). Therefore, it can easily bond to the azide of the linker of formula 3 via a click reaction. As will be described later, the linker of formula 3 can bind to an antibody. Therefore, unit 2 has the function of binding and immobilizing the antibody to the copolymer via the linker. Furthermore, regarding the latter, unit 2 is more hydrophobic than unit 1. Therefore, increasing the content of unit 2 in the copolymer can adjust the LCST to a lower temperature.

[0020] The LCST of a copolymer having a repeating unit content of Formula 2 of 1.0 to 30.0 mol % can be easily adjusted to between room temperature and 40°C. Furthermore, the amount of antibody bound thereto is sufficient for practical use. According to the nucleic acid extraction method of the second embodiment, the LCST is low, so that the antibody-copolymer conjugate bound to the antigen can be more easily aggregated (for example, by simply heating at body temperature) and nucleic acid can be extracted.

[0021] A third embodiment of the nucleic acid extraction method of the present disclosure is a nucleic acid extraction method according to the first or second embodiment, wherein the repeating unit represented by formula 2 above is at least one repeating unit selected from the group consisting of a repeating unit represented by formula 4 described below and a repeating unit represented by formula 5 described below.

[0022] As described above, unit 2 has two functions. When unit 2 is a unit represented by formula 4 or formula 5, the hydrophobicity can be more appropriately adjusted, and as a result, the LCST can be more easily adjusted to a more manageable range (room temperature to 40°C). Furthermore, binding to the linker on which the antibody is immobilized becomes easier, resulting in improved convenience and efficiency of the nucleic acid extraction method.

[0023] A fourth embodiment of the nucleic acid extraction method of the present disclosure is the nucleic acid extraction method of any one of the first to third embodiments, wherein the copolymer further contains a repeating unit represented by formula 6 described below.

[0024] The repeating unit represented by formula 6 (unit 6) is more hydrophilic than unit 2. Therefore, unit 6 has the effect of adjusting the LCST of the copolymer (and antibody-copolymer conjugate) to a higher temperature. Therefore, even when the content of unit 2 is high, the LCST is easily adjusted to a more manageable range (room temperature to 40°C). Therefore, both antigen-binding ability and ease of handling (convenience) are likely to be achieved at a higher level.

[0025] A fifth embodiment of the nucleic acid extraction method of the present disclosure is the nucleic acid extraction method of any one of the first to fourth embodiments, wherein the content of the repeating unit represented by formula 2 is 2.0 to 30.0 mol % when all repeating units of the copolymer are taken as 100 mol %.

[0026] The above-mentioned polymer and antibody-copolymer conjugate achieve both high levels of antigen binding ability and ease of handling (convenience), and as a result, nucleic acids can be extracted more reliably and easily by the nucleic acid extraction method of the fifth embodiment.

[0027] A sixth embodiment of the nucleic acid extraction method of the present disclosure is the nucleic acid extraction method of any one of the first to fifth embodiments, wherein the number average molecular weight of the copolymer is 5,000 to 50,000.

[0028] When the number-average molecular weight of the copolymer is 5,000 or more, better concentration efficiency is likely to be obtained (in other words, better temperature responsiveness is likely to be obtained), while when it is 50,000 or less, a conjugate with an antibody is more likely to be formed, or an antigen-antibody reaction is more likely to proceed. As a result, in the nucleic acid extraction method of the sixth embodiment using an antibody-copolymer conjugate based on the above copolymer, the antibody-copolymer conjugate is more likely to bind to an antigen, and nucleic acids can be extracted more efficiently.

[0029] A seventh embodiment of the nucleic acid extraction method of the present disclosure is any one of the first to sixth nucleic acid extraction methods, wherein the molar ratio of the content of the copolymer to the content of the antibody contained in the antibody-linker complex in the mixed solution (copolymer / antibody) is 0.5 to 30.0.

[0030] The copolymer and the antibody-linker conjugate are easily and reliably bonded by a click reaction. Furthermore, the copolymer itself has the same LCST as the antibody-copolymer conjugate. Therefore, even if the content of the copolymer in the mixed solution is lower than that of the antibody-linker conjugate, the copolymer that is not involved in the bond with the antibody-linker conjugate can contribute to aggregation due to temperature change (heating) and nucleic acid extraction. From this perspective, the lower limit of the numerical range of copolymer / antibody is preferably 1.0 or more, and more preferably 2.0 or more. Preferred embodiments of the numerical range of copolymer / antibody will be described later.

[0031] An eighth embodiment of the nucleic acid extraction method of the present disclosure is any one of the first to seventh nucleic acid extraction methods, wherein the antibody-copolymer conjugate comprises a repeating unit represented by Formula 1 described below and a repeating unit represented by Formula 7 described below.

[0032] The copolymer is typically obtained by binding an antibody-linker conjugate to a copolymer having unit 5. Unit 7 based on unit 5 has the function of adjusting the LCST to a manageable temperature (room temperature to 40°C). Therefore, according to the nucleic acid extraction method of the eighth embodiment using the antibody-copolymer conjugate, the antibody-copolymer conjugate with (or without) an antigen bound thereto and the copolymer can be more easily aggregated and precipitated, making it easier to extract nucleic acids.

[0033] A ninth embodiment of the nucleic acid extraction method of the present disclosure is any one of the first to eighth nucleic acid extraction methods, wherein the heating is heating the mixed solution to a temperature of 20 to 40°C.

[0034] Heating of the mixture to 20 to 40°C can also be achieved by, for example, warming the container containing the mixture with the body temperature of a laboratory technician. According to the nucleic acid extraction method of the ninth embodiment, nucleic acids can be easily extracted without using a heating device or the like.

[0035] A tenth embodiment of the nucleic acid extraction method of the present disclosure is any one of the first to ninth nucleic acid extraction methods, wherein the specimen is a membrane structure having a lipid bilayer.

[0036] When a sample has a lipid bilayer and nucleic acids contained therein are to be extracted, it has been experimentally confirmed that nucleic acids can be extracted simply by preparing a mixture containing the sample, a copolymer, and an antibody-linker complex in the absence of a protease to produce an antibody-copolymer conjugate, and then heating the mixture to aggregate the antibody-copolymer conjugate (and the copolymer, if present in the mixture) (as described below).The nucleic acid extraction method of the tenth embodiment allows the target nucleic acid to be extracted more easily with a very simple procedure and without using many reagents, compared to conventional methods.

[0037] An eleventh embodiment of the nucleic acid extraction method of the present disclosure is a nucleic acid extraction method according to any one of the first to ninth embodiments, wherein the specimen is an enveloped virus.

[0038] It has been experimentally confirmed that the nucleic acid extraction method of the present disclosure, when used with an enveloped virus as a sample, can more easily extract the target nucleic acid using a much simpler procedure than conventional methods, without using many reagents.

[0039] A twelfth embodiment of the nucleic acid extraction method of the present disclosure is a nucleic acid extraction method according to any one of the first to ninth embodiments, wherein the specimen is SARS-CoV-2 virus.

[0040] It has been experimentally confirmed that the nucleic acid extraction method of the present disclosure, when using SARS-CoV-2 as a sample, can more easily extract the target nucleic acid using a much simpler procedure than conventional methods, without using many reagents.

[0041] A first embodiment of the nucleic acid amplification method of the present disclosure is a nucleic acid amplification method including: extracting the nucleic acid from the sample using any one of the nucleic acid extraction methods 1 to 12; and amplifying the extracted nucleic acid by polymerase chain reaction.

[0042] The nucleic acid amplification method of the first embodiment includes a step of extracting nucleic acids using the nucleic acid extraction method described above, which reduces the labor required for the entire process and requires fewer reagents and less equipment than conventional methods.

[0043] A first embodiment of the nucleic acid extraction kit of the present disclosure is a nucleic acid extraction kit used to extract nucleic acids from at least one specimen selected from the group consisting of cells, extracellular vesicles, and virions in the absence of proteases, and comprises: a first agent containing a copolymer including a repeating unit represented by Formula 1 described below and a repeating unit represented by Formula 2 described below; and a second agent containing an antibody-linker complex obtained by bonding an antibody that binds to the specimen and a linker represented by Formula (3) above via an amide bond.

[0044] The nucleic acid extraction kit of the first embodiment contains a first agent containing a copolymer and a second agent containing an antibody-linker complex, and by mixing these agents as needed, an antibody-copolymer conjugate can be produced. Because the first and second agents are separate, the kit has high storage stability, and the operation required for use is simple, requiring only mixing. The nucleic acid extraction kit of the first embodiment allows nucleic acids to be extracted in the absence of protein enzymes and with simple procedures.

[0045] The PCR (Polymerase Chain Reaction) test kit of the present disclosure is a PCR test kit that includes a first embodiment of the nucleic acid extraction kit.

[0046] The PCR test kit allows nucleic acids to be easily extracted from specimens contained in biological samples, etc., in the absence of protease, and then the extracted nucleic acids can be amplified. By using this PCR test kit, the time and cost required for PCR testing can be significantly reduced.

[0047] Hereinafter, the nucleic acid extraction method and the like will be described in detail based on non-limiting embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​written before and after "to" as the lower and upper limits.

[0048] Furthermore, in this specification, when there are multiple substituents or linking groups, etc. (hereinafter referred to as substituents, etc.) represented by a specific symbol, or when multiple substituents, etc. are specified simultaneously, it means that the respective substituents, etc. may be the same or different from each other. This also applies to the specification of the number of substituents, etc. Furthermore, unless otherwise specified, when multiple substituents, etc. are close (particularly adjacent), they may be linked to each other or condensed to form a ring. Furthermore, for substituents, etc. not specified as substituted or unsubstituted in this specification, the group may further have a substituent within a range that does not impair the intended effect. This also applies to compounds not specified as substituted or unsubstituted.

[0049] As used herein, the term "virion" refers to a viral particle of an infectious virus. Infectious viruses can replicate in the cells of bacteria, plants, and animals, including humans (collectively referred to as "hosts"). In one form, a virion is a metabolically inactive infectious agent with a diameter of 20 to 300 nm, and comprises a nucleic acid (RNA or DNA) core and a protein coat. Furthermore, enveloped viruses, which have an envelope comprising a lipid bilayer, are also included in the above.

[0050] Examples of infectious viruses include influenza viruses (avian influenza virus, equine influenza virus, swine influenza virus, canine influenza virus, feline influenza virus, and human influenza virus), human immunodeficiency virus (HIV), flaviviruses (e.g., hepatitis virus, dengue virus, and Zika virus), human papillomavirus (HPV), bovine papillomavirus, herpes viruses (e.g., HSV-I, HSV-II, CMV, and VZV), rhinoviruses, and coronaviruses (e.g., SARS-CoV-2 ... -CoV-2, SARS coronavirus, and MERS coronavirus, etc.), enterovirus, polyomavirus, respiratory syncytial virus (RSV), hepatitis B virus, hepatitis C virus, rotavirus, measles virus, mumps virus, rubella virus, varicella virus, human metapneumovirus, Ebola virus, Marburg virus, alphavirus (e.g., chikungunya virus, Ross River virus, Sindbis virus, Mayaro virus, etc.), porcine epidemic diarrhea, porcine reproductive and respiratory syndrome virus, and foot-and-mouth disease virus.

[0051] As used herein, "extracellular vesicles" refer to particle-like structures released from cells into the extracellular environment by any mechanism. "Extracellular vesicles" include exosomes, microvesicles, and apoptotic bodies. Extracellular vesicles may contain proteins, nucleic acids, lipids, and other molecules derived from their host cells. In this nucleic acid detection method, nucleic acid-containing extracellular vesicles are preferred as the specimen. The extracellular vesicles may be derived from various cells, such as red blood cells, white blood cells, cancer cells, stem cells, dendritic cells, and macrophages.

[0052] As used herein, the term "cell" refers to either a prokaryotic cell or a eukaryotic cell, and is not particularly limited thereto. Examples include bacteria, archaea, yeast, plant cells, insect cells, and animal cells (e.g., human cells, non-human cells, non-mammalian vertebrate cells, invertebrate cells, etc.).

[0053] The "specimen" herein refers to at least one selected from the group consisting of cells, extracellular vesicles, and virions, with virions being preferred. Furthermore, the specimen is preferably a membrane structure having a lipid bilayer (such as a cell, vesicle, liposome, or enveloped virus), more preferably an enveloped virus, and even more preferably the SARS-CoV-2 virus. The specimen may be contained in a sample obtained by culture or the like, or may be contained in a biological sample collected from animals, including humans, plants, or the like.

[0054] For example, samples obtained from humans may be samples derived from the lower respiratory tract such as sputum, tracheal aspirate, and bronchoalveolar lavage fluid; nasopharyngeal swabs such as nasopharyngeal swabs and throat swabs; saliva; serum; whole blood; urine; stool; autopsy tissue; etc.

[0055] As used herein, the terms "antibody" and antibody residues represented by "Ab" refer to an immunoglobulin molecule or a portion (fragment) thereof that has the ability to bind to an epitope of an antigen molecule. "Antibody" also includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments.

[0056] As used herein, "nucleic acid" refers to DNA (deoxyribonucleic acid) molecules and RNA molecules, and may be single-stranded or double-stranded. As used herein, "protease" refers to an enzyme that recognizes a specific site in the amino acid sequence constituting a protein, cleaves the bond, and decomposes the protein into amino acids or peptides. Protease is also called "protease" or "proteinase." Protease is not particularly limited, but examples include proteases used for the extraction and / or purification of nucleic acids. Conventionally, non-specific proteases such as proteinase K (EC 3.4.21.64, etc.), protease (pronase), trypsin, and subtilisin have often been used to extract nucleic acids.

[0057] Nucleic acid extraction methods performed "in the presence" of a protease (in other words, nucleic acid extraction methods using a protease) are known to those skilled in the art and include those described in JP 2004-215676 A, JP 2006-087394 A, JP 2006-061041 A, JP 2011-525806 A, WO 2017 / 200249, and WO 2017 / 200249.

[0058] "In the absence of proteases" means that the extraction is carried out in an environment (extraction reaction system) in which proteases are not used. Typically, this means that nucleic acid extraction is carried out without the intentional use of proteases. This does not prevent the extraction reaction system from being unintentionally contaminated with proteases, for example, from the sample, but it is preferable that the extraction reaction system does not contain proteases.

[0059] As used herein, "carrier nucleic acid" refers to a compound that, when added to a sample, improves the efficiency of nucleic acid extraction from a specimen, and examples include polyadenylic acid (homopolymer). Generally, carrier nucleic acids are often used when extracting nucleic acids from pathogens and / or samples containing low concentrations of nucleic acids. Commercially available nucleic acid extraction kits, such as the "QIAamp Viral RNA Mini Kit," already contain carrier RNA.

[0060] [Nucleic Acid Extraction Method] An embodiment of the nucleic acid extraction method (hereinafter also referred to as "the present nucleic acid extraction method") comprises preparing a mixture containing at least one specimen (hereinafter also referred to simply as "specimen") selected from the group consisting of cells, extracellular vesicles, and virions, in the absence of a protease (and further without using a carrier nucleic acid), a copolymer (hereinafter also referred to as "specific copolymer") containing a repeating unit represented by Formula 1 described below and a repeating unit represented by Formula 2 described below, an antibody that binds to the specimen, and an antibody-linker conjugate obtained by binding, via an amide bond, a linker represented by Formula 3 described below to generate an antibody-copolymer conjugate, and heating the mixture to aggregate the antibody-copolymer conjugate, thereby extracting nucleic acid contained in the specimen.

[0061] <Step S1> Figure 1 is a flow diagram showing the procedure of the present nucleic acid extraction method. First, in step S1, a mixed solution containing a sample, a specific copolymer, and an antibody-linker complex is prepared to produce an antibody-copolymer conjugate. At this time, it is not necessary to treat impurities with a protease, but of course, this may be done.

[0062] The specific copolymer has a repeating unit represented by formula 1 (hereinafter also referred to as "unit 1") and a repeating unit represented by formula 2 (hereinafter also referred to as "unit 2"), which will be described in detail later. A polymer constituted solely by unit 1 exhibits temperature responsiveness with a LCST (Lower Critical Solution Temperature) of 32°C to water. The specific copolymer containing unit 1 has temperature responsiveness.

[0063] Furthermore, each repeating unit of unit 2 has one moiety (click reaction moiety) containing a cyclic alkyne (alkynylene group), and can be easily conjugated by bonding with an azide group of a linker (described later) through a click reaction. That is, the specific copolymer having both unit 1 and unit 2 has both the function of bonding with a linker through a click reaction and the function of changing its solubility in water upon heating (specifically, the solubility decreases and the unit aggregates).

[0064] On the other hand, the linker represented by formula 3 described below has an azide group and an active ester group in the molecule. An active ester refers to a carboxylic acid derivative that can react with an amino group. The linker has an N-hydroxysuccinimide group as the active ester group. Because the linker has the N-hydroxysuccinimide group, it can bond with an amino group in an antibody to form a conjugate (antibody-linker conjugate).

[0065] In this step, in a mixed solution containing the specific copolymer having the above characteristics and the antibody-linker conjugate, the click reaction site of the specific copolymer reacts with the azide group of the antibody-linker conjugate to produce an antibody-copolymer conjugate.

[0066] (Specific Copolymer) The specific copolymer is a copolymer containing a repeating unit (unit 1) represented by the following formula 1 and a repeating unit (unit 2) represented by the following formula 2.

[0067]

[0068] Unit 1 A polymer constituted solely by unit 1 exhibits temperature responsiveness in water, with an LCST (Lower Critical Solution Temperature) of 32°C. By including unit 1, the specific copolymer has temperature responsiveness. Specifically, its solubility in water changes with temperature.

[0069] The content of unit 1 contained in the specific copolymer is not particularly limited, and is typically preferably 1 to 99 mol% when the total amount of repeating units is 100 mol%. In particular, from the viewpoint of the specific copolymer having more sensitive temperature responsiveness and / or the ease of controlling the LCST within a temperature range that is easy to handle in nucleic acid extraction operations (specifically, the LCST is likely to be around room temperature to 40°C), the content of unit 1 in the specific copolymer is preferably more than 50 mol%, more preferably 60 mol% or more, and preferably 97 mol% or less.

[0070] The content of unit 1 in the specific copolymer is preferably 1 to 99 mol%, more than 50 mol% and 99 mol% or less, 60 to 99 mol%, 1 to 97 mol%, more than 50 mol% and 97 mol% or less, or 60 to 97 mol%.

[0071] In formula 1, X １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, and from the viewpoint of providing the specific copolymer with more sensitive temperature responsiveness, a hydrogen atom or a linear alkyl group having 1 to 4 carbon atoms is preferred, and a hydrogen atom or a methyl group is more preferred.

[0072] Although not particularly limited, unit 1 is preferably a unit based on a monomer represented by the following formula 1'.

[0073]

[0074] In formula 1', X １ is X in formula 1 １ The monomer represented by formula 1′ may be synthesized by a known method, for example, a method described in the synthesis examples below, or a commercially available product may be used.

[0075] Unit 2 The specific copolymer has unit 2 represented by formula 2. Unit 2 has one moiety (click reaction site) containing a cyclic alkyne (alkynylene group) per repeating unit, and can easily form a conjugate by binding to an azide group in the linker (described later) via a click reaction. By pre-binding the linker and antibody, the protein can be immobilized to the specific copolymer via the linker. In other words, an antibody-copolymer conjugate can be prepared. The prepared conjugate undergoes aggregation and precipitation at temperatures at or above the LCST due to the temperature responsiveness inherent in the specific copolymer. This can be utilized to concentrate the corresponding antigen and extract and concentrate the nucleic acid released as a result.

[0076] The content of unit 2 in the specific copolymer is not particularly limited, but is typically preferably 1 to 99 mol % when the total amount of repeating units in the specific copolymer is 100 mol %. In particular, in terms of obtaining better effects of the present invention, the content is preferably 1.0 mol % or more, more preferably 2.0 mol % or more, and is preferably 30 mol % or less, more preferably 20 mol % or less.

[0077] The content of unit 2 in the specific copolymer is preferably 1.0 to 99.0 mol%, 2.0 to 99.0 mol%, 1.0 to 30.0 mol%, 2.0 to 30.0 mol%, 1.0 to 20.0 mol%, or 2.0 to 20.0 mol%.

[0078] Unit 2 typically has a higher hydrophobicity than unit 1 or other repeating units described below. Therefore, by increasing the content of unit 2 in the specific polymer, the LCST can be lowered. Furthermore, the higher the content of unit 2, the easier it is to introduce an antibody. On the other hand, from the viewpoint of adjusting the LCST to 20 to 40°C, the content of unit 2 is preferably 1.0 mol% or more, more preferably 2.0 mol% or more, and preferably 30 mol% or less, and more preferably 20 mol% or less. From the viewpoint of adjusting the LCST to 20 to 40°C, the content of unit 2 is preferably 1.0 to 30.0 mol%, 1.0 to 20.0 mol%, 2.0 to 30.0 mol%, or 2.0 to 20.0 mol%.

[0079]

[0080] In formula 2, X ２ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, and from the viewpoint of providing the specific copolymer with more sensitive temperature responsiveness, a hydrogen atom or a linear alkyl group having 1 to 4 carbon atoms is preferred, and a hydrogen atom or a methyl group is more preferred.

[0081] In formula 2, L ２ represents a divalent group. The divalent group is not particularly limited, but includes —O—, —S—, —C(O)—, —C(O)O—, —OC(O)O—, —NR Ａ - (R Ａ is a hydrogen atom or a monovalent substituent), linear, branched or cyclic aliphatic hydrocarbon groups having 1 to 20 carbon atoms, monocyclic or fused ring aromatic hydrocarbon groups having 6 to 20 carbon atoms, and groups formed by combining these groups. Among these, from the viewpoint of obtaining a copolymer having the excellent effects of the present invention, -O-, -C(O)-, -NR Ａ More preferred are —O—, —C(O)—, —NR Ａ At least one group selected from the group consisting of - and linear alkylene groups having 1 to 10 carbon atoms is more preferred, and -O-, -C(O)-, or -NH- is particularly preferred.

[0082] In formula 2, R １ represents a hydrogen atom, a halogen atom, -OR ５ , -NO ２ , -CN, -S(O) ２ R ５ , an alkyl group having 1 to 24 carbon atoms, an alkenyl group having 2 to 24 carbon atoms, and a (hetero)aryl group having 6 to 24 carbon atoms; １ may be bonded to each other to form a ring, and R ５ represents a group selected from the group consisting of hydrogen, a halogen atom, an alkyl group having 1 to 24 carbon atoms, and a (hetero)aryl group having 6 to 24 carbon atoms. １may be the same or different. In Formula 2, Z is C(R １ ) ２ ,O,S,and ,NR １ In addition, Z represents a group selected from the group consisting of L ２ When a divalent group represented by the formula: １ ) ２ , or NR １ More specifically, ═C(R １ ) R Ｘ -L ２ - or =N-R ｘ -L ２ -, where R Ｘ represents a single bond, an alkylene group (having 1 to 24 carbon atoms), an alkenylene group (having 2 to 24 carbon atoms), an alkynylene group (having 2 to 24 carbon atoms), or an arylene group (having 6 to 24 carbon atoms) which may have a heteroatom. In Formula 2, a' is an integer of 0 to 8, a" is an integer of 0 to 8, and the sum of a' and a" is less than 10.

[0083] In order to obtain a more excellent effect of the present invention, the unit 2 is represented by the following formula 2 ａ The unit represented by the following formula is preferred.

[0084]

[0085] Formula 2 ａ Medium, X ２ , L ２ , R １ , a', and a" have the same meanings as the symbols in Formula 2, and the preferred embodiments are also the same. Formula 2 ａ In the middle, Z is CR １ or a nitrogen atom, ２ is a number equal to or less than a'+a". １ is R in formula 2 １ The same applies to the preferred embodiments.

[0086] Unit 2 is L ２ When the bonding site is marked with "*", it is preferable that the compound has a structure (click reaction site) represented by the following formula C.

[0087]

[0088] Unit 2 preferably has a structure represented by the above formula C as the bonding site (monovalent group) at the position of "*". Note that * in the monovalent group represented by formula C bonds to the wavy line portion in the following formula.

[0089] In the above formula, X ２ , and L ２ has the same meaning as the group represented by the same symbol in formula (2), and the preferred embodiments are also the same.

[0090] From the viewpoint of obtaining a copolymer having a more excellent effect of the present invention, the unit 2 is preferably at least one selected from the group consisting of units represented by the following formulas: ２１ represents a divalent group, and L in Formula 2 ２ The same applies to the preferred embodiments. ２ is X in formula 2 ２ The same applies to the preferred embodiments.

[0091]

[0092] Among these, from the viewpoint of obtaining a copolymer having more excellent effects of the present invention, the unit 2 is preferably the unit 4 or the unit 5 represented by the following formula 4 or 5.

[0093]

[0094] In Formula 4 and Formula 5, X ２ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; ４ is -O-, -S-, and -NR Ｂ -, and R Ｂ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, n represents an integer of 1 to 10, and in formula 5, L ２ represents a divalent group, and a preferred embodiment is ２ is the same as the divalent group represented by the formula:

[0095] The method for synthesizing unit 2 is not particularly limited, and any known synthesis method can be used. However, a method for obtaining unit 2 by binding a precursor compound to unit 2′ represented by the following formula 2′ is preferred, as it allows unit 2 to be obtained more easily.

[0096]

[0097] In formula 2', R Ｃ is a reactive substituent, and specific examples thereof include a hydroxy group, an amino group, a carboxy group, a glycidyl group, an epoxy group, a glycidyl ether group, a mercapto group, a hydroxysuccinimide ester, and a maleimide group, with a hydroxy group being preferred. ２ is X in Equations 4 and 5 ２ is synonymous with.

[0098] The precursor compound may be, for example, a compound having a click reaction site and the above R Ｃ A compound having a group capable of reacting with the above-mentioned formula C and a carboxy group can be used, and a commercially available product can be used, or a compound synthesized by a known method can be used. In particular, the specific copolymer can be synthesized more easily by using a precursor compound having a structure represented by the formula C (click reaction site) and a carboxy group.

[0099] As a synthesis method for the precursor compound, for example, if the click reaction site is a difluorinated cyclooctyne, the methods described in Schemes 1 and 2 of J. Am. Chem. Soc. 2008, 130, 34, 11486-11493 can be used. Furthermore, if the click reaction site is a dibenzoazacyclooctyne, the method described in Scheme 1 of Chemical Communications (2010), 46(1), 97-99 can be used.

[0100] In addition, compounds in which a substituent such as an amino group, a hydroxy group, or a carboxy group is added to the click reaction site such as BCN (bicyclo[6.1.0]nonyne) or DBCO (dibenzocyclooctyne), or compounds in which the click reaction site is maleimide- or hydroxysuccinimide-esterified, are commercially available, and these can also be used as precursor compounds.

[0101] Among these, the unit 2′ is preferably the unit 6 represented by the following formula 6, in that the unit 2 can be obtained more easily. In this case, the precursor compound may typically be a precursor compound having a carboxy group and a structure represented by formula C.

[0102]

[0103] When synthesizing the specific copolymer, if unreacted unit 6 exists, in other words, if the specific copolymer contains unit 1, unit 2, and unit 6, the LCST can be adjusted to a higher temperature. Since unit 6 has higher hydrophilicity than other repeating units, the LCST can be adjusted to a higher temperature by increasing the content of unit 6.

[0104] In the above formula 6, X ６ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, and from the viewpoint of providing the copolymer with more sensitive temperature responsiveness, a hydrogen atom or a linear alkyl group having 1 to 4 carbon atoms is preferred, and a hydrogen atom or a methyl group is more preferred.

[0105] The content of unit 6 in the specific copolymer is not particularly limited, but is preferably 0 to 15 mol % when the total amount of all repeating units in the specific copolymer is taken as 100 mol %.

[0106] The molecular weight of the specific copolymer is not particularly limited, but typically, the number average molecular weight is preferably 2,000 to 100,000, more preferably 5,000 to 50,000, and even more preferably 10,000 to 30,000. When the number average molecular weight is 5,000 or more, better concentration efficiency is likely to be obtained (in other words, better temperature responsiveness is likely to be obtained), while when it is 50,000 or less, a conjugate with an antibody is likely to be formed, or an antigen-antibody reaction is likely to proceed.

[0107] In particular, in terms of obtaining a better concentration efficiency when an antibody-copolymer conjugate prepared from the obtained specific copolymer is used, the number-average molecular weight is preferably 15,000 or more, and more preferably 20,000 or more. In general, antibodies are often highly hydrophilic, and in such cases, a copolymer with better concentration efficiency is required. When the number-average molecular weight of the copolymer is within the above numerical range, a better concentration efficiency is likely to be obtained.

[0108] From the above, the molecular weight of the specific polymer is preferably 2,000 to 100,000, 5,000 to 50,000, 10,000 to 30,000, 15,000 to 100,000, 15,000 to 50,000, 15,000 to 30,000, 20,000 to 100,000, 20,000 to 50,000, or 20,000 to 30,000.

[0109] (Other Units) The specific copolymer may have repeating units other than those described above. Examples of repeating units other than those described above include repeating units based on N-cyclopropylacrylamide (LCST = 46°C), N-n-propylacrylamide (LCST = 22°C), N-tetrahydrofurfuryl acrylamide (LCST = 28°C), N-ethoxyethylacrylamide (LCST = 35°C), N-methyl-N-ethylacrylamide (LCST = 56°C), N-methyl-N-isopropylacrylamide (LCST = 23°C), N-methyl-N-n-propylacrylamide (LCST = 20°C), N,N-diethylacrylamide (LCST = 32°C), N-cyclopropylmethacrylamide (LCST = 59°C), N-isopropylmethacrylamide (LCST = 44°C), N-n-propylmethacrylamide (LCST = 28°C), and N-tetrahydrofurfurylmethacrylamide (LCST = 35°C).

[0110] (Method for Producing Specific Copolymer) The method for producing the specific copolymer is not particularly limited, but from the viewpoint of more simply producing the specific copolymer, it is preferable to have the following steps (1) and (2) in this order.

[0111] Step (1): A step of copolymerizing monomers represented by the following formula 1' and formula 3' to obtain a copolymer (precursor of the specific copolymer).

[0112]

[0113] In the above formulas 1' and 3', X １ , and X ２ are X in formula 1, respectively. １ , and X in formula 2′ ２ The same applies to the preferred embodiments.

[0114] The method for copolymerizing the above-mentioned monomers is not particularly limited, and it is preferable to use a living polymerization method such as a living radical polymerization method, a living anionic polymerization method, or a living cationic polymerization method. Among them, the living radical polymerization method is preferred from the viewpoint of more easily obtaining a copolymer (or a precursor thereof).

[0115] Living radical polymerization is based on the rapid establishment of equilibrium between a small amount of growing radical (free radical) species and a large amount of dormant species in a propagation reaction through the action of heat, light, and metal catalysts, and various forms of living radical polymerization have been proposed.

[0116] For example, there are the ATRP method (atom transfer radical polymerization method) using an alkyl halide as a dormant, the RAFT method (reversible addition fragmentation chain transfer method) using a thioester, and the NMP method (nitroxide mediated polymerization method) using an alkoxyamine.

[0117] The RAFT method is a method in which a chain transfer agent having a high chain transfer constant, called a RAFT agent, is added to a conventional radical polymerization system to polymerize a vinyl monomer. Thioesters can be used as the RAFT agent.

[0118] The amount of the RAFT agent can be appropriately selected depending on the molecular weight of the target copolymer. That is, since the RAFT agent is bonded to the terminal of each copolymer, for example, when a 100-mer copolymer is the target, the RAFT agent may be used in an amount of 0.1 to 3 mol % relative to 100 mol % of the monomer.

[0119] The radical polymerization initiator used in the RAFT polymerization is not particularly limited, and may be appropriately selected from known initiators such as azo compounds, peroxides, and redox-type initiators.

[0120] Examples of azo compounds include 2,2'-azobisisobutyronitrile (AIBN), 2,2'-azobis2,4-dimethylvaleronitrile, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, and 4,4'-azobis(4-cyanovaleric acid).

[0121] Generally, the amount of polymerization initiator is preferably 0.1 to 50 mol% per mole of the RAFT agent. The reaction temperature in the RAFT method is determined by the radical polymerization initiator used, but is generally between 40°C and 150°C. Polymerization is often carried out under atmospheric pressure, but polymerization under pressure is also possible.

[0122] The RAFT method can be carried out in the absence of a solvent, but can also be carried out in the presence of a solvent. The solvent to be used as needed is not particularly limited, and known solvents can be used. The reaction can also be carried out in water, and the reaction can also proceed by emulsion polymerization. The emulsifier used in this case can be a nonionic emulsifier, a cationic emulsifier, or an anionic emulsifier that can be used in general emulsion polymerization.

[0123] Step (2): A step of reacting the obtained copolymer (precursor thereof) with a precursor compound represented by formula 10 to obtain a specific copolymer.

[0124]

[0125] In Formula 10, Z is a group containing a click reaction site (for example, a cyclic alkyne), and is preferably a group selected from the groups represented by Formula C already described above. １０ The bonding position of L １０represents a divalent group, and L in Formula 2 ２ The same applies to the preferred embodiments.

[0126] The desired specific copolymer is synthesized by forming (condensing) an ester bond between the hydroxy group of the copolymer (precursor) and the carboxy group of the precursor compound represented by formula 10.

[0127] The method for forming the ester bond is not particularly limited, and examples thereof include a method in which a copolymer (precursor) and a precursor compound are subjected to a condensation reaction in the presence of a condensing agent, a catalyst, and a solvent at 0 to 150°C (preferably 0 to 100°C) for 30 minutes to 24 hours.

[0128] Examples of condensing agents that can be used include triphenyl phosphite, N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-carbonyldiimidazole, dimethoxy-1,3,5-triazinylmethylmorpholinium, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and (2,3-dihydro-2-thioxo-3-benzoxazolyl)diphenyl phosphonate. Of these, N,N'-dicyclohexylcarbodiimide is preferred. In this case, N,N-dimethyl-4-aminopyridine or the like can be used as the catalyst, and dichloromethane or the like can be used as the solvent.

[0129] (Antibody-Linker Conjugate) The antibody-linker conjugate is a compound obtained by bonding an antibody that binds to an analyte and a linker represented by the following formula 3 via an amide bond.

[0130]

[0131] In formula 3, L ３represents a divalent hydrocarbon group which may have a heteroatom. The divalent hydrocarbon group is not particularly limited, but is preferably a (poly)oxyalkylene group (the alkylene group preferably has 1 to 6 carbon atoms) or a linear, branched, or cyclic hydrocarbon group having 1 to 20 carbon atoms, and more preferably a polyoxyalkylene group. The number of repeating units in the polyoxyalkylene group is preferably 2 to 10, and more preferably 2 to 8.

[0132] The linker represented by formula 3 has an N-hydroxysuccinimidyl ester (NHS ester) and can form an amide bond with a primary amine (e.g., a lysine residue) of a protein. ２ On the other hand, the linker has an azide group, which bonds with an alkynylene group (click reaction site) of the specific copolymer to form a triazole ring.

[0133] The method for preparing the antibody-linker conjugate is not particularly limited, but may be such that the linker represented by formula 3 is dissolved in an organic solvent or the like (for example, an aprotic polar solvent such as dimethyl sulfoxide), and the resulting solution is added to a solution in which the antibody is dispersed in a buffer solution (for example, a carbonate buffer solution) containing a buffering agent as needed. The reaction temperature is not particularly limited, but is preferably 1 to 20°C, and more preferably 1 to 10°C. The reaction time is not particularly limited, but is preferably 1 to 24 hours.

[0134] The ratio of antibody to linker added during production of the antibody-linker conjugate is not particularly limited, but the ratio of the amount of active ester groups in the linker to the amount of amino groups in the antibody (molar ratio, active ester groups / amino groups) based on the amount of substance is preferably 0.1 to 500. Furthermore, the amount of linker added per mole of antibody is not particularly limited, but is preferably 0.01 to 5,000 moles, more preferably 1 to 2,000 moles, and even more preferably 50 to 200 moles.

[0135] The antibody used is not particularly limited as long as it binds to the analyte (or a protein contained in the analyte) and has a primary amino group. For example, when the analyte is the SARS-CoV-2 virus, the antibody may be directed against the S (Spike) protein or the N (Nucleocapsid) protein. Furthermore, the antibody may be any of a monoclonal antibody, a polyclonal antibody, a multispecific antibody, and an antibody fragment.

[0136] (Antibody-Copolymer Conjugate) An antibody-copolymer conjugate is formed by bonding a click reaction site of a specific copolymer with an azide group of an antibody-linker complex to form a triazole ring structure. Examples of antibody-copolymer conjugates include compounds having repeating units represented by the following formulas 1 and 7.

[0137]

[0138] In formula 1, X １ is the X of the specific copolymer. １ The same applies to the preferred embodiments. ２ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; ２ The same applies to the preferred embodiments. ２ represents a divalent group, and L in Formula 2 ２ The same applies to the preferred embodiments. ３ represents a divalent hydrocarbon group which may have a heteroatom, and L in Formula 3 ３ The same applies to the preferred embodiments. Furthermore, Ab represents an antibody residue. That is, it represents a state in which an amide bond is formed between the primary amine of the antibody and the NHS ester of the linker, and the antibody is immobilized to a specific copolymer via the linker.

[0139] In the above-mentioned form, the antibody and unit 7 are bound in a one-to-one relationship; however, the antibody-copolymer conjugate is not limited to the above-mentioned form, and may have a structure in which multiple primary amines of the antibody are bound to alkynylene groups of the specific copolymer via linkers, i.e., a crosslinked structure centered on the antibody molecule.

[0140] Furthermore, the antibody-copolymer conjugate may contain unit 2 represented by formula 2 in addition to the above repeating units. That is, it may contain unreacted unit 2. In one embodiment, since the molecules of the specific copolymer are smaller than the molecules of the antibody, even if the specific copolymer has multiple binding sites, it may be difficult for the antibody to bind to each of the binding sites on the specific copolymer. In this case, unreacted unit 2 may remain in the antibody-copolymer conjugate.

[0141] The ratio of the antibody to the specific copolymer is not particularly limited, but in one embodiment, it is preferable that the ratio be 0.1 to 50 moles of the specific copolymer per mole of the antibody.

[0142] Furthermore, the antibody-copolymer conjugate may contain other units (units 5) that the specific copolymer may contain, and the preferred range of the content of each unit is the same as the preferred range of the corresponding unit in the specific copolymer.

[0143] The antibody is not particularly limited and any known antibody can be used, but among them, an anti-SARS-CoV-2 antibody is preferred, and for example, an anti-SARS-CoV-2 (COVID-19) nucleocapsid (protein) antibody and an anti-SARS-CoV-2 spike (protein) antibody are more preferred. The molar content ratio of the specific copolymer content to the antibody content in the antibody-copolymer conjugate is not particularly limited, but is preferably 0.1 to 100.0, 0.5 to 70.0, 0.5 to 50.0, 0.5 to 30.0, 1.0 to 50.0, 1.0 to 40.0, 2.0 to 100.0, 2.0 to 50.0, 5.0 to 100.0, 5.0 to 50.0, 10.0 to 100.0, 10.0 to 50.0, or 10.0 to 30.0.

[0144] The method for preparing the antibody-copolymer conjugate is not particularly limited, but may involve mixing and stirring the specific copolymer and the antibody-linker complex in water containing a buffer, if necessary. The reaction temperature is not particularly limited, but is preferably 1 to 20°C, more preferably 4 to 10°C. The reaction time is not particularly limited, but is preferably 1 to 24 hours.

[0145] Returning to the flow of Figure 1, the method for preparing the mixed solution in step S1 is not particularly limited, and examples include a method of mixing a biological sample, an antibody-linker conjugate, and a specific copolymer. When the biological sample is in liquid form, other components may be added to the biological sample. The order in which the antibody-linker conjugate and the specific copolymer are added is not particularly limited, and the antibody-linker conjugate and the specific copolymer may be added after the antibody-linker conjugate, or the specific copolymer may be added after the antibody-linker conjugate, or the antibody-linker conjugate and the specific copolymer may be added at the same time.

[0146] The amount of antibody-specific copolymer conjugate added to a biological sample is not particularly limited, but for example, the amount of antibody-specific copolymer conjugate added per 1 mg of specimen (antigen protein) is preferably 0.1 mg or more, more preferably 0.5 mg or more, and even more preferably 1.0 mg or more. There is no particular upper limit, but generally, 5.0 mg or less is preferred. More specifically, the amount of antibody-specific copolymer conjugate added per mL of specimen-containing liquid medium (e.g., including saliva) is preferably 0.1 to 10.0 mg.

[0147] As described above, the amount of antibody-specific copolymer conjugate added to a biological sample is preferably 0.1 to 5.0 mg, 0.5 to 5.0 mg, or 1.0 to 5.0 mg per 1 mg of specimen (antigen protein).

[0148] At this time, a specific copolymer in a free state (not conjugated with an antibody) may be further added to the mixture. Addition of the specific copolymer makes the response to heat more sensitive, and allows the sample to be concentrated more efficiently. The amount of the specific copolymer added is not particularly limited, but is preferably 0.1 to 20 times the molar content of the specific copolymer contained in the antibody-copolymer conjugate, where the molar content is taken as 1.

[0149] In this step, the analyte contained in the mixture is not particularly limited, but may be provided from a biological sample. In one embodiment, the biological sample containing the analyte may be added directly to the mixture. Generally, biological samples contain many contaminants. In general, nucleic acid extraction often involves treatment with protease or the like to inactivate or digest these contaminants. However, in the present nucleic acid extraction method, even in the absence of protease, the analyte is captured by the antibody-copolymer conjugate and can be easily separated from the contaminants in step S2 described below. Therefore, nucleic acid extraction can be performed with sufficient efficiency without treatment with protease or the like.

[0150] <Step S2> Returning to the flow diagram of Figure 1, next, in step S2, the mixture containing the specimen and the antibody-copolymer conjugate obtained in step S1 is heated to aggregate the antibody-copolymer conjugate that has captured the specimen, thereby extracting the nucleic acid contained in the specimen. At this time, it is not necessary to add a carrier nucleic acid, but it may be added. The heating temperature may be adjusted appropriately depending on the LCST of the specific copolymer, but a temperature of 20 to 40°C is preferred.

[0151] Typical procedures for extracting nucleic acids contained in a specimen include lysis of the tissue with proteolytic enzymes, inactivation of unnecessary enzymes, washing, filtering, and fractionation of components. Nucleic acid extraction for PCR testing of SARS-CoV-2 requires multiple steps, and the process typically takes approximately 90 minutes. Furthermore, the use of various reagents tends to increase the cost required for a single extraction procedure.

[0152] On the other hand, the present inventors have surprisingly found that the present nucleic acid extraction method can extract nucleic acids contained in a sample even in the absence of protease, and even without the use of carrier nucleic acid. Although the mechanism behind this is not entirely clear, it is presumed that the sample is captured by the interaction between the antibody and the specific copolymer conjugate, and then agglutinated, thereby simultaneously separating contaminants and concentrating the sample.

[0153] This nucleic acid extraction method allows for the extraction of nucleic acids contained in a sample with a simple procedure. Specifically, the antibody-specific copolymer conjugate that has captured the sample is agglutinated by heating, and the solids are collected by centrifugation or other methods, thereby simultaneously removing contaminants and concentrating the sample. The nucleic acids contained in the sample can then be released from the collected solids using a known method. Examples of such methods include, but are not limited to, a method using a nucleic acid-binding solid carrier such as silica particles and a chaotropic agent ( J. Clin. Microbiol., Vol. 28 No. 3, pp. 495-503 (1990)). Specifically, this method involves adsorbing nucleic acids to a nucleic acid-binding solid carrier in the presence of a chaotropic agent, washing the carrier with a wash solution, and then eluting the nucleic acids from the carrier using water or a low-salt buffer solution. These methods are known to those skilled in the art, and commercially available kits can also be used. Nucleic acids extracted by this nucleic acid extraction method can be subjected to nucleic acid amplification.

[0154] Methods for amplifying DNA extracted from a sample include methods that involve a thermal denaturation step of double-stranded DNA, such as PCR (Polymerase chain reaction) and LCR (Ligase chain reaction); LAMP (Loop-mediated isothermal amplification), SDA (Strand displacement amplification), ICAN (Isothermal and chimeric primer-initiated amplification of nucleic acids), SMAP (Smart amplification process), and 3SR (Self-sustained amplification). For example, an isothermal amplification method that does not include a heat denaturation step of double-stranded DNA, such as RT-PCR (RT-PCR sequence replication), can be used.

[0155] Methods that can be used to amplify RNA extracted from a sample include TMA (Transcription Mediated Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), and TRCR (Transcription-reverse transcription concentrated reaction).

[0156] [Nucleic Acid Amplification Method] An embodiment of the nucleic acid amplification method includes extracting nucleic acid from a specimen using the nucleic acid extraction method described above, and amplifying the extracted nucleic acid using polymerase chain reaction (PCR). Methods for amplifying DNA by PCR are known, and can be performed by referring to Science 239, 487-491 (1988), etc.

[0157] When the specimen is an RNA virus or the like, a reverse transcription reaction can be performed to synthesize DNA complementary to the extracted RNA before amplifying the DNA using PCR. Methods for reverse transcription are known, and the reaction temperature is a temperature at which the thermostable reverse transcriptase exhibits activity, preferably 35 to 90°C in one embodiment. The time for the reverse transcription reaction can be appropriately set taking into account the length of the cDNA to be synthesized, etc.

[0158] The RNA extraction from the sample, the reverse transcription reaction, and the target nucleic acid amplification reaction may be performed sequentially or continuously (in one step). When the reverse transcription reaction and the target nucleic acid amplification reaction are performed sequentially, it is preferable to use a heat-stable reverse transcriptase and a heat-stable DNA polymerase. As the heat-stable DNA polymerase, various polymerases that can be used in PCR can be used. Alternatively, a heat-stable DNA polymerase having heat-stable reverse transcriptase activity can also be used.

[0159] A primer pair for amplifying a target region of cDNA can also be used. In this case, one of the primers may also be used as a reverse transcription primer in the reverse transcription reaction. Furthermore, if there are multiple target nucleic acid regions, multiple primer pairs may be used in combination. According to this nucleic acid amplification method, nucleic acid extraction can be performed with a simple procedure without using protease, thereby eliminating bottlenecks and enabling rapid nucleic acid amplification.

[0160] [Nucleic Acid Extraction Kit] An embodiment of the nucleic acid extraction kit is a nucleic acid extraction kit used to extract nucleic acids from a sample in the absence of a protease, and includes a first agent containing a specific copolymer and a second agent containing an antibody-linker conjugate. The first agent is configured, for example, to include a container and the specific polymer contained in the container, and may be a solid (e.g., powder) containing the specific polymer, or may be a liquid. When the first agent is liquid, it may be in a form containing the specific polymer and water (aqueous solution containing a buffering agent).

[0161] The second agent may, for example, comprise a container and the antibody-linker conjugate contained in the container. The second agent may be in a liquid form, and may be in a form containing the antibody-linker conjugate and a buffer solution (e.g., phosphate-buffered saline, PBS). The content of the antibody-linker conjugate in the second agent is not particularly limited, but is preferably 0.01 to 10,000 μg / mL.

[0162] An antibody-copolymer conjugate can be produced by mixing the first and second agents. The mixing ratio of the first and second agents is not particularly limited, but is preferably adjusted so that the ratio of the molar content of the specific copolymer contained in the first agent to the molar content of the antibody (bound to a linker) contained in the second agent (antibody / specific copolymer) is 1 to 200, more preferably 10 to 50. The amounts of each agent may be adjusted so that the antibody / specific copolymer ratio falls within the above-mentioned numerical range when the entire amounts of the first and second agents contained in the nucleic acid extraction kit are mixed.

[0163] This nucleic acid extraction kit allows nucleic acids contained in a sample to be concentrated and extracted by preparing a mixture containing the first and second agents and a sample, generating an antibody-copolymer conjugate, and then heating the mixture. For example, if the sample is an RNA virus, the first and second agents are added to saliva or other samples containing the RNA virus, allowing the reaction to occur. Heating the mixture causes the RNA virus captured by the antibody-copolymer conjugate to aggregate and precipitate, along with the antibody-copolymer conjugate. Solid-liquid separation removes contaminants (liquid phase) and concentrates the sample. RNA is then extracted from the concentrated sample. This nucleic acid extraction kit allows nucleic acids to be extracted in the absence of protease and with simple procedures. Furthermore, even from biological samples containing only a small amount of the sample (e.g., RNA virus), the antibody-copolymer conjugate's ability to capture and aggregate the sample allows for efficient nucleic acid extraction.

[0164] [PCR Test Kit] As long as the PCR test kit includes the nucleic acid extraction kit, other components are not particularly limited. For example, if the sample is an RNA virus, other components may include a reverse transcriptase for cDNA synthesis, a probe for real-time detection, an intercalator, a buffer, deoxyribonucleotides such as dNTP, a surfactant, salts, and a primer. A plurality of these components may be mixed and provided as a premix.

[0165] This PCR test kit allows nucleic acids to be easily extracted from specimens contained in biological samples, etc., in the absence of protease, and then the extracted nucleic acids can be amplified. By using this PCR test kit, the time and cost required for PCR testing can be significantly reduced.

[0166] The following non-limiting examples will further illustrate the embodiments.

[0167] [Synthesis Example of Specific Copolymer] Synthesis of HIPAAm For the synthesis of the specific copolymer, "HIPAAm" was synthesized as a preparation according to the following procedure. "HIPAAm" is an abbreviation derived from "Hydroxy IsoPropyl AcrylAmide".

[0168] D,L-2-amino-1-propanol (0.15 mol) and triethylamine (0.15 mol) were thoroughly dissolved in anhydrous chloroform and stirred at 5°C for 20 minutes, after which acryloyl chloride (0.15 mol) was slowly added and the mixture was stirred at 5°C for 2 hours.

[0169] After evaporating the solvent, the residue was redissolved in 2-propanol and kept at -20°C for 24 hours or more. Finally, salts were removed by filtration, and the residue was concentrated and purified by column chromatography. The synthesis of HIPAAm was confirmed by thin layer chromatography (TLC) and １ H NMR (Nuclear Magnetic Resonance) (solvent: D ２ O) confirmed.

[0170]

[0171] Synthesis of P(NIPAAm-co-HIPAAm) As a polymer that serves as a precursor compound for the specific copolymer, "P(NIPAAm-co-HIPAAm)" was synthesized by the following procedure. P(NIPAAm-co-HIPAAm) was synthesized by RAFT polymerization of HIPAAm and NIPAAm (see the scheme below for the structure), as shown in the scheme below. A solution containing 1.89 g of NIPAAm, 0.11 g of HIPAAm, 1.31 mg of AIBN, 12.7 mg of CDT (Cyanomethyl Dodecyl Trithiocarbonate), and 17.6 ml of ethanol was stirred at 20°C for 20 hours, evaporated, and dried in vacuo.

[0172]

[0173] Synthesis of P(NIPAAm-co-HIPAAm-co-SAKIPAAm) P(NIPAAm-co-HIPAAm-co-SAKIPAAm) was synthesized as a specific polymer. As shown in the scheme below, dibenzylcyclooctynoic acid (DBCO-Acid) having a click reaction site was introduced to the hydroxyl group of HIPAAm by dehydration condensation to obtain the clickable responsive polymer P(NIPAAm-co-HIPAAm-co-SAKIPAAm). A solution containing 30 ml of DCM (Dichloromethane), 35.8 mg of DBCO acid, 12 mg of DMAP (4-Dimethylaminopyridine), 100 mg of P(NIPAAm-co-HIPAAm), and 20 mg of DCC (N',N'-Dicyclohexylcarbodiimid) was stirred overnight, evaporated, and dried in vacuo.

[0174] The number average molecular weight of the resulting P(NIPAAm-co-HIPAAm-co-SAKIPAAm) was 2.01 × 10 ４ The LCST was about 30°C. １The content of repeating units determined by H-NMR was NIPAAm:HIPAAm:SAKIPAAm (both on a molar basis) = 96.4:1.2:2.4. The notation "NIPAAm" in P(NIPAAm-co-HIPAAm-co-SAKIPAAm) means a unit based on NIPAAm, and corresponds to the unit represented by formula 1. The same applies to the notation "HIPAAm", which corresponds to the unit represented by formula 6. The same applies to the notation "SAKIPAAm", which corresponds to the unit represented by formula 2.

[0175]

[0176] [Synthesis of antibody-linker conjugate] Azido-PEG4-NHS ester (Azido-ethylene glycol (EG4)-NHS ester, Tokyo Chemical Industry Co., Ltd.) powder was dissolved in DMSO (10 mg / ml, dimethyl sulfoxide). Furthermore, instead of the anti-COVID-19 monoclonal antibody (>95%, manufactured by MyBioSource, anti-COVID-19 antibody: anti-Viral COVID 19 Nucleocapsid (NP) Humanized Coronavirus Monoclonal Antibody), "SARS-CoV-2 Spike Protein (S1-NTD) Antibody #56996" (Cell Signaling Technology) was dispersed in carbonate buffer (pH 8.6) and stirred at 4°C for 5 hours to synthesize an antibody-linker conjugate. The antibody / linker ratio was 1:100.

[0177] Biological sample collection: Biological samples were collected from 325 patients recruited between October 2021 and April 2022 at the outpatient clinic of the Egyptian Liver Research and Teaching Hospital. These patients included 92 COVID-19 patients (50 men and 42 women). Nasopharyngeal swabs were collected from all participants and stored in the designated transport medium for transporting SARS-CoV-2 specimens at -80°C until testing.

[0178] The participation criteria for the above study were as follows: - Age 18 or older - Willingness and ability to provide written informed consent - Those who have tested positive for COVID-19, tested negative, or are suspected of being positive by PCR or antigen test The exclusion criteria were as follows: - Age under 18 - Those who do not agree to provide informed consent

[0179] The study protocol was approved by the Research Ethics Committee of the Egyptian Liver Research and Teaching Hospital, and the study protocol and conduct were in accordance with the CIOMS / WHO International Ethical Guidelines for Biomedical Research Involving Human Subjects. Geneva: CIOMS. 1993, as amended, and written informed consent was obtained from all patients.

[0180] Example 1 Viral RNA was extracted from biological samples in the absence of protease using the synthesized specific copolymer and antibody-linker conjugate. The procedure was as follows.

[0181] (Procedure 1) First, 900 μL of the sample was mixed with an antibody-linker conjugate (10 μg / mL, 100 μL PBS solution) and incubated at 37°C for 1 hour. Next, P(NIPAAm-co-HIPAAm-co-SAKIPAAm) (antibody:specific copolymer ratio = 1:30, Mn = 2.01 × 10 ４ The antibody-linker complex (g / mol) was conjugated to the antibody-linker complex via a click reaction at 4°C for 1 hour. The mixture was then transferred to a 2.0 mL microtube and centrifuged at 37°C and 13,800 x g for 5 minutes. The concentrated precipitate was collected and the supernatant was discarded. Next, the concentrated precipitate and 200 μL of PBS were added to a lysis tube (LT).

[0182] (Procedure 2) 250 μL of ethanol (96-100%) was then added to the mixture, which was then stirred (vortexed) for approximately 15 seconds, incubated at room temperature (15-25°C) for 5 minutes, and then centrifuged to remove droplets.

[0183] The lysate was then transferred to a QIAamp MinElute column and centrifuged at 6000 x g for at least 1 minute. The wash tube containing the filtrate was then discarded, and 500 μL of Buffer AW1 (Qiagen) was added, followed by centrifugation at 6000 x g for at least 1 minute. The wash tube containing the filtrate was then discarded, and 500 μL of Buffer AW2 (Qiagen) was added, followed by centrifugation at 6000 x g for at least 1 minute.

[0184] The wash tube containing the filtrate was then discarded, and 500 μL of ethanol (96-100%) was added, followed by centrifugation at 6000 × g for ≥ 1 min. The wash tube containing the filtrate was then discarded, and the QIAamp MinElute column was transferred to a clean 2 ml wash tube (WT).

[0185] The membrane was then dried by centrifugation at approximately 20,000 x g for 3 minutes. The QIAamp MinElute column was then transferred to a new 2 ml wash tube (WT). The lid was then opened and the column was incubated at 56°C for 3 minutes to completely dry the membrane. The QIAamp MinElute column was then transferred to an elution tube (ET), and the filtrate was discarded. 20-150 μL of Buffer AVE (Qiagen) was then added to the center of the membrane, and the column was incubated at room temperature for 5 minutes. The column was then centrifuged at approximately 20,000 x g for 1 minute or longer.

[0186] The RNA extracted by the above procedure was subjected to RT-PCR (PCR with Reverse Transcription) testing. Specifically, cDNA was synthesized using "SARS-CoV-2 / SARS-CoV Multiplex (manufactured by DNA-Technology)" and quantified using "DTlite Real-Time PCR (manufactured by DNA-Technology)." The thermal cycler program and other settings were all in accordance with the kit manual.

[0187] Comparative Example 1 Viral RNA was extracted from the same biological sample as in Example 1 using a protease, without using a specific copolymer or an antibody-linker conjugate. The detailed procedure was the same as that in Example 1, except that (Procedure 1) was as follows.

[0188] First, 25 μL of protease was added to a lysis tube (LT). Next, 200 μL of the sample was added to the lysis tube. 200 μL of "Buffer AL (Qiagen)" containing 28 μg / mL of carrier RNA was then added and stirred for approximately 15 seconds. Next, the mixture was incubated at 56°C in a heating block for 15 minutes, and then centrifuged to remove droplets. The subsequent procedure (Procedure 2) was the same as in Example 1.

[0189] [Reference Example 1] Using a specific copolymer, an antibody-linker conjugate, and further using a protease, viral RNA was extracted from the same biological sample as in Example 1. The detailed procedure was the same as in Example 1, except that (Procedure 1) was changed as follows.

[0190] First, 900 μL of the sample was mixed with the antibody-linker conjugate (10 μg / mL, 100 μL PBS solution) and incubated at 37°C for 1 hour. Next, P(NIPAAm-co-HIPAAm-co-SAKIPAAm) (antibody:specific copolymer ratio = 1:30, Mn = 2.01 × 10 ４ The antibody-linker complex (g / mol) was conjugated to the antibody-linker complex via a click reaction for 1 hour at 4°C. The mixture was then transferred to a 2.0 mL microtube and centrifuged at 37°C and 13,800 x g for 5 minutes. The concentrated precipitate was collected, and the supernatant was discarded.

[0191] Next, 25 μL of protease was added to a lysis tube (LT). 200 μL of the sample containing the concentrated precipitate was added to this. 200 μL of "Buffer AL (Qiagen)" containing 28 μg / mL of carrier RNA was then added and stirred for approximately 15 seconds. The mixture was then incubated at 56°C in a heating block for 15 minutes, after which the mixture was centrifuged to remove droplets. The subsequent procedure (Procedure 2) was the same as in Example 1.

[0192] [Results] Figure 2 shows the quantification results obtained by real-time PCR ("SLAN-96P Real-Time PCR System") using nucleic acids extracted by the method of Comparative Example 1 and the method of Reference Example 1. The results in Figure 2 show that the quantification results obtained by Reference Example 1, which used a specific copolymer and an antibody-linker conjugate, were approximately 1.5 times the quantification results obtained by Comparative Example 1, demonstrating that the specific copolymer and the antibody-linker conjugate concentrate the sample.

[0193] 3 shows the results of quantification by real-time PCR using nucleic acids extracted by the method of Comparative Example 1 and the method of Example 1. The quantification result of Example 1 was about 30 times that of Comparative Example 1. The results of FIG. 3 demonstrate that target nucleic acids can be detected with sufficient sensitivity without using protease and carrier nucleic acid.

Claims

1. In the absence of proteolytic enzymes, At least one specimen selected from the group consisting of cells, extracellular vesicles, and virions; A copolymer including a repeating unit represented by the following formula 1 and a repeating unit represented by the following formula 2: preparing a mixture containing an antibody capable of binding to the analyte, and an antibody-linker conjugate obtained by binding a linker represented by the following formula 3 via an amide bond; forming an antibody-copolymer conjugate; heating the mixture to aggregate the antibody-copolymer conjugate, and extracting the nucleic acid contained in the sample. 【Chemistry 1】 (In formula 1, X １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; ２ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; L ２ represents a divalent group, R １ is a hydrogen atom, a halogen atom, -OR ５ , -NO ２ , -CN, -S(O) ２ R ５ a C1-24 alkyl group, a C2-24 alkenyl group, and a C6-24 (hetero)aryl group; １ may be the same or different, and two or more of them may be bonded to each other to form a ring; R ５ is selected from the group consisting of a hydrogen atom, a halogen atom, an alkyl group having 1 to 24 carbon atoms, and a (hetero)aryl group having 6 to 24 carbon atoms; Z is C(R １ ) ２ , O, S, and NR １ wherein a' is an integer from 0 to 8, a" is an integer from 0 to 8, and the sum of a' and a" is less than 10. 【Chemistry 2】 (In formula 3, L ３ represents a divalent hydrocarbon group which may have a heteroatom.

2. 2. The nucleic acid extraction method according to claim 1, wherein the content of the repeating unit represented by formula 2 is 1.0 to 30.0 mol % when all repeating units of the copolymer are taken as 100 mol %.

3. The nucleic acid extraction method according to claim 1, wherein the repeating unit represented by formula 2 is at least one repeating unit selected from the group consisting of a repeating unit represented by the following formula 4 and a repeating unit represented by the following formula 5: 【Chemistry 3】 (In Formula 4 and Formula 5, X ２ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; ４ is -O-, -S-, and -NR ２ -, R ２ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, n represents an integer of 1 to 10, and in formula 5, L ２ represents a divalent group.

4. The nucleic acid extraction method according to claim 1 , wherein the copolymer further comprises a repeating unit represented by the following formula 6: 【Chemistry 4】 (In formula 6, X ６ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms.

5. 2. The nucleic acid extraction method according to claim 1, wherein the content of the repeating unit represented by formula 2 is 2.0 to 30.0 mol % when all repeating units of the copolymer are taken as 100 mol %.

6. 2. The nucleic acid extraction method according to claim 1, wherein the copolymer has a number average molecular weight of 5,000 to 50,000.

7. 2. The nucleic acid extraction method according to claim 1, wherein a molar ratio of a content of the copolymer to a content of the antibody contained in the antibody-linker complex in the mixed solution is 0.5 to 30.

0.

8. The nucleic acid extraction method according to claim 1, wherein the antibody-copolymer conjugate comprises a repeating unit represented by the following formula 1 and a repeating unit represented by the following formula 7: 【Chemistry 5】 (In formula 1, X １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; ２ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; L ２ represents a divalent group; L ３ represents a divalent hydrocarbon group which may have a heteroatom, and Ab represents a residue of the antibody.

9. 2. The nucleic acid extraction method according to claim 1, wherein the heating is performed by heating the mixture to a temperature of 20 to 40°C.

10. The nucleic acid extraction method according to claim 1 , wherein the specimen is a membrane structure having a lipid bilayer.

11. The nucleic acid extraction method according to claim 1 , wherein the specimen is an enveloped virus.

12. The nucleic acid extraction method according to claim 1, wherein the specimen is SARS-CoV-2 virus.

13. Extracting the nucleic acid from the sample using the nucleic acid extraction method according to any one of claims 1 to 12; and amplifying the extracted nucleic acid by polymerase chain reaction.

14. A nucleic acid extraction kit used for extracting nucleic acid from at least one specimen selected from the group consisting of cells, extracellular vesicles, and virions in the absence of protease, comprising: A first agent including a copolymer including a repeating unit represented by the following formula 1 and a repeating unit represented by the following formula 2, A nucleic acid extraction kit comprising: an antibody that binds to the sample; and a second agent containing an antibody-linker complex obtained by binding a linker represented by the following formula 3 via an amide bond: 【Chemistry 6】 (In formula 1, X １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; ２ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; L ２ represents a divalent group, R １ is a hydrogen atom, a halogen atom, -OR ５ , -NO ２ , -CN, -S(O) ２ R ５ a C1-24 alkyl group, a C2-24 alkenyl group, and a C6-24 (hetero)aryl group; １ may be the same or different, and two or more of them may be bonded to each other to form a ring; R ５ is selected from the group consisting of a hydrogen atom, a halogen atom, an alkyl group having 1 to 24 carbon atoms, and a (hetero)aryl group having 6 to 24 carbon atoms; Z is C(R １ ) ２ , O, S, and NR １ wherein a' is an integer from 0 to 8, a" is an integer from 0 to 8, and the sum of a' and a" is less than 10. 【Chemistry 7】 (In formula 3, L ３ represents a divalent hydrocarbon group which may have a heteroatom.

15. A PCR test kit comprising the nucleic acid extraction kit according to claim 14.

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