Nucleic acid purification method and kit for purifying nucleic acid

Abstract

The present invention relates to a method for purifying a nucleic acid from a sample containing the nucleic acid in a simple, short-time and high-purity manner, and a kit for the purification. This method for purifying a nucleic acid comprises a step for bringing a sample containing a nucleic acid into contact with a zeolite and a proton acceptor.

Description

TECHNICAL FIELD

[0001] The present application relates to a nucleic acid purification method and a kit for purifying nucleic acid. BACKGROUND

[0002] At present, nucleic acid amplification techniques typified by PCR and LAMP have penetrated into all fields of biology represented by molecular biology and medicine, and are widely used in, for example, genetic diagnosis, DNA identification, food inspection, environmental health inspection, inspection of animals and plants, and the like. When amplifying nucleic acid in a sample, it is generally necessary to extract and purify nucleic acid from the sample. As a method for extracting and purifying nucleic acid from a sample, for example, a method using a commercially available nucleic acid extraction kit or the like can be cited. On the other hand, in the case of using a general commercially available nucleic acid extraction kit (for example, QIAamp Viral RNA Mini Kit (manufactured by Qiagen)), a nucleic acid of high purity can be obtained, but on the other hand, various devices such as a high-speed centrifuge and a heater are required, and it is difficult to use the kit in an environment where the work equipment is not complete. Furthermore, the operation steps are as many as several tens of steps, and thus are cumbersome, and it takes about 1 hour to extract and purify nucleic acid (for example, Non-Patent Documents 1 and 2). In nucleic acid amplification techniques, when the number of samples to be examined is large, it is desirable to be able to prepare a sample suitable for nucleic acid amplification in a short time, and thus an extraction and purification method that is cumbersome to operate is difficult to apply. In addition, in the case of using a commercially available nucleic acid extraction kit that is simpler to use (for example, Kanto Chemical Co., Inc. Easy DNA Extraction Kit, Template Prepper for DNA (manufactured by NIPPON GENE), ISOSPIN Viral RNA (manufactured by NIPPON GENE)), although nucleic acid can be extracted and purified in a short time, the purity of the nucleic acid is sometimes low. Furthermore, even in the case of using such a kit, there are cases where heating operation is required, and cases where the operation steps are as many as several tens of steps, and it is difficult to say that nucleic acid can be extracted and purified simply, in a short time, and with high purity. Therefore, there is a demand in the market for a method for extracting and purifying nucleic acid simply, in a short time, and with high purity, without requiring a large device.

[0003] Further, a method for isolating and purifying nucleic acids from a cell lysate or the like using zeolite is also known. For example, Patent Literature 1 discloses a method for preparing a sample for nucleic acid amplification for amplifying nucleic acids contained in a biological specimen, which includes an extraction step of adding a nucleic acid extraction reagent containing an anionic surfactant and / or a base to the biological specimen to obtain a nucleic acid extraction liquid, and a step of contacting the nucleic acid extraction liquid with a zeolite which is a proton type zeolite from which a substance adsorbed to the zeolite is removed. Patent Literature 2 discloses a pretreatment method for clarifying a lysate using a zeolite in a method for isolating nucleic acids from a biological solution such as a cell lysate, and describes that by adding a zeolite to a cell lysate treated with a basic reagent such as sodium dodecyl sulfate (SDS), the SDS or the like is adsorbed, and centrifugal separation is performed, whereby the cell lysate can be clarified.

[0004] Prior Art Documents

[0005] Patent Literature

[0006] Patent Literature 1: Japanese Patent No. 5290987

[0007] Patent Literature 2: Japanese Patent Application Laid-Open No. 2005-531329

[0008] Non-Patent Literature

[0009] Non-Patent Literature 1: National Institute of Infectious Diseases, “Manual for Pathogen Detection (Pathogen Detection Manual) 2019-nCoV Ver. 2.5”, February 15, 2020, https: / / www.niid.go.jp / niid / images / lab-manual / 2019-nCoV20200215.pdf

[0010] Non-Patent Literature 2: QIAGEN KK, “QIAamp (Registered Trademark) Viral RNA Mini Protocol and Troubleshooting”, April 2010, https: / / www.qiagen.com / jp / resources / download.aspx?id=7d6918b1-77dd-4dac-b694-a9ca8aa58b43&lang=ja-JP SUMMARY

[0011] Problems to be Solved by the Invention

[0012] An object of the present application is to provide a method for purifying nucleic acids simply, in a short time, and with high purity from a sample containing nucleic acids, and a kit for the purification.

[0013] Method for Solving the Problem

[0014] The present inventors have found that, when a sample containing nucleic acids is mixed with a zeolite and a proton acceptor such as tris-hydroxymethyl aminomethane, the effect of the zeolite on the removal (adsorption) of substances other than nucleic acids, such as nucleic acid amplification reaction inhibitors, in the sample is unexpectedly improved, thereby completing the present application.

[0015] That is, the present application relates to, for example, the following invention. [1]

[0017] A method for purifying nucleic acids, comprising a step of contacting a sample containing nucleic acids with a zeolite and a proton acceptor. [2]

[0019] The method according to [1], wherein the proton acceptor is a compound represented by General Formula (1),

[0020] (In the formula, R 1 , R 2 , and R 3 each independently are a hydrogen atom, an aliphatic hydrocarbon group having a carbon number of 1 to 6 which can have a substituent, a chain aliphatic hydrocarbon group having a carbon number of 2 to 10 which can have a substituent, and which has a nitrogen atom, a sulfur atom, or an oxygen atom interposed between carbon atoms, the substituent being selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, a sulfonic acid group, an imino group, a guanidine group, an aromatic hydrocarbon group having a carbon number of 6 to 12, and a 3- to 6-membered heterocyclic group which contains at least one atom selected from the group consisting of a nitrogen atom, a sulfur atom, and an oxygen atom as a ring-forming atom and which can have an aliphatic hydrocarbon group having a carbon number of 1 to 6 as a substituent, In the case where the aliphatic hydrocarbon group having a carbon number of 1 to 6 which can have a substituent has an aromatic hydrocarbon group having a carbon number of 6 to 12 as a substituent, the aliphatic hydrocarbon group having a carbon number of 1 to 6 is an aliphatic hydrocarbon group having a carbon number of 2 to 6, and the aromatic hydrocarbon group is not bonded to the carbon atom of the aliphatic hydrocarbon group having a carbon number of 2 to 6 which is closest to the N atom in General Formula (1), R 1 and R 2 , R 2 and R 3 , or R 1 and R 3 may be bonded to form a cyclic structure, in which case the cyclic structure is a 5- to 7-membered ring, and can further contain a nitrogen atom, a sulfur atom, or an oxygen atom, and in the case where the cyclic structure has a double bond, contains a nitrogen atom other than the N atom in General Formula (1), In General Formula (1), the total number of primary amino groups, secondary amino groups, or tertiary amino groups is greater than the total number of -COOH and -SO3H. [3]

[0022] The method according to [1] or [2], wherein the above-mentioned proton acceptor is tris-hydroxymethyl aminomethane and / or 2-morpholinoethanesulfonic acid. [4]

[0024] The method according to any one of [1] to [3], wherein the above-mentioned sample is derived from at least one sample selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal discharge, swab specimen, amniotic fluid and gargle. [5]

[0026] The method according to any one of [1] to [4], wherein at least a part of the above-mentioned nucleic acid is not adsorbed by the zeolite in the above-mentioned contacting step. [6]

[0028] The method according to any one of [1] to [5], wherein the above-mentioned zeolite is at least one selected from the group consisting of offretite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, β-type zeolite and mordenite-type zeolite. [7]

[0030] The method according to any one of [1] to [6], wherein the above-mentioned zeolite is at least one selected from the group consisting of offretite-NH4-type zeolite, offretite-K-type zeolite, ZSM-5-NH4-type zeolite, Y-Na-type zeolite, β-NH4-type zeolite, mordenite-H-type zeolite, mordenite-Na-type and offretite-H-type zeolite. [8]

[0032] The method according to any one of [1] to [7], wherein the above-mentioned nucleic acid is RNA and / or DNA. [9]

[0034] A kit for purifying nucleic acid, comprising a zeolite and a proton acceptor.

[10]

[0036] The kit according to [9], wherein the above-mentioned proton acceptor is a compound represented by the general formula (1),

[0037] (In the formula, R 1 , R 2 and R 3each independently is a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms which can have a substituent, a chain aliphatic hydrocarbon group having 2 to 10 carbon atoms which can have a substituent, and a nitrogen atom, a sulfur atom, or an oxygen atom is interposed between carbon atoms, the above-mentioned substituent is selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, a sulfonic acid group, an imino group, a guanidine group, an aromatic hydrocarbon group having 6 to 12 carbon atoms, and a 3- to 6-membered heterocyclic group, the 3- to 6-membered heterocyclic group contains at least one atom selected from the group consisting of a nitrogen atom, a sulfur atom, and an oxygen atom as a ring-forming atom and can have an aliphatic hydrocarbon group having 1 to 6 carbon atoms as a substituent, In the case where the aliphatic hydrocarbon group having 1 to 6 carbon atoms which can have a substituent has an aromatic hydrocarbon group having 6 to 12 carbon atoms as a substituent, the aliphatic hydrocarbon group having 1 to 6 carbon atoms is an aliphatic hydrocarbon group having 2 to 6 carbon atoms, and the aromatic hydrocarbon group is not bonded to the carbon atom of the aliphatic hydrocarbon group having 2 to 6 carbon atoms which is closest to the N atom in General Formula (1), R 1 R 2 , R 2 R 3 , or R 1 R 3 may be bonded to form a cyclic structure, in this case, the cyclic structure is a 5- to 7-membered ring, and can further contain a nitrogen atom, a sulfur atom, or an oxygen atom, in the case where the above-mentioned cyclic structure has a double bond, a nitrogen atom other than the N atom in General Formula (1) is contained, In General Formula (1), the total number of primary amino groups, secondary amino groups, or tertiary amino groups is greater than the total number of -COOH and -SO3H.

[11]

[0039] The kit according to [9] or

[10] , in which the above-mentioned proton acceptor is tris-hydroxymethyl aminomethane and / or 2-morpholinoethanesulfonic acid.

[12]

[0041] The kit according to any one of [9] to

[11] , in which the above-mentioned zeolite is at least one selected from the group consisting of offretite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, beta-type zeolite, and mordenite-type zeolite.

[13]

[0043] The kit according to any one of [9] to

[12] , in which the above-mentioned zeolite is at least one selected from the group consisting of offretite-NH4-type zeolite, offretite-K-type zeolite, ZSM-5-NH4-type zeolite, Y-Na-type zeolite, beta-NH4-type zeolite, mordenite-H-type zeolite, mordenite-Na-type, and offretite-H-type zeolite.

[14]

[0045] The kit according to any one of [9] to

[13] , wherein the kit is a kit for purifying nucleic acid derived from at least one sample selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal discharge, a swab specimen, amniotic fluid, and a gargle, and the kit further comprises a nucleic acid extraction reagent containing a surfactant.

[15]

[0047] The kit according to any one of [9] to

[14] , wherein the nucleic acid is RNA and / or DNA.

[0048] Effects of the Invention

[0049] According to the present application, a method for purifying nucleic acid from a sample containing the nucleic acid in a simple, short time, and with high purity, and a kit for performing the purification can be provided. In particular, according to the present application, nucleic acid can be purified from a sample containing the nucleic acid in a simple, short time, and with high purity without using various devices such as a high-speed centrifuge and a heater. In addition, the present application can be used for purification of either or both of DNA and RNA, and thus has high versatility. DETAILED DESCRIPTION

[0050] The following describes modes for carrying out the present application in detail. However, the present application is not limited by the following modes.

[0051] [Method for purifying nucleic acid]

[0052] The method for purifying nucleic acid of the present embodiment includes a step of bringing a sample containing nucleic acid into contact with a zeolite and a proton acceptor. The contact step is a step of bringing a sample containing nucleic acid into contact with a zeolite and a proton acceptor, and the zeolite adsorbs substances other than nucleic acid. As shown in the examples described later, by bringing a sample containing nucleic acid into contact with a zeolite and a proton acceptor, the effect of the zeolite adsorbing substances other than nucleic acid is improved, and thus nucleic acid can be purified from a sample containing the nucleic acid in a simple, short time, and with high purity.

[0053] [Sample containing nucleic acid]

[0054] The sample containing nucleic acid is an aqueous solution or an aqueous suspension in which nucleic acid and substances other than nucleic acid are dissolved or suspended. The kind of nucleic acid contained in the sample is not particularly limited, and can be either DNA or RNA, and can be either single-stranded or double-stranded. The origin of the nucleic acid is also not limited, and can be, for example, nucleic acid derived from animals, plants, fungi, bacteria, or viruses. The method of the present application is particularly capable of purifying RNA in a simple and high-purity manner. The pH of the sample containing nucleic acid is not particularly limited.

[0055] As a substance other than nucleic acid, there can be mentioned proteins (e.g., enzymes, glycoproteins, polypeptides, etc.), lipids, sugars (e.g., monosaccharides, disaccharides, oligosaccharides, polysaccharides, etc.), salts, etc.

[0056] In addition, the sample containing nucleic acid can be derived from a specimen obtained from an organism, and can be derived from a specimen obtained from an environment such as water, soil, air, etc. The sample containing nucleic acid is preferably derived from a specimen obtained from an organism, and more preferably derived from at least one specimen selected from the group consisting of blood (whole blood, plasma, or serum), cerebrospinal fluid, urine, feces, sputum, saliva, nasal discharge, swab specimen, amniotic fluid, and gargle. As the sample containing nucleic acid, there can be a diluent / suspension of the above-mentioned specimen or an extract obtained by an extraction step described later, etc. The substance other than nucleic acid can be a component other than nucleic acid derived from these specimens.

[0057] The sample containing nucleic acid can contain a nucleic acid extraction reagent containing a surfactant described later, and, as necessary, an inhibitor against a substance that decomposes nucleic acid. As such an inhibitor, there can be mentioned, for example, a nucleic acid-decomposing enzyme inhibitor, etc.

[0058] <Zeolite>

[0059] "Zeolite" is a general term for aluminosilicates having micropores in crystals, and a three-dimensional network structure in which SiO4 and AlO4 tetrahedra share oxygen is the basic skeleton. Zeolites contain cations such as alkali metal ions, alkaline earth metal ions, ammonium ions, hydrogen ions (protons), etc. in the crystals, and "proton-type zeolite" refers to a zeolite containing hydrogen ions as the cations in the crystals, and "non-proton-type zeolite" refers to a zeolite other than the proton-type zeolite. The zeolite in the purification method of the present embodiment does not adsorb at least a part of the nucleic acid in the sample containing nucleic acid.

[0060] The crystal structure of the zeolite is not particularly limited, and there can be mentioned, for example, A-type, ferrierite-type, MCM-22-type, ZSM-5-type, mordenite-type, L-type, Y-type, X-type, β-type, etc. The zeolite is preferably at least one selected from the group consisting of ferrierite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, β-type zeolite, and mordenite-type.

[0061] The cation in the crystal of the zeolite is not particularly limited. As such a cation, there can be, for example, alkali metal ions (e.g., sodium ions (Na + ), potassium ions (K + ), etc.), alkaline earth metal ions (e.g., magnesium ions (Mg 2+ ), calcium ions (Ca + ), etc.), ammonium ions (NH4 + ), hydrogen ions (H + ), etc. As the cation in the crystal of the zeolite, hydrogen ions (H+ ) other than the cation. That is, as the zeolite, a non-protic zeolite is preferred.

[0062] The zeolite is particularly preferably at least one selected from the group consisting of offretite-NH4 type zeolite, offretite-K type zeolite, ZSM-5-NH4 type zeolite, Y-Na type zeolite, β-NH4 type zeolite, mordenite-H type zeolite, mordenite-Na type, and offretite-H type zeolite, and more preferably at least one selected from the group consisting of offretite-NH4 type zeolite, offretite-K type zeolite, ZSM-5-NH4 type zeolite, Y-Na type zeolite, β-NH4 type zeolite, and mordenite-Na type. For example, the "offretite-NH4 type zeolite" refers to a zeolite having a crystal structure of offretite type and containing an ammonium ion as a cation.

[0063] <Protic Acceptor>

[0064] In the present specification, the "protic acceptor" refers to a compound capable of accepting a proton (hydrogen ion) in a neutral aqueous solution. For example, it refers to a compound capable of accepting (coordination bonding) a proton (H + ) more than the number of moles of the proton that can be supplied (released). In other words, it can be a compound in which the number of moles of the atom or functional group (proton-accepting group) that accepts a proton is more than the number of moles of the atom or functional group (proton-supplying group) that supplies a proton. The proton-accepting group (or electron-supplying group) and the proton-supplying group (or electron-accepting group) can be judged by a person skilled in the art according to the structure of the functional group, and as the proton-accepting group, for example, a primary amino group, a secondary amino group, a tertiary amino group can be cited, and as the proton-supplying group, for example, a carboxyl group (-COOH), a sulfonic acid group (-SO3H), and the like can be cited. For example, tris-hydroxymethyl aminomethane has one amino group as a proton-accepting group and does not have a proton-supplying group. Therefore, the compound as a whole can accept more moles of protons than it can supply, and can be called a protic acceptor. On the other hand, tricine has one amino group as a proton-accepting group and one carboxyl group as a proton-supplying group. Therefore, the compound as a whole can accept the same number of moles of protons as it can supply, and cannot be called a protic acceptor. Note that in the structure of a compound, the proton-accepting group and the proton-supplying group are sometimes difficult to accept or supply a proton due to interaction (e.g., resonance) with the nearby functional group, and therefore, the protic acceptor cannot sometimes be judged only by the number of moles of the proton-accepting group and the proton-supplying group. Therefore, a so-called "lone pair of electrons" is required in order to form the proton-accepting group.

[0065] The protic acceptor can be a compound represented by the following general formula (1).

[0066]

[0067] in the formula, R 1 , R 2 , and R 3 each independently is a hydrogen atom, a carbon number 1-6 aliphatic hydrocarbon group which can have a substituent, a carbon number 2-10 chain aliphatic hydrocarbon group which can have a substituent, and a substituent is selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, a sulfonic acid group, an imino group, a guanidine group, a carbon number 6-12 aromatic hydrocarbon group, and a 3-6 membered heterocyclic group which contains at least one atom selected from the group consisting of a nitrogen atom, a sulfur atom, and an oxygen atom as a ring-constituting atom and can have a carbon number 1-6 aliphatic hydrocarbon group as a substituent, in the case where the carbon number 1-6 aliphatic hydrocarbon group which can have a substituent has a carbon number 6-12 aromatic hydrocarbon group as a substituent, the carbon number 1-6 aliphatic hydrocarbon group is a carbon number 2-6 aliphatic hydrocarbon group, and the aromatic hydrocarbon group is not bonded to the carbon atom of the carbon number 2-6 aliphatic hydrocarbon group which is closest to the N atom in the general formula (1), R 1 and R 2 , R 2 and R 3 , or R 1 and R 3 may be bonded to form a cyclic structure, in this case, the cyclic structure is a 5-7 membered ring, and can further contain a nitrogen atom, a sulfur atom, or an oxygen atom, and in the case where the cyclic structure has a double bond, a nitrogen atom other than the N atom in the general formula (1) is contained. in the general formula (1), the total number of primary amino groups, secondary amino groups, or tertiary amino groups is more than the total number of -COOH and -SO3H.

[0068] R 1 , R 2 , and R 3 may be at least one hydrogen atom, at least two hydrogen atoms, or R 1 , R 2 , and R 3 may all be hydrogen atoms.

[0069] R 1 , R 2 , and R 3 each independently is a carbon number 1-6 aliphatic hydrocarbon group which can have a substituent, the carbon number of the aliphatic hydrocarbon group can be 1-5, 1-4, 1-3, 2, or 1. The aliphatic hydrocarbon group can be a saturated hydrocarbon group, or an unsaturated hydrocarbon group, and can be a chain hydrocarbon group, or a cyclic hydrocarbon group. In the case of a cyclic hydrocarbon group, it can be an alicyclic hydrocarbon group, or an aromatic hydrocarbon ring.

[0070] Examples of aliphatic hydrocarbon groups having 1 to 6 carbon atoms include alkyl groups having 1 to 6 carbon atoms, preferably 1 to 5 carbon atoms; alkenyl groups having 2 to 6 carbon atoms, preferably 2 to 5 carbon atoms; alkynyl groups having 2 to 6 carbon atoms, preferably 2 to 5 carbon atoms; cycloalkyl groups having 3 to 6 carbon atoms, preferably 3 to 5 carbon atoms; cycloalkenyl groups having 3 to 6 carbon atoms, preferably 3 to 5 carbon atoms; and cycloalkynyl groups having 3 to 6 carbon atoms, preferably 3 to 5 carbon atoms.

[0071] R 1 R 2 and R 3 When each aliphatic hydrocarbon group, which is a chain with 2 to 10 carbon atoms and may have substituents, is independently formed by nitrogen, sulfur, or oxygen atoms sandwiched between carbon atoms, the number of carbon atoms in the chain aliphatic hydrocarbon group can be 2 to 9, 3 to 9, 4 to 8, 5 to 7, or 6 to 7. An aliphatic hydrocarbon group with nitrogen, sulfur, or oxygen atoms sandwiched between carbon atoms refers to a group in which, for example, there is a nitrogen atom (-NH-, -NR-, or =N-), a sulfur atom (-S-), or an oxygen atom (-O-) covalently bonded to each carbon atom between the two carbon atoms. There can be one, two, or three nitrogen, sulfur, or oxygen atoms sandwiched between the carbon atoms; additionally, there can be one, two, or three of these. As a chain aliphatic hydrocarbon group, it can be a straight-chain hydrocarbon group or a branched-chain hydrocarbon group.

[0072] Examples of chain-like aliphatic hydrocarbon groups with 2 to 10 carbon atoms, in which nitrogen, sulfur, or oxygen atoms are sandwiched between carbon atoms, include alkyl groups with 2 to 10 carbon atoms (preferably 3 to 10, 4 to 10, 5 to 10, or 6 to 10), alkenyl groups with 2 to 10 carbon atoms (preferably 3 to 10, 4 to 10, 5 to 10, or 6 to 10), alkynyl groups with 2 to 10 carbon atoms (preferably 3 to 10, 4 to 10, 5 to 10, or 6 to 10), cycloalkyl groups with 3 to 10 carbon atoms (preferably 4 to 10, 5 to 10, or 6 to 10), cycloalkenyl groups with 3 to 10 carbon atoms (preferably 4 to 10, 5 to 10, or 6 to 10), and cycloalkynyl groups with 3 to 10 carbon atoms (preferably 4 to 10, 5 to 10, or 6 to 10).

[0073] The above substituent is selected from the group consisting of a hydroxy group, a carboxy group, an amino group, a sulfonic acid group, an imino group, a guanidine group, an aromatic hydrocarbon group having 6 to 12 carbons, and a 3- to 6-membered heterocyclic group having at least one atom selected from the group consisting of a nitrogen atom, a sulfur atom, and an oxygen atom as a ring-constituting atom and having an aliphatic hydrocarbon group having 1 to 6 carbons as a substituent. As the aromatic hydrocarbon group having 6 to 12 carbons, either a monocyclic aromatic hydrocarbon or a polycyclic aromatic hydrocarbon group can be used, and as the aromatic hydrocarbon group, for example, an aromatic hydrocarbon group having 6 to 12 carbons, preferably 6 to 10, 6 to 8 carbons, such as a phenyl group, a tolyl group, a naphthyl group, and the like can be used. As the 3- to 6-membered heterocyclic group having at least one atom selected from the group consisting of a nitrogen atom, a sulfur atom, and an oxygen atom as a ring-constituting atom and having an aliphatic hydrocarbon group having 1 to 6 carbons as a substituent, for example, an imidazolyl group and the like can be used.

[0074] In the case where the aliphatic hydrocarbon group having 1 to 6 carbons which can have a substituent has an aromatic hydrocarbon group having 6 to 12 carbons as a substituent, the aliphatic hydrocarbon group having 1 to 6 carbons is an aliphatic hydrocarbon group having 2 to 6 carbons, and the aromatic hydrocarbon group is not bonded to the carbon atom of the aliphatic hydrocarbon group having 2 to 6 carbons which is closest to the N atom in General Formula (1). In this case, the aromatic hydrocarbon group having 6 to 12 carbons does not resonate with the N atom in General Formula (1), and thus the N atom in General Formula (1) has a lone pair of electrons.

[0075] R 1 R 2 R 2 R 3 R 1 R 3 may be bonded to form a cyclic structure, and in this case, the cyclic structure is a 5- to 7-membered ring, and can further contain a nitrogen atom, a sulfur atom, or an oxygen atom. In the case where the cyclic structure has a double bond, the cyclic structure contains a nitrogen atom other than the N atom in General Formula (1). In the case where the cyclic structure has a double bond, the double bond can resonate with the N atom in General Formula (1), and thus the N atom in General Formula (1) can not have a lone pair of electrons, but by making the cyclic structure have a nitrogen atom other than the N atom in General Formula (1), that is, a proton-accepting group other than the N atom in General Formula (1), the compound of General Formula (1) can be made a proton acceptor. As such a compound, for example, an imidazole and the like can be used. R 1 R 2 R 2 R 3 R 1 R 3 For example, by making R 1 R 2 or R 3The single bond or double bond between two carbon atoms, the single bond between a carbon atom and an oxygen atom or a sulfur atom, or the single bond or double bond between a carbon atom and a nitrogen atom is closed to form a 5- to 7-membered ring structure. As the 5- to 7-membered ring structure, for example, an imidazole ring, a pyrrolidine ring, a piperazine ring, a piperidine ring, a thiomorpholine ring, a morpholine ring, and the like can be given. As specific examples of the compound, for example, imidazole or a derivative thereof, pyrrolidine or a derivative thereof, piperazine or a derivative thereof, piperidine or a derivative thereof, thiomorpholine or a derivative thereof, morpholine or a derivative thereof, and the like can be given.

[0076] In General Formula (1), the total number of primary, secondary, or tertiary amines is greater than the total number of -COOH and -SO3H, for example, by one or more, by two or more, or by three or more. The primary, secondary, or tertiary amines are proton-accepting groups, and -COOH and -SO3H are proton-donating groups. Therefore, when the total number of primary, secondary, or tertiary amines is greater than the total number of -COOH and -SO3H, the compound as a whole is a proton acceptor.

[0077] As described above, the compound represented by General Formula (1) is a proton acceptor, and from the viewpoint of chemical structure, contains a part of an amine or a derivative thereof, a part of an amino acid or a derivative thereof, and the like. As the amine, for example, a primary amine, a secondary amine, or a tertiary amine can be given, and an aliphatic amine, an aromatic amine, or a heterocyclic amine can be given.

[0078] As the aliphatic amine represented by General Formula (1), for example, trimethylolaminomethane, t-butylamine, ethylamine, isopropylamine, ethanolamine, 1,3-bis[tris(hydroxymethyl)methylamino]propane, diethylamine, trimethylamine, triethylamine, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), arginine, lysine, and the like can be given.

[0079] As the heterocyclic amine represented by General Formula (1), for example, imidazole or a derivative thereof, pyrrolidine or a derivative thereof, piperazine or a derivative thereof, piperidine or a derivative thereof, thiomorpholine or a derivative thereof, morpholine or a derivative thereof, histidine, and the like can be given.

[0080] As the derivative of imidazole, for example, 1-methylimidazole, 2-methylimidazole, 4-methylimidazole, 1-ethylimidazole, 2-ethylimidazole, 1-propylimidazole, 2-propylimidazole, 1-isopropylimidazole, 2-isopropylimidazole, 1-butylimidazole, 2-butylimidazole, 1-tert-butylimidazole, 2-tert-butylimidazole, 1,2-dimethylimidazole, 2-ethyl-4-methylimidazole, 2-hydroxymethyl-1-methylimidazole, 4-hydroxymethyl-5-methylimidazole, 2-(1H-imidazol-1-yl)ethanol, 2-mercapto-1-methylimidazole, 1,2,4,5-tetramethylimidazole, and the like can be given.

[0081] As derivatives of piperazine, for example, 1,4-dimethylpiperazine, 2-methylpiperazine, 1-ethylpiperazine (N-ethylpiperazine), 2,5-dimethylpiperazine, 2,6-dimethylpiperazine, 1-amino-4-methylpiperazine, 1-acetylpiperazine, 1-(2-aminoethyl)piperazine, 1-(2-hydroxyethyl)piperazine, 1-butylpiperazine, 1-(methylsulfonyl)piperazine, 1,4-bis(2-hydroxyethyl)piperazine, 1-(2-hydroxyethyl)piperazine-4-(2-ethanesulfonic acid), 1-(2-hydroxyethyl)piperazine-4-(3-propanesulfonic acid), 1-(2-hydroxyethyl)piperazine-4-(2-hydroxy-3-propanesulfonic acid), and the like can be given.

[0082] As derivatives of piperazine, for example, 1,4-dimethylpiperazine, 2-methylpiperazine, 1-ethylpiperazine (N-ethylpiperazine), 2,5-dimethylpiperazine, 2,6-dimethylpiperazine, 1-amino-4-methylpiperazine, 1-acetylpiperazine, 1-(2-aminoethyl)piperazine, 1-(2-hydroxyethyl)piperazine, 1-butylpiperazine, 1-(methylsulfonyl)piperazine, 1,4-bis(2-hydroxyethyl)piperazine, 1-(2-hydroxyethyl)piperazine-4-(2-ethanesulfonic acid), 1-(2-hydroxyethyl)piperazine-4-(3-propanesulfonic acid), 1-(2-hydroxyethyl)piperazine-4-(2-hydroxy-3-propanesulfonic acid), and the like can be given.

[0083] As derivatives of piperazine, for example, 1,4-dimethylpiperazine, 2-methylpiperazine, 1-ethylpiperazine (N-ethylpiperazine), 2,5-dimethylpiperazine, 2,6-dimethylpiperazine, 1-amino-4-methylpiperazine, 1-acetylpiperazine, 1-(2-aminoethyl)piperazine, 1-(2-hydroxyethyl)piperazine, 1-butylpiperazine, 1-(methylsulfonyl)piperazine, 1,4-bis(2-hydroxyethyl)piperazine, 1-(2-hydroxyethyl)piperazine-4-(2-ethanesulfonic acid), 1-(2-hydroxyethyl)piperazine-4-(3-propanesulfonic acid), 1-(2-hydroxyethyl)piperazine-4-(2-hydroxy-3-propanesulfonic acid), and the like can be given.

[0084] As derivatives of piperazine, for example, 1,4-dimethylpiperazine, 2-methylpiperazine, 1-ethylpiperazine (N-ethylpiperazine), 2,5-dimethylpiperazine, 2,6-dimethylpiperazine, 1-amino-4-methylpiperazine, 1-acetylpiperazine, 1-(2-aminoethyl)piperazine, 1-(2-hydroxyethyl)piperazine, 1-butylpiperazine, 1-(methylsulfonyl)piperazine, 1,4-bis(2-hydroxyethyl)piperazine, 1-(2-hydroxyethyl)piperazine-4-(2-ethanesulfonic acid), 1-(2-hydroxyethyl)piperazine-4-(3-propanesulfonic acid), 1-(2-hydroxyethyl)piperazine-4-(2-hydroxy-3-propanesulfonic acid), and the like can be given.

[0085] As derivatives of piperazine, for example, 1,4-dimethylpiperazine, 2-methylpiperazine, 1-ethylpiperazine (N-ethylpiperazine), 2,5-dimethylpiperazine, 2,6-dimethylpiperazine, 1-amino-4-methylpiperazine, 1-acetylpiperazine, 1-(2-aminoethyl)piperazine, 1-(2-hydroxyethyl)piperazine, 1-butylpiperazine, 1-(methylsulfonyl)piperazine, 1,4-bis(2-hydroxyethyl)piperazine, 1-(2-hydroxyethyl)piperazine-4-(2-ethanesulfonic acid), 1-(2-hydroxyethyl)piperazine-4-(3-propanesulfonic acid), 1-(2-hydroxyethyl)piperazine-4-(2-hydroxy-3-propanesulfonic acid), and the like can be given.

[0086] < Contacting Step >

[0087] In the contacting step, the sample containing the nucleic acid is brought into contact with the zeolite and the proton acceptor to adsorb substances other than the nucleic acid. Thus, in the purification method of the present embodiment, the nucleic acid can be purified from the sample containing the nucleic acid by the contacting step. In the present specification, "purification" means improving the purity of the nucleic acid from the sample containing the nucleic acid.

[0088] In the contacting step, the "contacting" can be mixing the sample containing the nucleic acid with the zeolite and the proton acceptor, and for example, the sample containing the nucleic acid can be added to a container in which the zeolite is contained, or the zeolite can be added to the sample containing the nucleic acid, and the order in which the sample containing the nucleic acid is brought into contact with the zeolite and the proton acceptor is not particularly limited.

[0089] The amount of zeolite in the contacting step can be, for example, 50 mg or more, 60 mg or more, 70 mg or more, 80 mg or more, 90 mg or more, 100 mg or more, 110 mg or more, or 120 mg or more per 1 mL of the nucleic acid-containing sample. The amount of zeolite in the contacting step can be, for example, 350 mg or less, 340 mg or less, 330 mg or less, 320 mg or less, 310 mg or less, or 300 mg or less per 1 mL of the nucleic acid-containing sample.

[0090] The amount of the proton acceptor in the contacting step can vary depending on the molar equivalent of the protons that the compound is capable of accepting, and can be, for example, 10 mM or more, 12 mM or more, 14 mM or more, 16 mM or more, 18 mM or more, 20 mM or more, 22 mM or more, 24 mM or more, or 25 mM or more per the total amount of the nucleic acid-containing sample, in the case of tris(hydroxymethyl)aminomethane (Tris). The amount of Tris in the contacting step can be, for example, 300 mM or less, 250 mM or less, 200 mM or less, 150 mM or less, 100 mM or less, or 80 mM or less per the total amount of the nucleic acid-containing sample.

[0091] The contacting time in the contacting step is not particularly limited as long as the nucleic acid-containing sample is sufficiently contacted with the zeolite and the proton acceptor, and can be, for example, 10 seconds to 10 minutes, 20 seconds to 10 minutes, or 30 seconds to 5 minutes.

[0092] By the contacting step, at least a part of the substances other than the nucleic acid in the nucleic acid-containing sample is adsorbed by the zeolite. "Adsorption" refers to a state in which a substance is bound to or encapsulated in the surface or the fine pores of the zeolite, and the adsorbed substance is removed together with the zeolite when the zeolite is removed. By the contacting step, at least 30% or more, at least 40% or more, at least 50% or more, at least 60% or more, at least 70% or more, at least 80% or more, or at least 90% or more of the substances other than the nucleic acid in the nucleic acid-containing sample is adsorbed by the zeolite.

[0093] <Extraction Step>

[0094] The purification method of the present embodiment can include a step (extraction step) of obtaining the sample containing the nucleic acid by mixing the sample with a nucleic acid extraction reagent containing a surfactant and / or by subjecting the sample to a heat treatment, before the above contact step. By the surfactant or the heat treatment, a cell membrane, an envelope of a virus, or the like in the sample is destroyed, and thus the nucleic acid can be dissolved or suspended in the sample containing the nucleic acid. In a case where both the step of mixing the sample with the nucleic acid extraction reagent containing the surfactant and the step of subjecting the sample to the heat treatment are performed, the order thereof is not particularly limited. In a case where the purification method of the present embodiment includes the extraction step, the purification method of the present embodiment can also be understood as an extraction method.

[0095] The surfactant is not particularly limited, and can be an ionic surfactant (anionic surfactant, cationic surfactant), nonionic surfactant, or amphoteric surfactant, and from the viewpoint of more easily destroying a cell membrane, an envelope of a virus, or the like, the surfactant is preferably an ionic surfactant, and more preferably an anionic surfactant. The surfactant included in the nucleic acid extraction reagent can be one or two or more.

[0096] As the anionic surfactant, for example, higher fatty acid salts, straight-chain alkylbenzene sulfonate salts, a-sulfo fatty acid methyl ester salts, a-olefin sulfonate salts, alkyl sulfate salts, polyoxyethylene alkyl sulfate salts, and the like can be exemplified, and the alkyl sulfate salts are preferred, and lithium dodecyl sulfate (LDS), sodium dodecyl sulfate (SDS), or sodium cholate is more preferred.

[0097] As the cationic surfactant, for example, alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkyl dimethyl benzyl ammonium salts, benzyltrimethylammonium salts, and the like can be exemplified, and benzalkonium chloride or the like can be used.

[0098] As the nonionic surfactant, for example, sorbitan fatty acid esters, sucrose fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, polyoxyalkylphenyl ethers, and the like can be exemplified, and TRITON (registered trademark)-X100, Tween (registered trademark) 40, or the like can be used.

[0099] As the amphoteric surfactant, for example, alkylamino fatty acid salts, alkyl betaines, alkyl amine oxides, and the like can be exemplified, and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) can be used.

[0100] The content of the surfactant in the nucleic acid extraction reagent is not particularly limited as long as it is a conventional content that enables extraction of nucleic acids, and can vary depending on the type of surfactant. For example, it can be 0.01 w / v% or more, 0.1 w / v% or more, 0.5 w / v% or more, or 1 w / v% or more, based on the total amount of the nucleic acid extraction reagent. The content of the surfactant in the nucleic acid extraction reagent can be 5 w / v% or less, 4 w / v% or less, or 3 w / v% or less, based on the total amount of the nucleic acid extraction reagent. Note that in the case where the nucleic acid extraction reagent contains a plurality of surfactants, the content of the surfactant in the nucleic acid extraction reagent is the total content of the plurality of surfactants.

[0101] The nucleic acid extraction reagent can contain a base in addition to the surfactant. As the base, for example, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, and aqueous ammonia can be mentioned, and sodium hydroxide is preferred. The base contained in the nucleic acid extraction reagent can be one or two or more.

[0102] The content of the base in the nucleic acid extraction reagent is not particularly limited, and for example, can be 10 mM or more, 25 mM or more, 50 mM or more, 75 mM or more, 100 mM or more, 125 mM or more, 150 mM or more, 175 mM or more, or 200 mM or more, based on the total amount of the nucleic acid extraction reagent, in the case where the base is sodium hydroxide. The content of the base in the nucleic acid-containing reagent is not particularly limited, and for example, can be 500 mM or less, 480 mM or less, 460 mM or less, 440 mM or less, 420 mM or less, or 400 mM or less, based on the total amount of the nucleic acid extraction reagent, in the case where the base is sodium hydroxide.

[0103] The pH of the nucleic acid extraction reagent is not particularly limited as long as it is a conventional pH that does not decompose nucleic acids, and for example, can be 5 to 9, 5 to 7, or 6 to 7.

[0104] The heating treatment is not particularly limited as long as it enables extraction of nucleic acids from the sample, dissolves or suspends the nucleic acids in the nucleic acid-containing sample, and the like, and for example, can be heating treatment of the sample using a heating block or the like. The temperature of the heating treatment can be, for example, 50°C to 200°C, 60°C to 150°C, or 70°C to 100°C. The time of the heating treatment can be, for example, 1 minute to 30 minutes, 5 minutes to 30 minutes, or 15 minutes to 30 minutes.

[0105] <Removal Step>

[0106] The purification method of the present embodiment can include a step of removing the zeolite after the contact step (removal step). In the removal step, the zeolite is removed, and a sample containing the purified nucleic acids is obtained.

[0107] The removal of the zeolite can be performed, for example, by subjecting the mixture of the sample containing the nucleic acid after the contact step, the zeolite, and the proton acceptor to centrifugal separation, filtration, or a combination of these, and the like. The removal by centrifugal separation can be performed, for example, using a commercially available centrifugal column, and thus can be easily performed.

[0108] The sample containing the purified nucleic acid obtained by the removal step can be used, for example, in a nucleic acid amplification analysis or the like.

[0109] [Nucleic acid amplification method]

[0110] The nucleic acid amplification method of the present embodiment includes a step of purifying a nucleic acid by the purification method of the present embodiment, and a step of performing a nucleic acid amplification reaction on the purified nucleic acid (nucleic acid amplification step).

[0111] <Nucleic acid amplification step>

[0112] The nucleic acid amplification step can be performed according to a conventional method. More specifically, it can be performed by incubating a reaction solution containing the purified nucleic acid, a primer set containing a base sequence specific to the nucleic acid, a nucleic acid synthesizing enzyme such as a DNA polymerase, deoxynucleotide triphosphates (dNTPs: dATP, dTTP, dCTP, and dGTP), a reverse transcriptase as needed, a labeled probe for detecting an amplification product, and the like.

[0113] In the case where the nucleic acid is DNA, the nucleic acid amplification reaction can be, for example, a polymerase chain reaction (PCR), a loop-mediated isothermal amplification (LAMP) reaction, a nicking endonuclease amplification reaction (NEAR), a transcription-reverse transcription cooperative (TRC) reaction, a whole genome amplification (WGA) reaction, a strand displacement amplification (SDA) reaction, a helicase-dependent amplification (HDA) reaction, a recombinase polymerase amplification (RPA) reaction, an isothermal chimeric primer amplification (ICAN) reaction, and the like. In the case where the nucleic acid is RNA, the nucleic acid amplification reaction can be a reverse transcription PCR (RT-PCR), a reverse transcription LAMP (RT-LAMP) reaction, a transcription-mediated amplification (TMA) reaction, a nucleic acid sequence-based amplification (NASBA) reaction, or the like.

[0114] [Kit for purifying nucleic acid]

[0115] The kit for purifying a nucleic acid of the present embodiment (hereinafter also referred to as "the purification kit of the present embodiment") includes a zeolite and a proton acceptor. By using the purification kit of the present embodiment, a nucleic acid can be purified simply and with high purity from a sample containing the nucleic acid. The sample containing the nucleic acid, the zeolite, and the proton acceptor are as described above.

[0116] In the case where a sample derived from a sample is used as a sample containing a nucleic acid, the purification kit of the present embodiment can include a nucleic acid extraction reagent containing a surfactant. The nucleic acid extraction reagent containing a surfactant is as described above.

[0117] In the purification kit of the present embodiment, in addition to the zeolite, the proton acceptor, and the nucleic acid extraction reagent containing a surfactant, a buffer, a nucleic acid-decomposing enzyme inhibitor, a device for purifying a nucleic acid (e.g., a centrifugal column, etc.), and the like can be provided.

[0118] [Kit for amplifying a nucleic acid]

[0119] The kit for amplifying a nucleic acid of the present embodiment (hereinafter also referred to as “the nucleic acid amplification kit of the present embodiment”) includes a zeolite, a proton acceptor, a DNA polymerase, and deoxyribonucleotide triphosphates.

[0120] In the nucleic acid amplification kit of the present embodiment, in addition to the reagents included in the purification kit of the present embodiment, various publicly known reagents for performing a nucleic acid amplification reaction can be mixed. In the case where the target nucleic acid is RNA, the nucleic acid amplification kit of the present embodiment can further include a reverse transcriptase. As various other publicly known reagents for performing a nucleic acid amplification reaction, a buffer that provides conditions suitable for an enzyme reaction, an enzyme such as dithiothreitol (DTT), a protecting agent that stabilizes a target nucleic acid sequence, a labeled probe for detecting an amplification product, an intercalating agent, and the like can be exemplified. In addition, the nucleic acid amplification kit of the present embodiment can include a device for detecting nucleic acid amplification.

[0121] Examples

[0122] The present application is more specifically described below based on examples. However, the present application is not limited by these examples.

[0123] [Example 1: Evaluation 1 of effect of zeolite adsorbing substances other than nucleic acids]

[0124] As the zeolite, a chabazite-NH4 type zeolite (Dow Corning Toray Co., Ltd., Model: 720NHA) was used. As the nucleic acid extraction reagent, 0.5% sodium dodecyl sulfate (SDS) or 0.5% SDS + 50 mM Tris-HCl (pH 7.0) was used. As the target nucleic acid solution, SARS-CoV-2 transcriptional RNA (250 copies per test) was used.

[0125] After adding 200 μL of the saliva sample to 800 μL of the nucleic acid extraction reagent and mixing, 240 mg of zeolite (720NHA) was added and further mixed, after which it was passed through a 0.45 μm filter to remove the zeolite and substances adsorbed thereon. The resulting filtrate was used as zeolite-treated samples 1-3 (SDS only) and 1-6 (SDS + Tris-HCl). To 9.5 μL of the zeolite-treated sample, 0.5 μL of the target nucleic acid solution was added, and the target nucleic acid was amplified using the Loopamp (registered trademark) New Coronavirus 2019 (SARS-CoV-2) Test Kit (manufactured by Eiken Chemical Co., Ltd.) at 62.5°C for 35 minutes. The turbidity value that increased as the target nucleic acid (cDNA of SARS-CoV-2 transcription RNA) was amplified was measured in real time using the Loopamp EXIA Amplification Unit (manufactured by Eiken Chemical Co., Ltd.).

[0126] As positive controls, samples 1-1 (SDS only) and 1-4 (SDS + Tris-HCl) obtained by adding distilled water (DW) to the nucleic acid extraction reagent instead of the saliva sample and mixing were used. In addition, as negative controls, zeolite-untreated samples 1-2 (SDS only) and 1-5 (SDS + Tris-HCl) obtained without adding zeolite were used. For these samples, too, amplification of the target nucleic acid was performed using the LAMP method as described above. The results are shown in Table 1. In Table 1, those for which no detection was made within the measurement time (35 minutes) are indicated as “N.D.”

[0127] [Table 1]

[0128] In the case of the sample not containing saliva (positive control), amplification of the target nucleic acid was confirmed regardless of the presence or absence of the buffer, and the detection time was also equivalent (samples 1-1, 1-4). Thus, it was confirmed that the presence or absence of the buffer did not affect the detection time of the target nucleic acid. On the other hand, in the case of the zeolite untreated sample containing the saliva sample (negative control), amplification of the target nucleic acid was not confirmed regardless of the presence or absence of the buffer (samples 1-2, 1-5). This indicates that the nucleic acid cannot be purified in the case where the zeolite is not used and only the buffer is used. Further, in the case of the zeolite treated sample containing the saliva sample (samples 1-3, 1-6), amplification of the target nucleic acid was confirmed when the nucleic acid extraction reagent containing Tris-HCl was used (sample 1-6), and the detection time equivalent to that of the positive control was shown, whereas amplification of the target nucleic acid was not confirmed when the nucleic acid extraction reagent not containing Tris-HCl was used (sample 1-3). According to these results, it was indicated that, in the presence of Tris-HCl, the zeolite can adsorb substances other than the nucleic acid in the sample and remove these substances. That is, it was indicated that, by bringing the sample containing the nucleic acid into contact with the zeolite and Tris-HCl, the nucleic acid can be purified simply, in a short time, and with high purity from the sample containing the nucleic acid.

[0129] [Experiment Example 2: Evaluation 2 of the Effect of Adsorption of Substances Other than Nucleic Acid by Zeolite]

[0130] As the nucleic acid extraction reagent, 0.5% SDS or 0.5% SDS + 50 mM Tris-HCl (pH 7.0) or 0.5% SDS + 50 mM tris(hydroxymethyl)aminomethane (pH 7.0) were used, and otherwise, various samples were prepared in the same manner as in Experiment Example 1. For the obtained samples, amplification of the target nucleic acid (cDNA of SARS-CoV-2 transcription RNA) was performed using Loopamp (registered trademark) New Coronavirus 2019 (SARS-CoV-2) Detection Kit (manufactured by Eiken Chemical Co., Ltd.) at 62.5°C for 35 minutes, and the turbidity value that increased with the amplification of the target nucleic acid was measured in real time using Loopamp EXIA Amplification Unit (manufactured by Eiken Chemical Co., Ltd.). The results thereof are shown in Table 2. In Table 2, those for which detection was not performed within the measurement time (35 minutes) are indicated as “N.D.”.

[0131] [Table 2]

[0132] In the case of the sample not containing saliva, amplification of the target nucleic acid was confirmed even without zeolite treatment (samples 2-1, 2-2, 2-3). The amplification of the nucleic acid was hardly affected by the presence or absence and the kind of the buffer. On the other hand, in the case of the sample containing saliva, amplification of the target nucleic acid was not confirmed in the sample not treated with zeolite regardless of the presence or absence of the buffer (samples 2-4, 2-5, 2-6). Further, in the case of the sample containing saliva treated with zeolite (samples 2-7, 2-8, 2-9), amplification of the target nucleic acid was confirmed when the nucleic acid extraction reagent containing Tris-HCl was used (sample 2-8), and the detection time was shown to be earlier than that of the sample 2-7 not containing the buffer and equivalent to that of the sample 2-2 not containing the saliva sample. On the other hand, when the nucleic acid extraction reagent containing tris(hydroxymethyl)aminomethane was used (sample 2-9), although amplification of the target nucleic acid was confirmed, the detection time was shown to be significantly later than that of the sample 2-3 not containing the saliva sample and equivalent to that of the sample 2-7 not containing the buffer. From these results, the effect of the substance other than the nucleic acid in the sample adsorbed by the zeolite was promoted by the presence of Tris-HCl, but the above-mentioned promoting effect was not found for tris(hydroxymethyl)aminomethane having the same buffering action (pH adjusting action) as Tris-HCl. That is, it was indicated that the promoting effect of the above-mentioned Tris-HCl was not simply based on the pH adjusting action.

[0133] [Experiment 3: Effect of each compound on the substance other than the nucleic acid adsorbed by the zeolite]

[0134] As the nucleic acid extraction reagent, in addition to using 0.5% SDS, a nucleic acid extraction reagent to which the compounds shown in Table 3 were added at 50 mM to 0.5% SDS and the pH was adjusted to 7.0 was used. After 200 μL of a saliva sample was added to 800 μL of each of the nucleic acid extraction reagents and mixed, 240 mg of a magnesium-zeolite-NH4 type zeolite (Tosoh Corporation, type: 720NHA) was added, further mixed, and then passed through a 0.45-μm filter to remove the zeolite and substances adsorbed thereon. The obtained filtrate was used as the zeolite-treated samples 3-3 to 3-40. Similarly, as a positive control, samples 3-1, 3-2 to which distilled water was added instead of the saliva sample were prepared, and as a negative control, zeolite-untreated samples 3-41 to 3-42 obtained without adding the zeolite were prepared. As in Test Example 1, 0.5 μL of SARS-CoV-2 transcriptional RNA (250 copies / test) as a target nucleic acid solution was added to 9.5 μL of each of the obtained samples, and amplification of the target nucleic acid was performed by using Loopamp (registered trademark) New Coronavirus 2019 (SARS-CoV-2) Detection Kit (manufactured by Eiken Chemical Co., Ltd.) at 62.5°C for 35 minutes, and the turbidity value that increased as the target nucleic acid (cDNA of SARS-CoV-2 transcriptional RNA) was amplified was measured in real time using a Loopamp EXIA amplification unit (manufactured by Eiken Chemical Co., Ltd.). The results are shown in Table 4. In Table 4, those for which no detection was made within the measurement time (35 minutes) are shown as “N.D.”

[0135] In the case of the samples 3-1, 3-2 that did not contain saliva, the amplification of the target nucleic acid could be confirmed even without the zeolite. In addition, in the case of Tris alone without zeolite treatment, almost no difference in the detection time from the case where Tris was not present was observed.

[0136] Note that it was confirmed that in the case of the zeolite-untreated samples using any of the nucleic acid extraction reagents, the reaction promotion or the reaction delay caused by the compounds was not observed (part of the data is not shown). Note that in the case of the zeolite-untreated samples containing the saliva sample, in the case of using any of the nucleic acid extraction reagents, no amplification of the nucleic acid (turbidity) was detected within the measurement time (part of the data is not shown).

[0137] [Table 3]

[0138] [Table 4]

[0139] The detection time when using a nucleic acid extraction reagent not containing the test compound (sample 3-3) was 22.1 minutes. Thus, it was shown that the effect of the test compound contained in the sample, which was shorter than the detection time of sample 3-3, i.e., 22.1 minutes, improved the zeolite adsorption of substances other than nucleic acids. As such a compound, the compounds contained in samples 3-4 to 3-13, 3-15 to 3-21, 3-23 to 3-29. These compounds are all proton acceptors, and are compounds represented by the above general formula (1). On the other hand, the compounds contained in samples 3-14, 3-22, 3-30 to 3-40 did not improve the above effect. These compounds are neither proton acceptors nor compounds represented by the above general formula (1).

[0140] Tert-butylamine has a partial structure other than the three hydroxyl groups of Tris. In addition, 1,1,1-tris(hydroxymethyl)ethane has a partial structure other than the amino group of Tris. In samples containing tert-butylamine and 1,1,1-tris(hydroxymethyl)ethane (samples 3-5, 3-36), the effect of improving the zeolite adsorption of substances other than nucleic acids was improved in the case of sample 3-5, and was not improved in the case of sample 3-36. It was thus considered that the reason why the sample containing Tris improved the above effect was related to the amino group possessed by Tris. The amino group is a functional group containing a nitrogen atom, and the nitrogen atom has a lone pair of electrons, so protons are coordinated bonded. In view of such a property, it was considered that one of the reasons why the above effect was improved was that Tris is a proton acceptor.

[0141] In fact, the compounds that improved the above effect were proton acceptors. In addition, aniline, which did not improve the above effect (see sample 3-14), has a nitrogen atom, and an aromatic hydrocarbon group is bonded to the carbon atom closest to the nitrogen atom, so it is not a compound represented by the above general formula (1). In addition, pyrrole (see sample 3-22) has a nitrogen atom, and forms a cyclic structure having a double bond, but does not contain a nitrogen atom other than this, so it is not a compound represented by the above general formula (1). The lone pair of electrons on the nitrogen atom of aniline or pyrrole is delocalized due to a resonance effect, so protons are difficult to coordinate bond, and cannot be called a proton acceptor. Furthermore, among cysteine, glycine, proline, and serine, which did not improve the above effect (see samples 3-30 to 3-33), the number of moles of the carboxylic acid group that donates protons is more than the number of moles of the nitrogen atom that accepts protons, so it is not a compound represented by the above general formula (1), and cannot be called a proton acceptor. In summary, it was considered that whether or not it is a proton acceptor is important in terms of whether or not the effect of the zeolite adsorption of substances other than nucleic acids is improved.

[0142] [Experiment 4: Confirmation of the presence or absence of adsorption of RNA to various zeolites]

[0143] In order to remove a substance that inhibits a nucleic acid amplification reaction (hereinafter referred to as a nucleic acid amplification inhibitor) contained in a nucleic acid extract solution prepared from a biological sample using a zeolite, it is presupposed that a target nucleic acid to be detected is not adsorbed to the zeolite. Therefore, whether or not the target nucleic acid is adsorbed to the zeolite was tested.

[0144] With respect to 1000 μL of a solution in which SARS-CoV-2 transcript RNA was added to a nucleic acid extraction reagent (0.5% SDS, 50 mM Tris-HCl (pH 7.0)), 480 mg of a zeolite described later was added, and mixed by inversion. The zeolite used was a zeolite of type 930NHA (β-NH4 type), 940NHA (β-NH4 type), 840NHA (ZSM-5-NH4 type), 720NHA (Ferrierite-NH4 type), 720KOA (Ferrierite-K type), or 320NAA (Y-Na type) of Toho Chemical Industry Co., Ltd. The suspension after the inversion mixing was passed through a 0.45 μm filter to remove the zeolite and the substance adsorbed thereto, and the obtained filtrate was used as a zeolite-treated sample 4-1 to 4-6. Using 5 μL of the zeolite-treated sample and 20 μL of a quantitative PCR reaction reagent, amplification of the target nucleic acid was performed by a quantitative PCR method. In addition, as a control, a sample 4-7 in which SARS-CoV-2 transcript RNA was added to the nucleic acid extraction reagent without adding the zeolite was prepared, and amplification of the target nucleic acid was similarly performed by the quantitative PCR method.

[0145] Amplification of the target nucleic acid by the quantitative PCR method was performed as follows: using the primer set shown in Table 5, a reaction was performed in a quantitative PCR reaction reagent liquid composed of the components shown in Table 6 at the temperatures and times shown in Table 7, and the fluorescence value that increased with the amplification of the target nucleic acid was measured in real time using a Thermal Cycler Dice (registered trademark) Real Time System III. Note that, in the probe shown in Table 5, the 5' end of the base sequence shown in SEQ ID NO: 3 was labeled with FAM, and the 3' end was labeled with BHQ1. The amplification confirmation results by the quantitative PCR method are shown in Table 8. Note that, in Table 8, the "ratio of the copy number" indicates the ratio when the "copy number without zeolite treatment" is taken as 100%.

[0146] [Table 5]

[0147] [Table 6]

[0148] [Table 7]

[0149] [Table 8]

[0150] In the case of the zeolite-treated samples 4-1 to 4-6, amplification of the target nucleic acid was confirmed, and amplification (copy number) was observed to the same extent as that of the sample 4-7 as a control. This indicates that RNA is hardly adsorbed to the zeolite in the presence of Tris-HCl.

[0151] [Experiment Example 5: Confirmation of presence or absence of adsorption of DNA to various zeolites]

[0152] DNA of Saccharomyces cerevisiae (hereinafter also referred to as “S. cerevisiae”) was added instead of SARS-CoV-2 transcriptional RNA, and otherwise, the same as in Experiment Example 4, to obtain zeolite-treated samples 5-1 to 5-6. In addition, as a control, a sample 5-7 was prepared in which S. cerevisiae DNA was added to the nucleic acid extraction reagent without adding the zeolite. For these samples, amplification of the target nucleic acid (S. cerevisiae DNA) was performed by the quantitative PCR method using the primer and probe set shown in Table 9, the quantitative PCR reaction reagent solution shown in Table 10, and the temperature and time shown in Table 11, and otherwise, the same as in Experiment Example 4, and measurement of the fluorescence value that increases with amplification of the target nucleic acid was performed. Note that, in the probe shown in Table 9, the 5’-terminal of the base sequence shown in SEQ ID NO: 6 was labeled with FAM, and the 3’-terminal was labeled with BHQ1. The amplification confirmation results by the quantitative PCR method are shown in Table 12. Note that, in Table 12, “copy number ratio” indicates the ratio when the “copy number without zeolite treatment” is set to 100%.

[0153] [Table 9]

[0154] [Table 10]

[0155] [Table 11]

[0156] [Table 12]

[0157] In the case of DNA, the same results as in the case of RNA were obtained. In the case of the zeolite-treated samples 5-1 to 5-6, amplification of the target nucleic acid was confirmed, and amplification (copy number) was observed to the same extent as that of the sample 5-7 as a control. This indicates that DNA is hardly adsorbed to the zeolite in the presence of Tris-HCl.

[0158] [Experiment Example 6: Evaluation of extraction and purification of RNA using various zeolites]

[0159] Investigation of whether other types of zeolite can also obtain the promotion of the adsorption effect by Tris-HCl confirmed by zeolite 720NHA.

[0160] As the zeolite, in addition to 720NHA, 690HOA, 642NAA, 840NHA, 722HOA, or 720KOA of Toho Chemical Industry Co., Ltd. was used, and otherwise the same as Test Example 1, zeolite-treated samples 6-5 to 6-16 were obtained. In addition, as a positive control, samples 6-1 to 6-2 were prepared by adding distilled water (DW) instead of the saliva sample and mixing. Further, as a negative control, zeolite-untreated samples 6-3 to 6-4 were prepared without adding zeolite and mixing. For the obtained samples, the same as Test Example 1, Loopamp (registered trademark) New Coronavirus 2019 (SARS-CoV-2) Test Kit (manufactured by Eiken Chemical Co., Ltd.) was used to amplify the target nucleic acid of the SARS-CoV-2 transcription RNA, and the turbidity value that increases with the amplification of the target nucleic acid was measured in real time. The results thereof are shown in Table 13. In Table 13, those for which no detection was made within the measurement time (90 minutes) are indicated as “N.D.”

[0161] [Table 13]

[0162] In the case of samples containing no saliva, amplification of the target nucleic acid could be confirmed even without zeolite treatment (samples 6-1, 6-2). In addition, the presence or absence of the buffer had little effect on the nucleic acid amplification. On the other hand, in the case of the zeolite-untreated samples containing the saliva sample, amplification of the target nucleic acid could not be confirmed regardless of the presence or absence of the buffer (samples 6-3, 6-4). Further, in the case of the zeolite-treated samples (6-5 to 6-16) containing the saliva sample, the detection time of the target nucleic acid was significantly shortened when Tris-HCl was present compared to when Tris-HCl was not present. According to this result, it was shown that by contacting a test sample containing nucleic acid with a zeolite and Tris-HCl, the effect of the zeolite adsorbing substances other than nucleic acid is improved, and nucleic acid can be extracted and purified simply, in a short time, and with high purity.

[0163] [Test Example 7: Evaluation of DNA extraction and purification using various zeolites]

[0164] As the target nucleic acid, DNA of Bordetella pertussis (hereinafter also referred to as "B. pertussis") was used instead of SARS-CoV-2 transcriptional RNA, and 720NHA or 840NHA was used as the zeolite in addition to the same as in Test Example 6, to obtain zeolite-treated samples 7-5 to 7-8. In addition, as a positive control, samples 7-1 to 7-2 were prepared by adding distilled water (DW) instead of a saliva sample and mixing. Further, as a negative control, zeolite-untreated samples 7-3 to 7-4 were prepared without adding the zeolite and mixing. To 11.5 μL of the obtained sample, 11.5 μL of distilled water and 2 μL of a target nucleic acid solution (2000 copies / test) were added, and amplification of the target nucleic acid was performed using Simprova (registered trademark) Respiratory Infection Panel (manufactured by RIKEN CHEMICAL CO., LTD.) at 64°C for 50 minutes, and the fluorescence value that increased with amplification of the target nucleic acid was measured using a Thermal Cycler Dice (registered trademark) Real Time System III. The results are shown in Table 14. In Table 14, those for which no detection was made within the measurement time (50 minutes) are shown as "N.D.".

[0165] [Table 14]

[0166] In the case of a sample containing no saliva, amplification of the target nucleic acid was confirmed even without zeolite treatment (samples 7-1, 7-2). In addition, the presence or absence of the buffer had little effect on nucleic acid amplification. On the other hand, in the case of the zeolite-untreated sample containing a saliva sample, amplification of the target nucleic acid was not confirmed regardless of the presence or absence of the buffer (samples 7-3, 7-4). Further, in the case of the zeolite-treated sample containing a saliva sample (7-5 to 7-8), the detection time of the target nucleic acid was significantly shortened in the presence of Tris-HCl compared to the absence of Tris-HCl. From this result, it was shown that by bringing a test sample containing nucleic acid into contact with a zeolite and Tris-HCl, the adsorption effect of the zeolite on substances other than nucleic acid is improved, and nucleic acid can be extracted and purified simply, in a short time, and with high purity.

[0167] [Test Example 8: Application of RNA purified using a zeolite to PCR]

[0168] The solution 200 μL obtained by adding the influenza A virus to the saliva sample was mixed with the nucleic acid extraction reagent containing 0.5% SDS or the nucleic acid extraction reagent containing 0.5% SDS + 50 mM Tris-HCl (pH 7.0). Next, 240 mg of zeolite of model number 720NHA manufactured by Tosoh Corporation was added, and mixed by inversion. The suspension after the inversion mixing was passed through a 0.45 μm filter to obtain zeolite-treated samples 8-6 and 8-8. In addition, as a positive control, samples 8-1 to 8-4 were prepared by adding physiological saline instead of the saliva sample and mixing. Further, as a negative control, zeolite-unprocessed samples 8-5 and 8-7 were prepared without adding the zeolite and mixing. Using 5 μL of each of the obtained samples and 20 μL of the quantitative PCR reagent, amplification of the target nucleic acid was performed by the quantitative PCR method.

[0169] Amplification of the target nucleic acid by the quantitative PCR method was performed as follows: using the primer set shown in Table 15, the reaction was performed in the quantitative PCR reagent solution shown in Table 6 above at the temperatures and times shown in Table 7 above, and the increase in the fluorescence value with the amplification of the target nucleic acid was measured in real time using a LightCycler (registered trademark) 480 system. Note that the probe composed of the base sequence shown by SEQ ID NO: 9 in Table 15 was labeled with FAM at the 5' end and with MGB at the 3' end. The results are shown in Table 16. Note that in Table 16, the "ratio of the copy number" indicates the ratio when the copy number of sample 8-1 is taken as 100%.

[0170] [Table 15]

[0171] [Table 16]

[0172] In the case of the samples containing physiological saline without the saliva sample (samples 8-1 to 8-4), amplification of the target nucleic acid was confirmed regardless of the presence or absence of the zeolite treatment and Tris-HCl. In the case of the zeolite non-treated samples (samples 8-5 and 8-7) as negative controls, about half of the amplification of the positive control was observed regardless of the presence or absence of Tris-HCl. According to these results, it was indicated that the saliva sample inhibited the nucleic acid amplification. In addition, the sample containing only the influenza virus A and the saliva sample (sample 8-6) detected the target nucleic acid at about half of the copy number of the sample containing physiological saline without the saliva sample (samples 8-1 to 8-4). That is, the sample 8-6 detected the target nucleic acid at the copy number equivalent to that of the negative control (sample 8-5). On the other hand, the sample containing the influenza virus A, the saliva sample, and Tris-HCl (sample 8-8) detected the target nucleic acid at a copy number reaching almost the same level as that of the sample containing physiological saline without the saliva sample (samples 8-1 to 8-4). According to this result, it was indicated that by bringing the test sample containing the nucleic acid into contact with the zeolite and Tris-HCl, the adsorption effect of the zeolite on the substance other than the nucleic acid was improved, and the nucleic acid could be extracted and purified simply, in a short time, and with high purity.

[0173] [Experiment 9: Application of DNA purified using zeolite to PCR]

[0174] A solution obtained by adding Streptococcus pneumoniae (hereinafter also referred to as "S. pneumoniae") to the saliva sample was mixed with a nucleic acid extraction reagent containing 0.5% SDS or a nucleic acid extraction reagent containing 0.5% SDS + 50 mM Tris-HCl (pH 7.0), and 240 mg of zeolite of the type 720NHA manufactured by Tosoh Corporation was added thereto, and the mixture was inverted. The suspension after the inversion was passed through a 0.45 μm filter to obtain zeolite-treated samples 9-6 and 9-8. In addition, as positive controls, samples 9-1 to 9-4 to which physiological saline was added instead of the saliva sample were prepared. Further, as negative controls, zeolite non-treated samples 9-5 and 9-7 to which the zeolite was not added were prepared. Using 5 μL of each of the obtained samples and 20 μL of a quantitative PCR reagent, amplification of the target nucleic acid was performed by a quantitative PCR method.

[0175] The amplification of the target nucleic acid using the quantitative PCR method was performed as follows: using the primer and probe set shown in Table 17, in a quantitative PCR reaction reagent solution consisting of the components shown in Table 18, at the temperatures and times shown in Table 19, the increase in the fluorescence value with the amplification of the target nucleic acid was measured in real time using a LightCycler (registered trademark) 480 system. Note that in the probe shown in Table 17, the 5' end of the base sequence shown as SEQ ID NO: 12 was labeled with FAM and the 3' end was labeled with TAMRA. The results of the amplification confirmation using the quantitative PCR method are shown in Table 20. Note that in Table 20, the "ratio of copy number" indicates the ratio when the copy number of sample 9-1 is taken to be 100%.

[0176] [Table 17]

[0177] [Table 18]

[0178] [Table 19]

[0179] [Table 20]

[0180] In the case of the samples containing physiological saline without the saliva sample (samples 9-1 to 9-4), amplification of the target nucleic acid was confirmed regardless of the presence or absence of zeolite treatment and Tris-HCl. In the case of the zeolite-untreated samples (samples 9-5 and 9-7) as negative controls, the ratio of amplification (copy number) was lower compared to the positive controls, regardless of the presence or absence of Tris-HCl. From these results, it was shown that the saliva sample inhibited nucleic acid amplification. In addition, in the sample containing only S. pneumoniae and the saliva sample in the nucleic acid extraction reagent (sample 9-6), the target nucleic acid was detected only at a low copy number compared to the samples containing physiological saline without the saliva sample (samples 9-1 to 9-4) as positive controls. On the other hand, in the sample containing S. pneumoniae, the saliva sample, and Tris-HCl (sample 9-8), the target nucleic acid was detected at a copy number that was almost the same as that in the samples containing physiological saline without the saliva sample (samples 9-1 to 9-4). From this result, it was also shown that by bringing the sample containing the nucleic acid into contact with the zeolite and Tris-HCl, the adsorption effect of the zeolite on substances other than nucleic acids was improved, and the nucleic acid could be extracted and purified simply, in a short time, and with high purity.

[0181] [Experiment 10: Application of purification of RNA using zeolite in other samples to PCR]

[0182] As the zeolite, mordenite-H type zeolite or ferrierite-NH4 type zeolite (Dioss, type: 690HOA or 720NHA) was used, and the nucleic acid extraction reagent was mixed with various samples (serum, urine, sputum, nasal swab specimen, cerebrospinal fluid, feces, whole blood), and otherwise the same as in Test Example 1, a zeolite-treated sample was obtained. In addition, as a positive control, a sample to which distilled water (DW) was added instead of a saliva sample and mixed was prepared. Further, as a negative control, a zeolite-untreated sample to which no zeolite was added and mixed was prepared. For the obtained sample, the amplification of the target nucleic acid was performed using Loopamp (registered trademark) New Coronavirus 2019 (SARS-CoV-2) Detection Kit (manufactured by Eiken Chemical Co., Ltd.) for SARS-CoV-2 transcription RNA (250 copies / test) as in Test Example 1, and the turbidity value that increased with the amplification of the target nucleic acid was measured in real time. The results thereof are shown in Tables 21 to 26. In Tables 21 to 26, those for which no detection was made within the measurement time (35 minutes) are shown as “N.D.”

[0183] [Table 21]

[0184] [Table 22]

[0185] [Table 23]

[0186] [Table 24]

[0187] [Table 25]

[0188] [Table 26]

[0189] The detection time of the target nucleic acid of the zeolite-treated sample containing Tris-HCl was shortened using any sample.

Claims

1. A method for purifying nucleic acids, comprising the step of contacting a sample containing nucleic acids with zeolite and a proton acceptor.

2. The method according to claim 1, wherein, The proton acceptor is a compound represented by general formula (1). In the formula, R 1 R 2 and R 3 Each of the following is independently composed of a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms that may have substituents, and a chain-like aliphatic hydrocarbon group having 2 to 10 carbon atoms that may have substituents, with nitrogen, sulfur, or oxygen atoms sandwiched between the carbon atoms. The substituents are selected from the group consisting of hydroxyl, carboxyl, amino, sulfonic acid, imino, guanidine, aromatic hydrocarbon groups having 6 to 12 carbon atoms, and 3 to 6-membered heterocyclic groups. The 3 to 6-membered heterocyclic group contains at least one atom selected from the group consisting of nitrogen, sulfur, and oxygen atoms as a constituent atom of the ring and may have an aliphatic hydrocarbon group having 1 to 6 carbon atoms as a substituent. When an aliphatic hydrocarbon group having 1 to 6 carbon atoms as a substituent has an aromatic hydrocarbon group having 6 to 12 carbon atoms as a substituent, the aliphatic hydrocarbon group having 1 to 6 carbon atoms is an aliphatic hydrocarbon group having 2 to 6 carbon atoms, and the aromatic hydrocarbon group is not bonded to the carbon atom of the aliphatic hydrocarbon group having 2 to 6 carbon atoms closest to the N atom in general formula (1). R 1 With R 2 R 2 With R 3 or R 1 With R 3 They can bond to form a ring structure, in which case the ring structure is a 5- to 7-membered ring, and may also contain nitrogen atoms, sulfur atoms or oxygen atoms. If the ring structure has double bonds, it contains nitrogen atoms other than the N atom in general formula (1). In general formula (1), the total number of primary, secondary or tertiary amino groups is greater than the total number of -COOH and -SO3H.

3. The method according to claim 1, wherein, The proton acceptor is tris(hydroxymethyl)aminomethane and / or 2-morpholinoethanesulfonic acid.

4. The method according to any one of claims 1 to 3, wherein, The sample is derived from at least one sample selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal mucus, swab specimens, amniotic fluid, and mouthwash.

5. The method according to any one of claims 1 to 3, wherein, During the contact step, at least a portion of the nucleic acid is not adsorbed by the zeolite.

6. The method according to any one of claims 1 to 3, wherein, The zeolite is selected from at least one group consisting of magnesium-alkali zeolite, ZSM-5 zeolite, Y-type zeolite, β-type zeolite, and mordenite.

7. The method according to any one of claims 1 to 3, wherein, The zeolite is selected from at least one group consisting of magnesium-alkali zeolite-NH4 type zeolite, magnesium-alkali zeolite-K type zeolite, ZSM-5-NH4 type zeolite, Y-Na type zeolite, β-NH4 type zeolite, mordenite-H type zeolite, mordenite-Na type zeolite, and magnesium-alkali zeolite-H type zeolite.

8. The method according to any one of claims 1 to 3, wherein, The nucleic acid is RNA and / or DNA.

9. A kit for purifying nucleic acids, comprising zeolite and a proton acceptor.

10. The kit according to claim 9, wherein, The proton acceptor is a compound represented by general formula (1). In the formula, R 1 R 2 and R 3 Each of the following is independently composed of a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms that may have substituents, and a chain-like aliphatic hydrocarbon group having 2 to 10 carbon atoms that may have substituents, with nitrogen, sulfur, or oxygen atoms sandwiched between the carbon atoms. The substituents are selected from the group consisting of hydroxyl, carboxyl, amino, sulfonic acid, imino, guanidine, aromatic hydrocarbon groups having 6 to 12 carbon atoms, and 3 to 6-membered heterocyclic groups. The 3 to 6-membered heterocyclic group contains at least one atom selected from the group consisting of nitrogen, sulfur, and oxygen atoms as a constituent atom of the ring and may have an aliphatic hydrocarbon group having 1 to 6 carbon atoms as a substituent. When an aliphatic hydrocarbon group having 1 to 6 carbon atoms as a substituent has an aromatic hydrocarbon group having 6 to 12 carbon atoms as a substituent, the aliphatic hydrocarbon group having 1 to 6 carbon atoms is an aliphatic hydrocarbon group having 2 to 6 carbon atoms, and the aromatic hydrocarbon group is not bonded to the carbon atom of the aliphatic hydrocarbon group having 2 to 6 carbon atoms closest to the N atom in general formula (1). R 1 With R 2 R 2 With R 3 or R 1 With R 3 They can bond to form a ring structure, in which case the ring structure is a 5- to 7-membered ring, and may also contain nitrogen atoms, sulfur atoms or oxygen atoms. If the ring structure has double bonds, it contains nitrogen atoms other than the N atom in general formula (1). In general formula (1), the total number of primary, secondary or tertiary amino groups is greater than the total number of -COOH and -SO3H.

11. The kit according to claim 9, wherein, The proton acceptor is tris(hydroxymethyl)aminomethane and / or 2-morpholinoethanesulfonic acid.

12. The kit according to any one of claims 9 to 11, wherein, The zeolite is selected from at least one group consisting of magnesium-alkali zeolite, ZSM-5 zeolite, Y-type zeolite, β-type zeolite, and mordenite.

13. The kit according to any one of claims 9 to 11, wherein, The zeolite is selected from at least one group consisting of magnesium-alkali zeolite-NH4 type zeolite, magnesium-alkali zeolite-K type zeolite, ZSM-5-NH4 type zeolite, Y-Na type zeolite, β-NH4 type zeolite, mordenite-H type zeolite, mordenite-Na type zeolite, and magnesium-alkali zeolite-H type zeolite.

14. The kit according to any one of claims 9 to 11, wherein, The kit is for purifying nucleic acids from at least one sample selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal mucus, swab specimens, amniotic fluid, and mouthwash. The kit also includes a nucleic acid extraction reagent containing a surfactant.

15. The kit according to any one of claims 9 to 11, wherein, The nucleic acid is RNA and / or DNA.

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