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

[Technical Field]

[0001] This disclosure relates to a nucleic acid extraction method, a nucleic acid amplification method, a nucleic acid extraction kit, and a PCR test kit. [Background technology]

[0002] Biological diagnostic, testing, and analytical procedures require the detection of nucleic acids contained in cells and / or virus particles within biological samples (e.g., swabs). Generally, such tests involve pretreatment, including the isolation, concentration, and purification of nucleic acids from the target substance (such as a virus) contained in the sample.

[0003] Nucleic acids in samples can form complex aggregates with impurities, and concentration and purification often require digestion of impurities using proteolytic enzymes and yield increase using "carrier nucleic acids" such as polyadenylic acid (hereinafter also referred to as "concentration"). This tendency was particularly pronounced when the sample concentration was low. Here, "sample" refers to cells and / or virus particles contained in a biological sample that have nucleic acids to be extracted.

[0004] For example, in PCR (Polymerase Chain Reaction) testing (hereinafter also referred to as "PCR testing") performed for the isolation and identification of SARS-CoV-2 infection, pretreatment with proteolytic enzymes and / or concentration of the sample and / or nucleic acid with carrier nucleic acids was sometimes performed as a pretreatment for reverse transcription and amplification of viral RNA (ribonucleic acid) (see, for example, Patent Document 1). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2004-215676 [Overview of the project] [Problems that the invention aims to solve]

[0006] The processing of samples with proteolytic enzymes and concentration by adding carrier nucleic acids (hereinafter also referred to as "processing with proteolytic enzymes, etc.") requires expensive reagents, complex experimental procedures, and a certain amount of time. Therefore, processing with proteolytic enzymes, etc. can become a bottleneck in PCR testing. Furthermore, the complicated inventory management of the various pre-treatment reagents used in processing with proteolytic enzymes, etc., and the high cost of these reagents, have contributed to increased testing costs.

[0007] The objective of this disclosure is to provide a nucleic acid extraction method that can easily extract nucleic acids contained in a sample even in the absence of proteolytic enzymes and without concentration using carrier nucleic acids. In other words, the objective is to provide a nucleic acid extraction method that allows for the purification and concentration of a sample for nucleic acid extraction in a single step. Furthermore, the objective of this disclosure is to provide a nucleic acid amplification method, a nucleic acid extraction kit, and a PCR test kit. [Means for solving the problem]

[0008] One embodiment of the nucleic acid extraction method of the present disclosure is a nucleic acid extraction method comprising: preparing a mixture containing at least one sample selected from the group consisting of cells, extracellular vesicles, and virions, an antibody-linker complex obtained by linking a repeating unit represented by the following formula 1, a copolymer containing a repeating unit represented by the following formula 2, an antibody bound to the sample, and a linker represented by the following formula 3 via an amide bond, in the absence of a protease, thereby generating an antibody-copolymer conjugate; and heating the mixture to agglutinate the antibody-copolymer conjugate and extract the nucleic acid contained in the sample. [ka]

[0009] In formula 1, X 1 represents a hydrogen atom, or a linear or branched alkyl group having 1 to 6 carbon atoms, in formula 2, X 2represents a hydrogen atom, or a linear or branched alkyl group having 1 to 6 carbon atoms, and L 2 represents a divalent group, and R 1 represents a hydrogen atom, a halogen atom, -OR 5 , -NO2, -CN, -S(O)2R 5 is selected from the group consisting of a hydrogen atom, a halogen atom, -OR, -NO2, -CN, -S(O)2R, 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, and a plurality of Rs 1 may be the same or different from each other, and two or more of them may be bonded to each other to form a ring, and 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 selected from the group consisting of C(R 1 )2, O, S, and NR 1 and 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] [Chemical formula] In Formula 3, L 3 represents a divalent hydrocarbon group that may have a heteroatom.

[0011] Also, one embodiment of the nucleic acid extraction kit of the present disclosure is a nucleic acid extraction kit used for extracting nucleic acids from at least one sample selected from the group consisting of cells, extracellular vesicles, and virions in the absence of a proteolytic enzyme, and includes a first agent containing a copolymer containing 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 sample and an antibody-linker complex obtained by binding a linker represented by the following Formula (3) via an amide bond. [Chemical formula]

[0012] In Formula 1, X 1represents a hydrogen atom, or a linear or branched alkyl group having 1 to 6 carbon atoms, in formula 2, X 2 L represents a hydrogen atom, or a linear or branched alkyl group having 1 to 6 carbon atoms. 2 represents a divalent group, R 1 This includes hydrogen atoms, halogen atoms, and -OR 5 -NO2, -CN, -S(O)2R 5 Multiple R groups are selected from the group consisting of alkyl groups with 1 to 24 carbon atoms, alkenyl groups with 2 to 24 carbon atoms, and (hetero)aryl groups with 6 to 24 carbon atoms. 1 These may be identical or different, and two or more of them may be bonded together to form a ring, R 5 Z is selected from the group consisting of hydrogen atoms, halogen atoms, alkyl groups having 1 to 24 carbon atoms, and (hetero)aryl groups having 6 to 24 carbon atoms, and Z is C(R 1 )2, O, S, and NR 1 A value is selected from the group consisting of the following: a' is an integer between 0 and 8, a'' is an integer between 0 and 8, and the sum of a' and a'' is less than 10.

[0013] [ka] In formula 3, L 3 This represents a divalent hydrocarbon group which may have a heteroatom. [Effects of the Invention]

[0014] This disclosure provides a nucleic acid extraction method that allows for easy extraction of nucleic acids contained in a sample even in the absence of proteolytic enzymes and without concentration using carrier nucleic acids. In other words, it provides a nucleic acid extraction method that allows for the purification and concentration of a sample for nucleic acid extraction in a single step. This disclosure also provides a nucleic acid amplification method, a nucleic acid extraction kit, and a PCR test kit. [Brief explanation of the drawing]

[0015] [Figure 1] This is a flowchart illustrating the procedure for the nucleic acid extraction method of the present invention. [Figure 2] These are the quantitative results obtained by real-time PCR using nucleic acids extracted by the method of Comparative Example 1 and the method of Reference Example 1. [Figure 3] These are the quantitative results obtained by real-time PCR using nucleic acids extracted by the method of Comparative Example 1 and the method of Example 1. [Modes for carrying out the invention]

[0016] A first embodiment of the nucleic acid extraction method of the present disclosure is a nucleic acid extraction method comprising: preparing a mixture containing at least one sample selected from the group consisting of cells, extracellular vesicles, and virions, a copolymer containing repeating units represented by formula 1 and formula 2 described later, an antibody bound to the sample, and a linker represented by formula 3 described later, linked via an amide bond, in the absence of a protease, thereby generating an antibody-polymer conjugate; and heating the mixture to agglutinate the antibody-polymer conjugate and extract the nucleic acid contained in the sample.

[0017] The antibody-polymer conjugate described above possesses water solubility and a Lower Critical Solution Temperature (LCST) due to its copolymer structure. Therefore, heating reduces its solubility in water, making it prone to precipitation and aggregation. The sample (antigen) is specifically recognized and bound by the antibody through antigen-antibody interaction. When heated in this state, the sample agglutinates and concentrates while bound to the conjugate. According to the nucleic acid extraction method of the first embodiment, the target nucleic acid can be easily extracted even from a sample with a low concentration of antigen, and / or without pretreatment using proteolytic enzymes.

[0018] A second embodiment of the nucleic acid extraction method of the present disclosure is a nucleic acid extraction method of the first embodiment in which, when the total repeating units of the copolymer are set to 100 mol%, the content of the repeating units represented by Formula 2, described later, is 1.0 to 30.0 mol%.

[0019] The repeating unit represented by formula 2 (unit 2) has two functions in the copolymer (and antibody-polymer conjugate). One is its function as an antibody binding site, and the other is its function in regulating the LCST. Regarding the former, unit 2 has one site containing a cyclic alkyne (alkynylene group) (click reaction site). Therefore, it can easily bind to the azide of the linker in formula 3 by a click reaction. As will be described later, the linker in formula 3 can bind to antibodies. Thus, unit 2 has the function of binding and immobilizing antibodies 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] Copolymers having a repeating unit content of 1.0 to 30.0 mol% represented by Equation 2 are easily adapted to have an LCST of room temperature to 40°C. Furthermore, the amount of antibody bound is practically sufficient. According to the nucleic acid extraction method of the second embodiment, because the LCST is low, the antibody-polymer conjugate to which the antigen is bound can be aggregated more easily (for example, by simply warming it with body temperature) and nucleic acids can be extracted.

[0021] A third embodiment of the nucleic acid extraction method of the present disclosure is a nucleic acid extraction method of the first or second embodiment in which the repeating unit represented by formula 2 is at least one repeating unit selected from the group consisting of the repeating unit represented by formula 4 (described later) and the repeating unit represented by formula 5 (described later).

[0022] As described above, unit 2 has two functions. When unit 2 is the unit represented by formulas 4 and 5, hydrophobicity is more easily adjusted, and as a result, LCST is more easily adjusted to a range that is easier to handle (room temperature to 40°C). In addition, binding to the linker on which the antibody is immobilized becomes easier, and as a result, the convenience and efficiency of the nucleic acid extraction method are further improved.

[0023] A fourth embodiment of the nucleic acid extraction method of this disclosure is a nucleic acid extraction method of any of the first to third embodiments, wherein the copolymer further includes repeating units represented by formula 6, which will be described later.

[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-polymer conjugate) to a higher temperature range. As a result, even with a high content of unit 2, the LCST is more easily adjusted to a more manageable range (room temperature to 40°C). Therefore, a higher level of compatibility between antigen binding and ease of handling (convenience) is more easily achieved.

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

[0026] The polymers and antibody-polymer conjugates described above achieve a higher level of balance between antigen binding and ease of handling (convenience). As a result, nucleic acids can be extracted more reliably and easily according to the nucleic acid extraction method of the fifth embodiment.

[0027] A sixth embodiment of the nucleic acid extraction method of this disclosure is a nucleic acid extraction method of any 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 5000 or more, better concentration efficiency is more easily obtained (in other words, better temperature responsiveness is more easily obtained), while when it is 50000 or less, it is easier to form a conjugate with the antibody, or the antigen-antibody reaction proceeds more easily. As a result, in the nucleic acid extraction method of the sixth embodiment using the antibody-polymer conjugate based on the above copolymer, the antibody-polymer conjugate binds to the antigen more easily, and nucleic acids can be extracted more efficiently.

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

[0030] The copolymer and the antibody-linker complex bind easily and reliably via a click reaction. Furthermore, the copolymer possesses the same LCST (Low-Copolymer Saturation Scale) as the antibody-copolymer conjugate. Therefore, even when the copolymer content in the mixture is lower than that of the antibody-linker complex, the copolymer that does not participate in binding to the antibody-linker complex can contribute to aggregation due to temperature changes (heating) and nucleic acid extraction. From this perspective, the lower limit of the copolymer / antibody numerical range is preferably 1.0 or higher, and more preferably 2.0 or higher. Preferred forms of the copolymer / antibody numerical range will be described later.

[0031] An eighth embodiment of the nucleic acid extraction method of this disclosure is a nucleic acid extraction method in which, in any of the first to seventh nucleic acid extraction methods, the antibody-polymer conjugate includes a repeating unit represented by Formula 1, described later, and a repeating unit represented by Formula 7, described later.

[0032] The above copolymer is typically obtained by conjugating an antibody-linker complex 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 above antibody-polymer conjugate, the antibody-polymer conjugate with (or without) the antigen, and the copolymer can be aggregated and precipitated more easily, and nucleic acids can be extracted more easily.

[0033] A ninth embodiment of the nucleic acid extraction method of this disclosure is a nucleic acid extraction method in which, in any of the first to eighth nucleic acid extraction methods, the heating is performed by heating the temperature of the mixed solution to 20 to 40°C.

[0034] Heating the mixture to 20-40°C can be achieved, for example, by warming the container holding 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 heating equipment or the like.

[0035] A tenth embodiment of the nucleic acid extraction method of this disclosure is a nucleic acid extraction method in which the sample is a membrane structure having a lipid bilayer, in any of the first to ninth nucleic acid extraction methods.

[0036] When the sample has a lipid bilayer and the nucleic acids contained within it 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 proteolytic enzymes to generate an antibody-polymer conjugate, and then heating this mixture to agglutinate the antibody-polymer conjugate (and the copolymer, if present in the mixture) (as described below). The nucleic acid extraction method of the 10th embodiment allows for easier extraction of target nucleic acids with a much simpler procedure and without the use of many reagents, compared to conventional methods.

[0037] An eleventh embodiment of the nucleic acid extraction method of this disclosure is a nucleic acid extraction method in which the sample is an enveloped virus, as in the nucleic acid extraction methods of the first to ninth embodiments.

[0038] The nucleic acid extraction method described herein has been experimentally confirmed to allow for the easier extraction of target nucleic acids using a much simpler procedure and without the need for numerous reagents, compared to conventional methods, when using enveloped viruses as samples.

[0039] A twelfth embodiment of the nucleic acid extraction method of this disclosure is a nucleic acid extraction method of the first to ninth embodiments, wherein the sample is the SARS-CoV-2 virus.

[0040] The nucleic acid extraction method described herein has been experimentally confirmed to allow for the easier extraction of target nucleic acids using a much simpler procedure and without the need for numerous reagents, compared to conventional methods, when using SARS-CoV-2 as a sample.

[0041] A first embodiment of the nucleic acid amplification method of this disclosure is a nucleic acid amplification method comprising: extracting the nucleic acid from the sample using any of the first to twelve nucleic acid extraction methods; 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, thus reducing the overall labor involved in the process. Furthermore, compared to conventional methods, it requires fewer reagents and fewer pieces of equipment.

[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 sample selected from the group consisting of cells, extracellular vesicles, and virions in the absence of proteolytic enzymes, comprising: a first agent comprising a copolymer comprising repeating units represented by formula 1 and repeating units represented by formula 2, described later; and a second agent comprising an antibody bound to the sample and an antibody-linker complex obtained by linking a linker represented by formula (3) via an amide bond.

[0044] According to the nucleic acid extraction kit of the first embodiment, since it contains a first agent containing a copolymer and a second agent containing an antibody-linker complex, an antibody-polymer conjugate can be prepared by mixing these two agents as needed. Because the first and second agents are separated, they have high shelf life, and the operation during use is simple, requiring only mixing. Using the nucleic acid extraction kit of the first embodiment, nucleic acids can be extracted in the absence of protein enzymes and with simple operations.

[0045] The PCR (Polymerase Chain Reaction) test kit described herein is a PCR test kit comprising a first embodiment of a nucleic acid extraction kit.

[0046] According to the above PCR test kit, nucleic acids can be easily extracted from biological samples and other specimens in the absence of proteolytic enzymes, and furthermore, these nucleic acids can be amplified. By using this PCR test kit, the time and cost required for PCR testing can be significantly reduced.

[0047] The nucleic acid extraction method and other related methods will be described in detail below based on non-limiting embodiments. In this specification, a numerical range represented by "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively.

[0048] Furthermore, in this specification, when there are multiple substituents or linking groups, etc. (hereinafter referred to as substituents, etc.) indicated by a specific symbol, or when multiple substituents, etc. are specified simultaneously, it means that each substituent, etc. may be identical or different from the others. The same applies to the specification of the number of substituents, etc. Furthermore, unless otherwise specified, when multiple substituents are in close proximity (especially adjacent), they may be linked to each other or fused to form a ring. Furthermore, substituents that are not specified as substituted or unsubstituted in this specification may have additional substituents on their base, to the extent that they do not impair the intended effect. The same applies to compounds that are not specified as substituted or unsubstituted.

[0049] In this specification, “virion” means 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”). One form of virion is a metabolically inactive infectious pathogen with a diameter of 20–300 nm, comprising a nucleic acid (RNA or DNA) core and a protein coat. Furthermore, enveloped viruses having an envelope containing a lipid bilayer are also included above.

[0050] Infectious viruses include, for example, 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, herpesviruses (e.g., HSV-I, HSV-II, CMV, and VZV), rhinoviruses, and coronaviruses (e.g., SARS). Examples include CoV-2, SARS coronavirus, and MERS coronavirus, enteroviruses, polyomaviruses, polynuclear respiratory viruses (RSV), hepatitis B virus, hepatitis C virus, rotavirus, measles virus, mumps virus, rubella virus, varicella virus, human metapneumovirus, Ebola virus, Marburg virus, alphaviruses (e.g., chikungunya virus, Ross River virus, Sindbis virus, Mayaro virus, etc.), porcine epidemic diarrhea, porcine genitourinary respiratory syndrome virus, and foot-and-mouth disease virus.

[0051] In this specification, "extracellular vesicles" refer to particulate 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, and extracellular vesicles containing nucleic acids are preferred as the sample in this nucleic acid detection method. Extracellular vesicles may originate from various cells, such as red blood cells, white blood cells, cancer cells, stem cells, dendritic cells, and macrophages.

[0052] In this specification, "cell" may be a prokaryotic cell or a eukaryotic cell, and is not particularly limited. Examples include bacteria, archaea, yeast, plant cells, insect cells, and animal cells (e.g., human cells, non-human cells, non-mammalian vertebrate cells, and invertebrate cells, etc.).

[0053] In this specification, "specimen" is 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 (cells, vesicles, liposomes, enveloped viruses, etc.), more preferably an enveloped virus, and even more preferably a SARS-CoV-2 virus. The specimen may be one contained in a sample obtained by culture or other means, or it may be one contained in a biological sample collected from animals, including humans, or plants, etc.

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

[0055] In this specification, "antibody" and antibody residues represented by "Ab" mean an immunoglobulin molecule or a part (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] In this specification, "nucleic acid" means DNA (deoxyribonucleic acid) molecules and RNA molecules, which may be single-stranded or double-stranded. In this specification, "proteinase" refers to an enzyme that recognizes specific sites in the amino acid sequence that make up a protein, cleaves those bonds, and breaks it down into amino acids or peptides. Proteinases are also called "proteases" or "proteinases." Examples of proteolytic enzymes, though not particularly limited, include those used for the extraction and / or purification of nucleic acids. In nucleic acid extraction, nonspecific proteolytic enzymes such as proteinase K (EC3.4.21.64, etc.), protease (pronase), trypsin, and subtilisin are commonly used.

[0057] Nucleic acid extraction methods performed "in the presence" of proteolytic enzymes (in other words, nucleic acid extraction methods using proteolytic enzymes) include Japanese Patent Publication No. 2004-215676, Japanese Patent Publication No. 2006-087394, Japanese Patent Publication No. 2006-061041, Japanese Patent Publication No. 2011-525806, International Publication No. 2017 / 200249, and International Publication No. 2017 / 200249, and are known to those skilled in the art.

[0058] "In the absence of proteolytic enzymes" means that the process is carried out in an environment (extraction reaction system) where proteolytic enzymes are not used. Typically, this means that nucleic acid extraction is performed without the intentional use of proteolytic enzymes. This does not prevent unintentional contamination of the extraction reaction system with proteolytic enzymes, for example, from the sample, but it is preferable that the extraction reaction system does not contain proteolytic enzymes.

[0059] In this specification, "carrier nucleic acid" refers to a compound that, when added to a sample, has the function of improving the efficiency of nucleic acid extraction from the sample, such as polyadenylic acid (homopolymer). Generally, carrier nucleic acids are often used when performing nucleic acid extraction from pathogens and / or samples with low nucleic acid concentrations. 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 "this nucleic acid extraction method") includes, in the absence of proteolytic enzymes (and furthermore without using carrier nucleic acids), preparing a mixture containing at least one sample selected from the group consisting of cells, extracellular vesicles, and virions (hereinafter also simply referred to as "sample"), a copolymer containing repeating units represented by formula 1 described later and repeating units represented by formula 2 described later (hereinafter also referred to as "specific copolymer"), an antibody bound to the sample, and an antibody-linker complex obtained by linking an antibody represented by formula 3 described later via an amide bond, thereby generating an antibody-copolymer conjugate, and heating the mixture to agglutinate the antibody-copolymer conjugate and extract the nucleic acid contained in the sample.

[0061] <Step S1> Figure 1 is a flowchart illustrating the procedure for this nucleic acid extraction method. First, in step S1, a mixture containing the sample, a specific copolymer, and an antibody-linker complex is prepared to generate an antibody-polymer conjugate. While it is not necessary to treat impurities with proteolytic enzymes at this stage, it is of course possible to do so.

[0062] As will be explained in detail later, 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"). A polymer composed solely of unit 1 exhibits a temperature response to water, with an LCST (Lower Critical Solution Temperature) of 32°C. The specific copolymer containing unit 1 is temperature responsive.

[0063] Furthermore, each repeating unit of unit 2 has one site containing a cyclic alkyne (alkynylene group) (click reaction site), which can be easily combined with the azide group of the linker described later via a click reaction to form a complex. In other words, a specific copolymer possessing both unit 1 and unit 2 has the function of bonding with a linker through a click reaction, and the function of changing its solubility in water upon heating (specifically, its solubility decreases and it aggregates).

[0064] On the other hand, the linker represented by formula 3, described later, has an azide group and an active ester group in its 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 an N-hydroxysuccinimide group, it can bind to the amino group of the antibody to form a complex (antibody-linker complex).

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

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

[0067] [ka]

[0068] Unit 1 Unit 1, when composed solely of this unit, exhibits a temperature-responsive property in water, with a Lower Critical Solution Temperature (LCST) of 32°C. The inclusion of Unit 1 gives a specific copolymer temperature responsiveness; specifically, its solubility in water changes with temperature.

[0069] The content of unit 1 in the specific copolymer is not particularly limited, and typically, 1 to 99 mol% is preferred when the total repeating units are considered to be 100 mol%. In particular, from the viewpoint of the specific copolymer having a more sensitive temperature response and / or being able to easily control the temperature range of the LCST to be manageable in nucleic acid extraction operations (specifically, the LCST tends 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%; greater than 50 mol% and 99 mol% or less; 60 to 99 mol%; 1 to 97 mol%; greater than 50 mol% and 97 mol% or less; or 60 to 97 mol%.

[0071] In formula 1, X 1 The group represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms. From the viewpoint of having a more sensitive temperature response in the specific copolymer, 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] While not particularly limited, unit 1 is preferably a unit based on a monomer represented by the following formula 1'.

[0073] [ka]

[0074] In formula 1′, X 1 X in Equation 1 1 This is synonymous with the preferred form, and the same applies to the monomer represented by formula 1'. The monomer represented by formula 1' may be synthesized, for example, by a known method, such as the 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. Each repeating unit of unit 2 has one site containing a cyclic alkyne (alkynylene group) (click reaction site), which can easily form a complex by clicking with the azide group of the linker described later. If the linker and antibody are pre-bound, the protein can be immobilized on the specific copolymer via the linker. In other words, an antibody-polymer conjugate can be prepared. The resulting conjugate undergoes aggregation and precipitation at LCST or higher temperatures due to its temperature-responsive properties derived from a specific copolymer. This property allows for the concentration of the corresponding antigen and the extraction and concentration of the nucleic acid released as a result.

[0076] The content of unit 2 in the specific copolymer is not particularly limited, but when the total repeating units of the specific copolymer are considered to be 100 mol%, typically 1 to 99 mol% is preferred. In particular, for obtaining the best possible effects of the present invention, a concentration of 1.0 mol% or more is preferred, 2.0 mol% or more is more preferred, 30 mol% or less is preferred, and 20 mol% or less is even more preferred.

[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 is typically more hydrophobic than Unit 1 or other repeating units described later. Therefore, increasing the content of Unit 2 in a particular polymer can lower the LCST (Low Cellular Temperature). Furthermore, the higher the content of Unit 2, the easier it is for antibodies to be introduced. On the other hand, from the perspective of adjusting the LCST to 20-40°C, the content of Unit 2 is preferably 1.0 mol% or more, more preferably 2.0 mol% or more, preferably 30 mol% or less, and more preferably 20 mol% or less. From the perspective of adjusting the LCST temperature to 20-40°C, 1.0-30.0 mol%, 1.0-20.0 mol%, 2.0-30.0 mol%, or 2.0-20.0 mol% are preferred.

[0079] [ka]

[0080] In formula 2, X 2 The group represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms. From the viewpoint of having a more sensitive temperature response in the specific copolymer, 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 The symbol represents a divalent group. There are no particular restrictions on what constitutes a divalent group, but these include -O-, -S-, -C(O)-, -C(O)O-, -OC(O)O-, and -NR. A -(R A Preferably, the group consists of at least one selected from the group comprising a hydrogen atom or a monovalent substituent, a linear, branched, or cyclic aliphatic hydrocarbon group having 1 to 20 carbon atoms, a monocyclic or fused aromatic hydrocarbon group having 6 to 20 carbon atoms, and combinations thereof. In particular, from the viewpoint of obtaining copolymers that exhibit the superior effects of the present invention, -O-, -C(O)-, and -NR A - More preferably, one group selected from the group consisting of linear alkylene groups having 1 to 10 carbon atoms, and groups formed by combining these, is -O-, -C(O)-, -NR A -, and at least one group selected from the group consisting of linear alkylene groups having 1 to 10 carbon atoms are more preferred, and -O-, -C(O)-, or -NH- are particularly preferred.

[0082] In formula 2, R 1 This includes hydrogen atoms, halogen atoms, and -OR 5 -NO2, -CN, -S(O)2R 5 , selected from the group consisting of alkyl groups with 1 to 24 carbon atoms, alkenyl groups with 2 to 24 carbon atoms, and (hetero)aryl groups with 6 to 24 carbon atoms, and two or more R 1 They may be bonded to each other to form a ring, R 5 R represents a group selected from the group consisting of hydrogen, halogen atoms, alkyl groups with 1 to 24 carbon atoms, and (hetero)aryl groups with 6 to 24 carbon atoms. Note that there are multiple R groups. 1 They may be the same or different from each other. In equation 2, Z is C(R 1 )2, O, S, and NR 1 This represents a group selected from the group consisting of [Z]. 2 When a divalent group represented by is bonded, Z is C(R 1 )2, or NR 1 It is a group from which one of the hydrogen atoms, halogen atoms, or carbon atoms has been removed. More specifically, =C(R 1 )R X -L 2 - A structure represented by -, or =NR x -L 2 -This is the structure represented by R. X This represents a single bond, or an alkylene group (1-24 carbon atoms), an alkenylene group (2-24 carbon atoms), an alkylylene group (2-24 carbon atoms), or an arylene group (6-24 carbon atoms) which may have a heteroatom. In equation 2, a' is an integer between 0 and 8, a'' is an integer between 0 and 8, and the sum of a' and a'' is less than 10.

[0083] In terms of obtaining a better effect of the present invention, unit 2 is given by the following formula 2 a A unit represented by is preferred.

[0084] [ka]

[0085] formula 2 a Medium, X 2 , L 2 , R 1 , a′, and a″ are synonymous with the respective symbols in Equation 2, and the preferred form is also the same. Equation 2 a Middle, Z is CR 1 , or representing a nitrogen atom, a 2 is a number less than or equal to a' + a''. Note that R 1 R in Equation 2 is 1 This is synonymous with the preferred form, and the same applies to the preferred form.

[0086] Unit 2 is L 2 When the binding site is denoted as "*", it is preferable to have a structure (click reaction site) represented by the following formula C.

[0087] [ka]

[0088] Unit 2 preferably has the structure represented by formula C above as the site (monovalent group) to which it is bonded at the position of "*". The * in the monovalent group represented by formula C is bonded to the wavy line portion in the following formula. [ka]

[0089] Note that in the above formula, X 2 , and L 2The symbol in formula (2) is equivalent to the base represented by the same symbol, and the preferred form is also the same.

[0090] From the viewpoint of obtaining a copolymer having a more superior effect of the present invention, it is preferable that unit 2 is at least one selected from the group consisting of units represented by the following formula. Note that in the following formula, L 21 represents a divalent group, and L in formula 2 2 This is synonymous with the preferred form, and the same applies to X. 2 X in equation 2 2 This is synonymous with the preferred form, and the same applies to the preferred form.

[0091] [ka]

[0092] In particular, from the viewpoint of obtaining a copolymer having a more superior effect of the present invention, unit 2 is preferably unit 4 or unit 5 represented by the following formula 4 or 5.

[0093] [ka]

[0094] In equations 4 and 5, X 2 L represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, in formula 4. 4 -O-, -S-, and -NR B - is a type of group selected from the group consisting of R B represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, n represents an integer from 1 to 10, and in formula 5, L 2 represents a divalent group, and the preferred form is L in formula 2. 2 It is similar to the divalent group of .

[0095] There are no particular restrictions on the method of synthesizing unit 2, and known synthesis methods can be used. However, a method of obtaining unit 2 by attaching a precursor compound to unit 2', represented by the following formula 2', is preferred because it allows for a simpler acquisition of unit 2.

[0096] [ka]

[0097] In formula 2′, R C X is a reactive substituent, specifically including hydroxyl groups, amino groups, carboxyl groups, glycidyl groups, epoxy groups, glycidyl ether groups, mercapto groups, hydroxysuccinimide esters, and maleimide, with hydroxyl groups being preferred. 2 X in equations 4 and 5 is 2 It is synonymous with [the above].

[0098] Examples of precursor compounds include the click reaction site and the above R C Any compound having a group that can react with the compound can be used, and commercially available products can be used, or they can be synthesized by known methods. In particular, using a precursor compound having the structure represented by formula C above (click reaction site) and a carboxyl group allows for the synthesis of a specific copolymer more easily.

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

[0100] Furthermore, compounds in which substituents such as amino groups, hydroxyl groups, and carboxyl groups are added to BCN (bicyclo[6.1.0]nonyne) and DBCO (Dibenzocyclooctyne), which are the click reaction sites, or which have been esterified with maleimide or hydroxysuccinimide are commercially available, and these can also be used as precursor compounds.

[0101] In particular, unit 2' is preferably unit 6 represented by the following formula 6, as it allows for the simpler acquisition of unit 2. In this case, the precursor compound can typically be a precursor compound having a carboxyl group and a structure represented by formula C.

[0102] [ka]

[0103] Furthermore, when synthesizing a specific copolymer, if there are unreacted units 6, in other words, if the specific copolymer contains units 1, 2, and 6, the LCST can be adjusted to the higher temperature side. Since unit 6 has higher hydrophilicity compared to other repeating units, increasing the content of unit 6 can adjust the LCST to the higher temperature side.

[0104] In the above formula 6, X 6 The group represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms. From the viewpoint of the copolymer having a more sensitive temperature response, 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] There are no particular restrictions on the content of unit 6 in the specific copolymer, but it is preferably 0 to 15 mol% when the total repeating units of the specific copolymer are set to 100 mol%.

[0106] The molecular weight of the specific copolymer is not particularly limited, but typically, a number-average molecular weight of 2,000 to 100,000 is preferred, 5,000 to 50,000 is more preferred, and 10,000 to 30,000 is even more preferred. When the number-average molecular weight is 5000 or higher, better concentration efficiency is more likely to be obtained (in other words, better temperature responsiveness is more likely to be obtained), while when it is 50000 or lower, it is more likely to form conjugates with antibodies or the antigen-antibody reaction will proceed more easily.

[0107] In particular, when using antibody-polymer conjugates prepared with the obtained specific copolymer, a number-average molecular weight of 15,000 or more is preferred, and more preferably 20,000 or more, in that superior concentration efficiency can be obtained. Generally, antibodies are often highly hydrophilic, and in such cases, a copolymer with superior concentration efficiency is required. When the number-average molecular weight of the copolymer is within the above numerical range, superior concentration efficiency is more easily obtained.

[0108] Based on the above, the molecular weight of the specific polymer is preferably 2000-100000, 5000-50000, 10000-30000, 15000-100000, 15000-50000, 15000-30000, 20000-100000, 20000-50000, or 20000-30000.

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

[0110] (Method for manufacturing specific copolymers) The method for producing the specific copolymer is not particularly limited, but from the viewpoint of producing the specific copolymer more easily, 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 formulas 1' and 3' to obtain a copolymer (a precursor of a specific copolymer).

[0112] [ka]

[0113] In the above formulas 1' and 3', X 1 , and, X 2 These are the X values ​​in equation 1, respectively. 1 , and X in equation 2' 2 These are synonymous with each other, and the same applies to the preferred form.

[0114] The method for copolymerizing the above monomers is not particularly limited, but it is preferable to use living polymerization methods such as living radical polymerization, living anion polymerization, and living cation polymerization. Among these, living radical polymerization is preferred from the viewpoint of obtaining copolymers (or their precursors) more easily.

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

[0116] Examples include the ATRP method (atomic transfer radical polymerization) using alkyl halides as dormants, the RAFT method (reversible addition fragmentation chain transfer) using thioesters, and the NMP method (nitroxide mediated polymerization) using alkoxyamines.

[0117] The RAFT method is a method for polymerizing vinyl monomers by adding a chain transfer agent with a high chain transfer constant, called a RAFT agent, to a conventional radical polymerization system. Thioesters can be used as RAFT agents.

[0118] The amount of RAFT agent can be appropriately selected depending on the molecular weight of the target copolymer. That is, since the RAFT agent is bound to the ends of each copolymer, for example, if the target product is a 100-mer copolymer, then 0.1 to 3 mol% of RAFT agent should be used per 100 mol% monomer.

[0119] The radical polymerization initiator used in RAFT polymerization is not particularly limited and can 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′-azobis-2,4-dimethylvaleronitrile, 2,2′-azobis(2-methylpropionamidine) dihydrochloride, and 4,4′-azobis(4-cyanovaleric acid).

[0121] The polymerization initiator is generally preferably present in an amount of 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 it is generally carried out at 40°C to 150°C. Polymerization is often performed under atmospheric pressure, but polymerization is also possible under pressure.

[0122] The RAFT method can be carried out in the absence of a solvent, but it can also be carried out in the presence of a solvent. The solvent used is not particularly limited and any known solvent can be used. Furthermore, the reaction can be carried out in water, and emulsion polymerization will also proceed. The emulsifiers used in this case can be nonionic, cationic, or anionic emulsifiers commonly used in emulsion polymerization.

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

[0124] [ka]

[0125] In formula 10, Z is a group containing a click reaction site (e.g., a cyclic alkyne), and is preferably a group selected from the groups represented by formula C described earlier. In formula C, * is L 10 This will be the connection point. Also, L 10 represents a divalent group, and L in formula 2 2 This is synonymous with the preferred form, and the same applies to the preferred form.

[0126] A desired specific copolymer is synthesized by forming an ester bond (condensation) between the hydroxyl group of the copolymer (precursor) and the carboxyl group of the precursor compound represented by formula 10.

[0127] The method for forming the ester bond is not particularly limited, but one example is to carry out a condensation reaction between a copolymer (precursor) and a precursor compound 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] As condensing agents, 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)phosphonate diphenyl can be used, among which N,N'-dicyclohexylcarbodiimide is preferred. In this case, N,N-dimethyl-4-aminopyridine can be used as a catalyst, and dichloromethane can be used as a solvent.

[0129] (Antibody-linker complex) An antibody-linker complex is a compound obtained by linking an antibody that binds to a sample with a linker represented by formula 3 below via an amide bond.

[0130] [ka]

[0131] In formula 3, L 3 This represents a divalent hydrocarbon group which may have a heteroatom. The divalent hydrocarbon group is not particularly limited, but (poly)oxyalkylene groups (preferably with 1 to 6 carbon atoms in the alkylene group) and linear, branched, or cyclic hydrocarbon groups with 1 to 20 carbon atoms are preferred, with polyoxyalkylene groups being more preferred. The number of repeating polyoxyalkylene groups 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 the primary amine of the protein (e.g., a lysine residue). That is, it binds to the -NH2 group of the antibody to form a complex. On the other hand, the linker has an azide group and binds to the alkylylene group (click reaction site) of the specific copolymer to form a triazole ring.

[0133] The method for preparing the antibody-linker complex is not particularly limited, but it is possible to dissolve the linker represented by formula 3 in an organic solvent (for example, an aprotic polar solvent such as dimethyl sulfoxide) and add it to a solution in which the antibody is dispersed in a buffer solution containing a buffering agent (for example, a carbonate buffer solution) as needed. The reaction temperature is not particularly limited, but 1 to 20°C is preferred, and 1 to 10°C is more preferred. The reaction time is not particularly limited, but 1 to 24 hours is preferred.

[0134] The ratio of antibody to linker added during the production of the antibody-linker complex is not particularly limited, but the molar ratio (active ester group / amino group) of the active ester groups of the linker to the amino group content of the antibody is preferably 0.1 to 500. Furthermore, while there are no particular restrictions on the amount of linker added per mole of antibody, 0.01 to 5000 moles is preferred, 1 to 2000 moles is more preferred, and 50 to 200 moles is even more preferred.

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

[0136] (Antibody-polymer conjugate) The antibody-copolymer conjugate is formed by binding the click reaction site of a specific copolymer to the azide group of the antibody-linker complex to form a triazole ring structure. Examples of the antibody-copolymer conjugate include compounds having repeating units represented by the following formula 1 and formula 7.

[0137] [Chemical formula]

[0138] In formula 1, X 1 is synonymous with X 1 of the specific copolymer, and the preferred forms are the same. Also, in formula 7, X 2 represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, and is synonymous with X 2 in formula 2, and the preferred forms are the same. Also, L 2 represents a divalent group, and is synonymous with L 2 in formula 2, and the preferred forms are the same. Also, L 3 represents a divalent hydrocarbon group which may have a heteroatom, and is synonymous with L 3 in formula 3, and the preferred forms are the same. Also, Ab represents an antibody residue. That is, an amide bond is formed between the primary amine of the antibody and the NHS ester of the linker, representing a state where the antibody is fixed to the specific copolymer via the linker.

[0139] In the above form, the antibody and unit 7 are bonded in a 1:1 ratio. However, the antibody-copolymer conjugate is not limited to the above form, and a state where a plurality of primary amines of the antibody are each bonded to an alkynylene group of the specific copolymer via a linker, that is, a structure similar to a crosslinked structure centered on the antibody molecule may be formed.

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

[0141] The ratio of antibody to specific copolymer is not particularly limited, but as one form, 0.1 to 50 moles of the specific copolymer per mole of antibody is preferred.

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

[0143] There are no particular restrictions on the antibody used, and any known antibody can be used, but anti-SARS-CoV-2 antibodies are preferred, and for example, anti-SARS-CoV-2 (COVID19) nucleocapsid (protein) antibodies and anti-SARS-CoV-2 spike (protein) antibodies are more preferred. While there are no particular restrictions on the molar ratio of the specific copolymer content to the antibody content in the antibody-copolymer conjugate, preferred values ​​are 0.1-100.0, 0.5-70.0, 0.5-50.0, 0.5-30.0, 1.0-50.0, 1.0-40.0, 2.0-100.0, 2.0-50.0, 5.0-100.0, 5.0-50.0, 10.0-100.0, 10.0-50.0, or 10.0-30.0.

[0144] The method for preparing the antibody-polymer conjugate is not particularly limited, but the specific copolymer and the antibody-linker complex can be mixed and stirred in water containing a buffering agent as needed. The reaction temperature is not particularly limited, but 1 to 20°C is preferred, and 4 to 10°C is more preferred. The reaction time is not particularly limited, but 1 to 24 hours is preferred.

[0145] Returning to the flow chart in Figure 1, the method for preparing the mixture in step S1 is not particularly limited, but one method is to mix the biological sample, the antibody-linker complex, and the specific copolymer. If the biological sample is in liquid form, the other components can be added to the biological sample. The order in which the antibody-linker complex and the specific copolymer are added is not particularly limited; the antibody-linker complex may be added first and then the specific copolymer, or the specific copolymer may be added first and then the antibody-linker complex, or both 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, it is preferable to add 0.1 mg or more of antibody-specific copolymer conjugate per 1 mg of sample (antigen protein), 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, it is preferable to add 0.1 to 10.0 mg of antibody-specific copolymer conjugate per 1 mL of liquid medium containing the sample (e.g., saliva).

[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 the sample (antigen protein).

[0148] At this time, a specific copolymer in a free state (one that has not formed a conjugate with the antibody) may be further added to the mixture. Adding the specific copolymer makes the response to heat more sensitive and allows for more efficient concentration of the sample. There are no particular restrictions on the amount of specific copolymer to add, but it is preferably 0.1 to 20 times the molar content of the specific copolymer contained in the antibody-polymer conjugate, which is set to 1.

[0149] In this step, the sample included in the mixture is not particularly limited, but may be provided from a biological sample. In one form, the biological sample containing the sample may be added directly to the mixture. Generally, biological samples contain many impurities. In typical nucleic acid extraction, these are often treated with proteolytic enzymes to inactivate or digest them. However, in this nucleic acid extraction method, even in the absence of proteolytic enzymes, the sample is captured by the antibody-polymer conjugate and can be easily separated from contaminants in step S2 described below. Therefore, nucleic acid extraction can be performed with sufficient efficiency without treatment with proteolytic enzymes or the like.

[0150] <Step S2> Returning to the flowchart in Figure 1, in step S2, the mixture obtained in step S1, containing the sample and antibody-polymer conjugate, is heated to agglutinate the antibody-polymer conjugate that has captured the sample, and the nucleic acid contained in the sample is extracted. At this point, it is not necessary to add a carrier nucleic acid, but it may be added. The heating temperature can be adjusted as appropriate according to the LCST of the specific copolymer, but 20-40°C is preferred.

[0151] General procedures for extracting nucleic acids from a sample include tissue lysis with proteolytic enzymes, inactivation of unwanted enzymes, washing, filtering, and fractionation of components. This process requires multiple steps, and nucleic acid extraction for SARS-CoV-2 PCR testing can typically take around 90 minutes. Furthermore, the use of various reagents tends to increase the cost of a single extraction operation.

[0152] On the other hand, the inventors have discovered that, surprisingly, this nucleic acid extraction method allows for the extraction of nucleic acids contained in a sample even in the absence of proteolytic enzymes, and even without the use of carrier nucleic acids. Although the mechanism is not entirely clear, it is presumed that the sample is captured by the interaction between the antibody-specific copolymer conjugate and the antibody, and by agglutinating this captured sample, the separation of impurities and the concentration of the sample can be performed simultaneously.

[0153] This nucleic acid extraction method allows for the extraction of nucleic acids contained within a sample through simple operations. Specifically, the antibody-specific copolymer conjugate that has captured the sample is agglutinated by heating, and the solid is obtained by centrifugation or the like, thereby removing impurities and concentrating the sample. Subsequently, the nucleic acids contained in the sample can be released from the obtained solid using a known method. Such a method is not particularly limited, but examples include a method using a nucleic acid-binding solid support such as silica particles and a chaotropic agent (J. Clin. Microbiol., vol.28 No.3, p.495-503 (1990)). In other words, this method involves adsorbing nucleic acids onto a nucleic acid-binding solid support in the presence of a chaotropic agent, then washing the support with a washing solution, and finally eluting the nucleic acids from the support using water or a low-salt buffer solution. These methods are well known to those skilled in the art, and commercially available kits can also be used. The nucleic acids extracted by this nucleic acid extraction method can be used for 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); and isothermal amplification methods that do not involve a thermal denaturation step of double-stranded DNA, such as 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 sequence replication).

[0155] Methods for amplifying RNA extracted from a sample include TMA (Transcription-Mediated Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), and TRCR (Transcription-reverse transcription concerted reaction).

[0156] [Nucleic acid amplification methods] Embodiments of the nucleic acid amplification method include extracting nucleic acids from a sample using the nucleic acid extraction method described above, and amplifying the extracted nucleic acids using polymerase chain reaction (PCR). Methods for amplifying DNA by PCR are well known and can be carried out by referring to Science 239, 487-491 (1988), etc.

[0157] If the sample 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. While the reverse transcription method is well-known, the reaction temperature should be such that the heat-stable reverse transcriptase is active, preferably between 35 and 90°C. The duration of the reverse transcription reaction can be appropriately set considering the length of the synthesized cDNA chain, etc.

[0158] The RNA extraction from the sample, the reverse transcription reaction, and the target nucleic acid amplification reaction may be performed sequentially or consecutively (in one step). When the reverse transcription reaction and the target nucleic acid amplification reaction are performed consecutively, it is preferable to use a thermostable reverse transcriptase and a thermostable DNA polymerase. Various polymerases usable in PCR can be used as the thermostable DNA polymerase. Alternatively, a thermostable DNA polymerase with thermostable reverse transcriptase activity can also be used.

[0159] Additionally, a primer pair can be used to amplify the target region of the cDNA. In this case, one of the primers may be shared with the reverse transcription primer used in the reverse transcription reaction. Furthermore, if there are multiple target nucleic acid regions, multiple primer pairs may be used in combination. This nucleic acid amplification method allows for nucleic acid extraction with simple operations without the need for proteolytic enzymes, thus 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 proteolytic enzymes, comprising a first agent containing a specific copolymer and a second agent containing an antibody-linker complex. The first agent comprises, for example, a container and a specific polymer contained in the container, and may be in solid form (e.g., powder form) or liquid form. When the first agent is liquid, it may be in a form comprising the specific polymer and water (an aqueous solution containing a buffering agent).

[0161] The second agent comprises, for example, a container and an antibody-linker complex contained in the container. The second agent may be in liquid form and may include an antibody-linker complex and a buffer (e.g., phosphate-buffered saline, PBS). The content of the antibody-linker complex in the second agent is not particularly limited, but 0.01 to 10000 μg / mL is preferred.

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

[0163] This nucleic acid extraction kit allows for the concentration and extraction of nucleic acids from a sample by preparing a mixture containing the first and second agents and the sample, generating an antibody-polymer conjugate, and then heating it. For example, if the sample is an RNA virus, the first and second agents are added to saliva or other sample containing the RNA virus and reacted. After heating, the RNA virus captured by the antibody-polymer conjugate agglutinates and precipitates together with the conjugate. Solid-liquid separation removes impurities (liquid phase) and concentrates the sample. RNA is then extracted from the concentrated sample. This nucleic acid extraction kit allows for the extraction of nucleic acids in the absence of proteolytic enzymes and with simple procedures. Furthermore, even from biological samples with low concentrations of the sample (e.g., RNA virus), nucleic acids can be efficiently extracted due to the sample capture and agglutination functions of the antibody-polymer conjugate.

[0164] [PCR test kit] The embodiment of the PCR test kit is not particularly limited in terms of other components, as long as it includes the nucleic acid extraction kit described above. Other components may include, for example, if the sample is an RNA virus, a reverse transcriptase for cDNA synthesis, a probe for real-time detection, an intercalator, a buffer, deoxyribonucleotides such as dNTPs, surfactants, salts, and primers. Some of these ingredients may be mixed together and provided as a premix.

[0165] This PCR test kit allows for easy extraction of nucleic acids from biological samples and other specimens in the absence of proteolytic enzymes, and further amplification of these nucleic acids. Using this PCR test kit significantly reduces the time and cost required for PCR testing. [Examples]

[0166] The embodiments will be described in more detail below with reference to non-limiting examples.

[0167] [Examples of synthesis of specific copolymers] • Synthesis of HIPAAm As preparation for the synthesis of a specific copolymer, "HIPAAm" was synthesized using 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. Then, 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 solution was redissolved in 2-propanol and kept at -20°C for at least 24 hours. Finally, the salts were removed by filtration, and the solution was concentrated and purified by column chromatography. The synthesis of HIPAAm was confirmed by thin-layer chromatography (TLC) and 1Confirmed by 1H NMR (Nuclear Magnetic Resonance) (solvent: D2O).

[0170] [ka]

[0171] • Synthesis of P(NIPAAm-co-HIPAAm) As a polymer that serves as a precursor compound for a specific copolymer, "P(NIPAAm-co-HIPAAm)" was synthesized using the following procedure. P(NIPAAm-co-HIPAAm) was synthesized by RAFT polymerization of HIPAAm and NIPAAm (see the scheme below for 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 then vacuum-dried.

[0172] [ka]

[0173] Synthesis of P(NIPAAm-co-HIPAAm-co-SAKIPAAm) As a specific polymer, P(NIPAAm-co-HIPAAm-co-SAKIPAAm) was synthesized. As shown in the scheme below, dibenzylcyclooctic acid (DBCO-Acid), which has a click reaction site, was introduced to the hydroxyl group of HIPAAm by dehydration condensation to obtain a 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′-Dicyclohexylcarbodiimide) was stirred overnight, evaporated, and then vacuum-dried.

[0174] The number-average molecular weight of the obtained P(NIPAAm-co-HIPAAm-co-SAKIPAAm) is 2.01 × 10⁻⁶. 4 The LCST was approximately 30°C. Also, 1 The repeating unit content determined by 1H-NMR was NIPAAm:HIPAAm:SAKIPAAm (all on a molar basis) = 96.4:1.2:2.4. In P(NIPAAm-co-HIPAAm-co-SAKIPAAm), "NIPAAm" refers to a unit based on NIPAAm, corresponding to the unit represented by Equation 1. Similarly, "HIPAAm" corresponds to the unit represented by Equation 6, and "SAKIPAAm" corresponds to the unit represented by Equation 2.

[0175] [ka]

[0176] [Synthesis of antibody-linker complexes] 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 anti-COVID-19 monoclonal antibody (>95%, 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), stirred at 4°C for 5 hours, and the antibody-linker complex was synthesized. The antibody / linker ratio was set to 1:100.

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

[0178] The criteria for participation in the above examination were as follows: • Age 18 or older • Having the willingness and ability to provide written informed consent. • Individuals who have tested positive for COVID-19, those who have tested negative, or those suspected of being positive, based on PCR or antigen testing. The exclusion criteria were as follows: • Under 18 years of age • Those who do not consent to informed consent

[0179] The above research protocol was approved by the Research Ethics Committee of the Egyptian Liver Research and Teaching Hospital. The research protocol and implementation were carried out in accordance with the "CIOMS / WHO. International Ethical Guidelines for Biomedical Research Involving Human Subjects. Geneva: CIOMS. 1993." and its modified version, with written informed consent obtained from all patients.

[0180] [Example 1] Viral RNA was extracted from biological samples in the absence of proteolytic enzymes using a synthesized specific copolymer and an antibody-linker complex. The procedure is as follows:

[0181] (Step 1) First, 900 μL of the sample was mixed with an antibody-linker complex (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 was conjugated by a click reaction at 4°C for 1 hour using g / mol. The mixture was then transferred to a 2.0 mL microcentrifuge tube and centrifuged at 37°C and 13,800 × g for 5 minutes. The concentrated precipitate was collected, and the supernatant was discarded. Next, the concentrated precipitate and 200 μL PBS were added to a lysis tube (LT).

[0182] (Step 2) Furthermore, 250 μL of ethanol (96-100%) was added to this mixture. After stirring (vortexing) for approximately 15 seconds, the mixture was incubated at room temperature (15-25°C) for 5 minutes, and then droplets were removed by centrifugation.

[0183] Next, the lysate was transferred to a "QIAamp MinElute column" and centrifuged at 6000×g for more than 1 minute. Next, the washing tube containing the filtrate was discarded, 500 μL of "Buffer AW1 (Qiagen)" was added, and the mixture was centrifuged at 6000 × g for at least 1 minute. Then, the washing tube containing the filtrate was discarded, 500 μL of "Buffer AW2 (Qiagen)" was added, and the mixture was centrifuged at 6000 × g for at least 1 minute.

[0184] Next, the wash tube containing the filtrate was discarded, 500 μL of ethanol (96-100%) was added, and the mixture was centrifuged at 6000 × g for at least 1 minute. Then, the wash tube containing the filtrate was discarded, and the QIAamp MinElute column was transferred to a clean 2 ml wash tube (WT).

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

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

[0187] [Comparative Example 1] Viral RNA was extracted from a biological sample similar to that of Example 1 using a proteolytic enzyme without using a specific copolymer or an antibody-linker complex. The detailed procedure was the same as that of 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. Further, 200 μL of "Buffer AL (Qiagen)" containing 28 μg / mL of carrier RNA was added and stirred for approximately 15 seconds. Next, after incubating at 56 °C for 15 minutes in a heating block, centrifugation was performed to remove droplets. The subsequent (Procedure 2) was the same as that of Example 1.

[0189] [Reference Example 1] Viral RNA was extracted from a biological sample similar to that of Example 1 using a proteolytic enzyme, a specific copolymer, and an antibody-linker complex. The detailed procedure was the same except that (Procedure 1) in Example 1 was changed as follows.

[0190] First, 900 μL of the sample was mixed with an antibody-linker complex (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 g / mol) was conjugated to the antibody-linker complex by click reaction at 4 °C for 1 hour. Thereafter, the mixture was transferred to a 2.0 mL microtube and centrifuged at 37 °C, 13,800×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). Then, 200 μL of the sample containing the concentrated precipitate was added. Furthermore, 200 μL of "Buffer AL (Qiagen)" containing 28 μg / mL of carrier RNA was added and the mixture was stirred for approximately 15 seconds. Next, the mixture was incubated in a heating block at 56°C for 15 minutes, and then the droplets were removed by centrifugation. The subsequent steps (Step 2) were carried out in the same manner as in Example 1.

[0192] [result] Figure 2 shows the quantitative 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 quantitative results from Reference Example 1, using the specific copolymer and antibody-linker complex, were approximately 1.5 times higher than those from Comparative Example 1, clearly indicating that the specific copolymer and antibody-linker complex concentrate the sample.

[0193] Figure 3 shows the quantitative results obtained by real-time PCR using nucleic acids extracted by the method of Comparative Example 1 and the method of Example 1. The quantitative results from Example 1 were approximately 30 times higher than those from Comparative Example 1. From the results in Figure 3, it is clear that target nucleic acids can be detected with sufficient sensitivity even without using proteolytic enzymes and carrier nucleic acids.

Claims

1. In the absence of proteolytic enzymes, A specimen selected from the group consisting of cells, extracellular vesicles, and virions, A copolymer comprising a repeating unit represented by the following formula 1 and a repeating unit represented by the following formula 2-1, A mixture is prepared containing an antibody that binds to the aforementioned sample, and an antibody-linker complex obtained by attaching a linker represented by the following formula 3 via an amide bond. To generate antibody-polymer conjugates, A method for extracting nucleic acids, comprising heating the aforementioned mixture to agglutinate the antibody-polymer conjugate and extracting the nucleic acids contained in the sample. 【Chemistry 1】 (In formula 1, X １ (This represents a hydrogen atom, or a linear or branched alkyl group having 1 to 6 carbon atoms.) 【Chemistry 2】 (In formula 2-1, X ２ (where represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, L 22 represents a divalent group, and the monovalent group bonded at the position of * after the wavy line has a structure selected from the following formula C.) 【Transformation 3】 【Chemistry 4】 (In formula 3, L ３ (This represents a divalent hydrocarbon group which may have a heteroatom.)

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

3. The nucleic acid extraction method according to claim 1, wherein the repeating unit represented by formula 2-1 is at least one repeating unit selected from the group consisting of the repeating unit represented by formula 4 and the repeating unit represented by formula 5 below. 【Transformation 5】 (In equations 4 and 5, X ２ L represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, in formula 4, ４ -O-, -S-, and -NR ２ - is a type of group selected from the group consisting of R ２ (where represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, n represents an integer from 1 to 10, and in formula 5, L 23 represents a divalent group.)

4. Furthermore, the nucleic acid extraction method according to claim 1, wherein the copolymer includes repeating units represented by the following formula 6. 【Transformation 6】 (In formula 6, X ６ (This represents a hydrogen atom, or a linear or branched alkyl group having 1 to 6 carbon atoms.)

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

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

7. The nucleic acid extraction method according to claim 1, wherein the molar ratio of the copolymer content to the antibody content 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-polymer conjugate includes a repeating unit represented by the following formula 1 and a repeating unit represented by the following formula 7. 【Transformation 7】 (In Formula 1, X １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms. In Formula 7, X ２ represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms. L23 represents a divalent group. L6 represents a divalent hydrocarbon group which may have a hetero atom. Ab represents the residue of the antibody.)

9. The nucleic acid extraction method according to claim 1, wherein the heating is to heat the temperature of the mixed solution to 20 to 40°C.

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

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

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

13. Extracting the nucleic acid from the sample using the nucleic acid extraction method described in any one of claims 1 to 12, A method for amplifying nucleic acids, comprising amplifying the extracted nucleic acid by a polymerase chain reaction.

14. A nucleic acid extraction kit used for extracting nucleic acids from at least one sample selected from the group consisting of cells, extracellular vesicles, and virions in the absence of proteolytic enzymes, A first agent comprising a copolymer containing repeating units represented by the following formula 1 and repeating units represented by the following formula 2-1, A nucleic acid extraction kit comprising: an antibody that binds to the aforementioned sample; and a second agent containing an antibody-linker complex obtained by attaching a linker represented by the following formula 3 via an amide bond. 【Transformation 8】 (In formula 1, X １ (This represents a hydrogen atom, or a linear or branched alkyl group having 1 to 6 carbon atoms.) 【Chemistry 9】 (In formula 2-1, X ２ (where represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, L 22 represents a divalent group, and the monovalent group bonded at the position of * after the wavy line has a structure selected from the following formula C.) 【Chemistry 10】 【Chemistry 11】 (In formula 3, L ３ (This represents a divalent hydrocarbon group which may have a heteroatom.)

15. A PCR test kit comprising the nucleic acid extraction kit described in claim 14.

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