Nucleic acid purification method and kit for nucleic acid purification

Abstract

The present invention addresses the problem of providing a method for purifying a nucleic acid easily, in a short time and with a high level of purity, from a sample containing said nucleic acid, and of providing a kit for the purification. A method for purifying a nucleic acid according to the present invention comprises a step for bringing a sample containing a nucleic acid into contact with a zeolite and a proton acceptor.

Description

Nucleic acid purification method and kit for nucleic acid purification

[0001] The present invention relates to a method for purifying nucleic acids and a kit for purifying nucleic acids.

[0002] Currently, nucleic acid amplification techniques, such as PCR and LAMP, have penetrated all fields of biology, including molecular biology and medicine, and are widely used in, for example, genetic diagnosis, DNA testing, food testing, environmental hygiene testing, and animal and plant testing. When amplifying nucleic acids in a sample, it is usually necessary to extract and purify the nucleic acid from the sample. Examples of methods for extracting and purifying nucleic acids from a sample include methods using commercially available nucleic acid extraction kits. On the other hand, when using a typical commercially available nucleic acid extraction kit (e.g., QIAamp Viral RNA Mini Kit (manufactured by Qiagen)), highly pure nucleic acids can be obtained, but multiple devices such as a high-speed centrifuge and a heater are required, making it difficult to use the kit unless the operating equipment is well-equipped. In addition, the operation is complicated, requiring several tens of steps, and it takes approximately one hour to extract and purify nucleic acids (e.g., Non-Patent Documents 1 and 2). In nucleic acid amplification technology, when the number of test samples is large, it is desirable to prepare samples that can be used for nucleic acid amplification in a short time, making it difficult to apply extraction and purification methods that require complicated operations. Furthermore, when using simpler commercially available nucleic acid extraction kits (e.g., Kaneka Simple DNA Extraction Kit (manufactured by Kaneka Corporation), Template Preparer for DNA (manufactured by Nippon Gene Co., Ltd.), ISOSPIN Viral RNA (manufactured by Nippon Gene Co., Ltd.)), nucleic acids can be extracted and purified in a short time, but the purity of the nucleic acids may be low. Furthermore, even when using such kits, heating operations may be required or the operation steps may amount to several tens of steps, making it difficult to say that nucleic acids can be extracted and purified easily and with high purity in a short time. For these reasons, there is a demand in the market for a method for extracting and purifying nucleic acids easily and with high purity in a short time without requiring large equipment.

[0003] Also known are methods for isolating and purifying nucleic acids from cell lysates using zeolites. For example, Patent Document 1 discloses a method for preparing a sample for nucleic acid amplification to be used for amplifying nucleic acids contained in a biological sample, comprising an extraction step of adding a nucleic acid extraction reagent containing an anionic surfactant and / or alkali to a biological sample to obtain a nucleic acid extract, and a step of contacting the nucleic acid extract with zeolite, where the zeolite is a proton-type zeolite and substances adsorbed to the zeolite are removed. Patent Document 2 discloses a method for isolating nucleic acids from a biological solution (e.g., a cell lysate) by pre-treating the clarified lysate with zeolite, which describes that adding zeolite to a cell lysate treated with an alkaline reagent such as sodium dodecyl sulfate (SDS) adsorbs SDS and the like, and then centrifuging the cell lysate to clarify it.

[0004] Patent No. 5290987 Publication Special Publication No. 2005-531329

[0005] National Institute of Infectious Diseases, "Pathogen Detection Manual 2019-nCoV Ver. 2.5," February 15, 2020, https: / / www.niid.go.jp / niid / images / lab-manual / 2019-nCoV20200215.pdf; Qiagen, Inc., "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

[0006] An object of the present invention is to provide a method for purifying nucleic acids to high purity from a sample containing nucleic acids simply and quickly, and a kit for such purification.

[0007] The present inventors unexpectedly discovered that when a sample containing nucleic acids is mixed with zeolite and a proton acceptor such as trishydroxymethylaminomethane, the zeolite is more effective in removing (adsorbing) substances other than nucleic acids, such as inhibitors of nucleic acid amplification reactions, from the sample, and thus completed the present invention.

[0008] That is, the present invention relates to, for example, the following inventions: [1] A method for purifying nucleic acid, the method comprising a step of contacting a sample containing nucleic acid with zeolite and a proton acceptor. [2] The proton acceptor is represented by general formula (1): (In the formula, R １ , R ２ and R ３ are each independently a hydrogen atom, an aliphatic hydrocarbon group of 1 to 6 carbon atoms which may have a substituent, or a chain aliphatic hydrocarbon group of 2 to 10 carbon atoms which may have a substituent and which has a nitrogen atom, a sulfur atom or an oxygen atom interposed between carbon atoms, wherein the 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 guanidyl group, an aromatic hydrocarbon group of 6 to 12 carbon atoms, 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-constituting atom and which may have a substituent which is an aliphatic hydrocarbon group of 1 to 6 carbon atoms; When the optionally substituted aliphatic hydrocarbon group having 1 to 6 carbon atoms has a substituent which is an aromatic hydrocarbon group having 6 to 12 carbon atoms, 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), and R １ and R ２ And, R ２ and R ３ and, or R １ and R ３ and may be bonded to form a cyclic structure, in which case the cyclic structure is a 5- to 7-membered ring and may further contain a nitrogen atom, a sulfur atom or an oxygen atom, and when the cyclic structure has a double bond, it contains a nitrogen atom other than the N atom in general formula (1), and in general formula (1), the total number of primary, secondary or tertiary amino groups is -COOH and -SO ３[3] The method according to [1], wherein the proton acceptor is a compound represented by the formula (I) (wherein the number of protons is greater than the total number of protons of the proton acceptor and the number of protons of the proton acceptor). [3] The method according to [1] or [2], wherein the proton acceptor is trishydroxymethylaminomethane and / or 2-morpholinoethanesulfonic acid. [4] The method according to any one of [1] to [3], wherein the sample is derived from at least one specimen selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal discharge, swab, amniotic fluid, and gargle. [5] The method according to any one of [1] to [4], wherein at least a portion of the nucleic acid is not adsorbed to the zeolite in the contacting step. [6] The method according to any one of [1] to [5], wherein the zeolite is at least one selected from the group consisting of ferrierite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, beta-type zeolite, and mordenite-type zeolite. [7] The zeolite is ferrierite-NH ４ type zeolite, ferrierite-K type zeolite, ZSM-5-NH ４ type zeolite, Y-Na type zeolite, beta-NH ４

[10] The method according to any one of [1] to [6], wherein the zeolite is at least one selected from the group consisting of zeolite type, mordenite-H type, mordenite-Na type, and ferrierite-H type.

[11] The method according to any one of [1] to

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

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

[13] The kit according to any one of [1] to

[17] , wherein the proton acceptor is a zeolite represented by general formula (1): (In the formula, R １ , R ２ and R ３are each independently a hydrogen atom, an aliphatic hydrocarbon group of 1 to 6 carbon atoms which may have a substituent, or a chain aliphatic hydrocarbon group of 2 to 10 carbon atoms which may have a substituent and which has a nitrogen atom, a sulfur atom or an oxygen atom interposed between carbon atoms, wherein the 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 guanidyl group, an aromatic hydrocarbon group of 6 to 12 carbon atoms, 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-constituting atom and which may have a substituent which is an aliphatic hydrocarbon group of 1 to 6 carbon atoms; When the optionally substituted aliphatic hydrocarbon group having 1 to 6 carbon atoms has a substituent which is an aromatic hydrocarbon group having 6 to 12 carbon atoms, 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), and R １ and R ２ And, R ２ and R ３ and, or R １ and R ３ and may be bonded to form a cyclic structure, in which case the cyclic structure is a 5- to 7-membered ring and may further contain a nitrogen atom, a sulfur atom or an oxygen atom, and when the cyclic structure has a double bond, it contains a nitrogen atom other than the N atom in general formula (1), and in general formula (1), the total number of primary, secondary or tertiary amino groups is -COOH and -SO ３

[11] The kit according to [9], wherein the proton acceptor is a compound represented by the formula (I) (wherein the number of protons is greater than the total number of H).

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

[11] , wherein the zeolite is at least one selected from the group consisting of ferrierite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, beta-type zeolite, and mordenite-type zeolite.

[13] The kit according to [9], wherein the zeolite is ferrierite-NH ４type zeolite, ferrierite-K type zeolite, ZSM-5-NH ４ type zeolite, Y-Na type zeolite, beta-NH ４

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

[13] , wherein the nucleic acid is at least one selected from the group consisting of zeolite type, mordenite-H type, mordenite-Na type, and ferrierite-H type.

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

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

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

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

[0009] According to the present invention, a method for purifying nucleic acids to high purity from a sample containing nucleic acids can be provided simply and quickly, and a kit for said purification can be provided. In particular, according to the present invention, nucleic acids can be purified to high purity from a sample containing nucleic acids simply and quickly without using multiple devices such as a high-speed centrifuge and a heater. Furthermore, the present invention is highly versatile because it can be applied to the purification of either or both DNA and RNA.

[0010] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0011] [Method for purifying nucleic acid] The method for purifying nucleic acid according to this embodiment includes a step of contacting a sample containing nucleic acid with zeolite and a proton acceptor. The contacting step is a step of contacting the sample containing nucleic acid with zeolite and a proton acceptor, and the zeolite adsorbs substances other than nucleic acid. As will be shown in the examples below, contacting the sample containing nucleic acid with zeolite and a proton acceptor improves the adsorption effect of zeolite on substances other than nucleic acid, thereby enabling nucleic acid to be purified to high purity from the sample containing nucleic acid simply and in a short time.

[0012] <Sample containing nucleic acid> A sample containing nucleic acid is an aqueous solution or suspension in which nucleic acids and substances other than nucleic acids are dissolved or suspended. The type of nucleic acid contained in the sample is not particularly limited, and may be DNA or RNA, and may be single-stranded or double-stranded. The origin of the nucleic acid is also not limited, and may be, for example, nucleic acid derived from animals, plants, fungi, bacteria, or viruses. The method of the present invention can particularly easily purify RNA with high purity. The pH of the sample containing nucleic acid is not particularly limited.

[0013] Substances other than nucleic acids include proteins (for example, enzymes, glycoproteins, polypeptides, etc.), lipids, sugars (for example, monosaccharides, disaccharides, oligosaccharides, polysaccharides, etc.), salts, and the like.

[0014] Furthermore, the sample containing nucleic acid may be derived from, for example, a specimen obtained from a living organism, or may be derived from a specimen obtained from the environment, such as water, soil, or air. The sample containing nucleic acid is preferably derived from a specimen obtained from a living organism, and more preferably 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, amniotic fluid, and gargle. The sample containing nucleic acid may be a dilution / suspension of the above specimen or an extract obtained by the extraction step described below. The substance other than nucleic acid may be a component other than nucleic acid derived from these specimens.

[0015] The sample containing nucleic acid may contain a nucleic acid extraction reagent containing a surfactant as described below, and may also contain an inhibitor of a substance that degrades nucleic acid, if necessary. Examples of such inhibitors include nuclease inhibitors.

[0016] <Zeolite> "Zeolite" is a general term for aluminosilicates that have micropores in their crystals, and is a type of SiO ４ and AlO ４The basic skeleton of this zeolite is a three-dimensional network structure in which regular tetrahedra of the above structure are bonded by sharing oxygen. Zeolites contain cations, such as alkali metal ions, alkaline earth metal ions, ammonium ions, and hydrogen ions (protons), in their crystals. A "proton-type zeolite" refers to a zeolite that contains hydrogen ions as cations in its crystals, and a "non-proton-type zeolite" refers to a zeolite other than a proton-type zeolite. The zeolite used in the purification method according to this embodiment does not adsorb at least a portion of the nucleic acids in a sample containing nucleic acids.

[0017] The crystal structure of the zeolite is not particularly limited, and examples thereof include A-type, ferrierite-type, MCM-22-type, ZSM-5-type, mordenite-type, L-type, Y-type, X-type, and beta-type zeolite. The zeolite is preferably at least one selected from the group consisting of ferrierite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, beta-type zeolite, and mordenite-type zeolite.

[0018] The cations in the zeolite crystals are not particularly limited. Examples of such cations include alkali metal ions (e.g., sodium ions (Na ＋ ), potassium ions (K ＋ ) and the like), alkaline earth metal ions (e.g., magnesium ions (Mg ２＋ ), calcium ions (Ca ＋ ) etc.), ammonium ion (NH ４ ＋ ), hydrogen ions (H ＋ ) etc. The cations in the zeolite crystals may be hydrogen ions (H ＋ In other words, the zeolite is preferably an aprotic zeolite.

[0019] Zeolites are particularly ferrierite-NH ４ type zeolite, ferrierite-K type zeolite, ZSM-5-NH ４ type zeolite, Y-Na type zeolite, beta-NH ４It is preferable that the zeolite is at least one selected from the group consisting of ferrierite-NH type zeolite, mordenite-H type zeolite, mordenite-Na type zeolite, and ferrierite-H type zeolite. ４ type zeolite, ferrierite-K type zeolite, ZSM-5-NH ４ type zeolite, Y-Na type zeolite, beta-NH ４ It is more preferable that the zeolite is at least one selected from the group consisting of ferrierite-NH type zeolite and mordenite-Na type zeolite. ４ The term "ferrierite-type zeolite" refers to a zeolite having a ferrierite-type crystal structure and containing ammonium ions as cations.

[0020] <Proton Acceptor> In this specification, the term "proton acceptor" refers to a compound that can accept a proton (hydrogen ion) in a neutral aqueous solution. For example, a proton acceptor that can accept (coordinate bond) a proton (H ＋ ) is a compound in which the number of moles of atoms or functional groups that accept protons (proton-accepting groups) is greater than the number of moles of protons that can be donated (released). In other words, the compound may be one in which the number of moles of atoms or functional groups that accept protons (proton-accepting groups) is greater than the number of moles of atoms or functional groups that donate protons (proton-donating groups). A person skilled in the art can determine the proton-accepting group (or electron-donating group) and the proton-donating group (or electron-accepting group) from the structure of the functional group. Examples of proton-accepting groups include primary amino groups, secondary amino groups, and tertiary amino groups, and examples of proton-donating groups include carboxyl groups (-COOH), sulfonic acid groups (-SO ３Examples of functional groups include tris(hydroxymethyl)aminomethane, which has one amino group as a proton-accepting group and no proton-donating groups. Therefore, the number of moles of protons that the compound as a whole can accept is greater than the number of moles of protons that it can donate, and it can be said to be a proton acceptor. On the other hand, tricine has one amino group as a proton-accepting group and one carboxyl group as a proton-donating group. Therefore, the number of moles of protons that the compound as a whole can accept is the same as the number of moles of protons that it can donate, and it cannot be said to be a proton acceptor. Note that in the structure of a compound, proton-accepting groups and proton-donating groups may have difficulty accepting or donating protons due to interactions (e.g., resonance) with nearby functional groups, so the proton acceptor may not be determined simply by the number of moles of proton-accepting groups and proton-donating groups. Therefore, in order to be a proton-accepting group, they must be a so-called "lone pair."

[0021] The proton acceptor may be a compound represented by the following general formula (1): In the formula, R １ , R ２ and R ３ are each independently a hydrogen atom, an aliphatic hydrocarbon group of 1 to 6 carbon atoms which may have a substituent, or a chain aliphatic hydrocarbon group of 2 to 10 carbon atoms which may have a substituent and which has a nitrogen atom, a sulfur atom or an oxygen atom interposed between carbon atoms, wherein the 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 guanidyl group, an aromatic hydrocarbon group of 6 to 12 carbon atoms, 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-constituting atom and which may have a substituent which is an aliphatic hydrocarbon group of 1 to 6 carbon atoms; When the optionally substituted aliphatic hydrocarbon group having 1 to 6 carbon atoms has a substituent which is an aromatic hydrocarbon group having 6 to 12 carbon atoms, 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), and R１ and R ２ And, R ２ and R ３ and, or R １ and R ３ and may be bonded to form a cyclic structure, in which case the cyclic structure is a 5- to 7-membered ring and may further contain a nitrogen atom, a sulfur atom or an oxygen atom, and when the cyclic structure has a double bond, it contains a nitrogen atom other than the N atom in general formula (1), and in general formula (1), the total number of primary, secondary or tertiary amino groups is -COOH and -SO ３ More than the total number of H.

[0022] R １ , R ２ and R ３ At least one or at least two of R １ , R ２ and R ３ may all be hydrogen atoms.

[0023] R １ , R ２ and R ３ are each independently an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may have a substituent, the number of carbon atoms in the aliphatic hydrocarbon group may be 1 to 5, 1 to 4, 1 to 3, 2, or 1. The aliphatic hydrocarbon group may be a saturated hydrocarbon group or an unsaturated hydrocarbon group, a chain hydrocarbon group, or a cyclic hydrocarbon group. When it is a cyclic hydrocarbon group, it may be an alicyclic hydrocarbon group or an aromatic hydrocarbon ring.

[0024] Examples of the aliphatic hydrocarbon group having 1 to 6 carbon atoms include an alkyl group having 1 to 6, preferably 1 to 5, carbon atoms, an alkenyl group having 2 to 6, preferably 2 to 5, carbon atoms, an alkynyl group having 2 to 6, preferably 2 to 5, carbon atoms, a cycloalkyl group having 3 to 6, preferably 3 to 5, carbon atoms, a cycloalkenyl group having 3 to 6, preferably 3 to 5, carbon atoms, and a cycloalkynyl group having 3 to 6, preferably 3 to 5, carbon atoms.

[0025] R １ , R ２ and R ３are each independently a chain aliphatic hydrocarbon group having 2 to 10 carbon atoms, optionally having a substituent, in which a nitrogen atom, sulfur atom, or oxygen atom is interposed between the carbon atoms, the number of carbon atoms in the chain aliphatic hydrocarbon group may be 2 to 9, 3 to 9, 4 to 8, 5 to 7, or 6 to 7. An aliphatic hydrocarbon group in which a nitrogen atom, sulfur atom, or oxygen atom is interposed between the carbon atoms means, for example, that between two carbon atoms in the aliphatic hydrocarbon group, there is present a nitrogen atom (—NH—, —NR—, or ═N—), a sulfur atom (—S—), or an oxygen atom (—O—) covalently bonded to each carbon atom. The number of nitrogen atoms, sulfur atoms, or oxygen atoms interposed between the carbon atoms may be one, two, or three, and may be one, two, or three types of nitrogen atoms, sulfur atoms, or oxygen atoms. The chain aliphatic hydrocarbon group may be a linear hydrocarbon group or a branched hydrocarbon group.

[0026] Examples of the chain aliphatic hydrocarbon group having 2 to 10 carbon atoms in which a nitrogen atom, sulfur atom, or oxygen atom is interposed between carbon atoms include an alkyl group having 2 to 10 carbon atoms, preferably 3 to 10, 4 to 10, 5 to 10, or 6 to 10 carbon atoms; an alkenyl group having 2 to 10 carbon atoms, preferably 3 to 10, 4 to 10, 5 to 10, or 6 to 10 carbon atoms; an alkynyl group having 2 to 10 carbon atoms, preferably 3 to 10, 4 to 10, 5 to 10, or 6 to 10 carbon atoms; a cycloalkyl group having 3 to 10 carbon atoms, preferably 4 to 10, 5 to 10, or 6 to 10 carbon atoms; a cycloalkenyl group having 3 to 10 carbon atoms, preferably 4 to 10, 5 to 10, or 6 to 10 carbon atoms; and a cycloalkynyl group having 3 to 10 carbon atoms, preferably 4 to 10, 5 to 10, or 6 to 10 carbon atoms, in which a nitrogen atom, sulfur atom, or oxygen atom is interposed between carbon atoms.

[0027] The 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 guanidyl group, an aromatic hydrocarbon group having 6 to 12 carbon atoms, and a 3- to 6-membered heterocyclic group containing 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 optionally having a substituent that is an aliphatic hydrocarbon group having 1 to 6 carbon atoms. The aromatic hydrocarbon group having 6 to 12 carbon atoms may be a monocyclic aromatic hydrocarbon or a polycyclic aromatic hydrocarbon group, and the aromatic hydrocarbon group may be, for example, an aromatic hydrocarbon group having 6 to 12 carbon atoms, preferably 6 to 10 or 6 to 8 carbon atoms, such as a phenyl group, a toluyl group, or a naphthyl group. An example of a 3- to 6-membered heterocyclic group containing 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 optionally having a substituent that is an aliphatic hydrocarbon group having 1 to 6 carbon atoms, is an imidazole group.

[0028] When an optionally substituted aliphatic hydrocarbon group having 1 to 6 carbon atoms has a substituent that is an aromatic hydrocarbon group having 6 to 12 carbon atoms, 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 that is closest to the N atom in general formula (1). In this case, the aromatic hydrocarbon group having 6 to 12 carbon atoms does not resonate with the N atom in general formula (1), and therefore the N atom in general formula (1) has a lone pair.

[0029] R １ and R ２ And, R ２ and R ３ and, or R １ and R ３and may be bonded to form a cyclic structure, in which case the cyclic structure is a 5- to 7-membered ring and may further contain a nitrogen atom, a sulfur atom, or an oxygen atom. When the cyclic structure has a double bond, it contains a nitrogen atom other than the N atom in general formula (1). When the cyclic structure has a double bond, the double bond may resonate with the N atom in general formula (1), so the N atom in general formula (1) may not have a lone pair. However, when the cyclic structure has a nitrogen atom other than the N atom in general formula (1), i.e., a proton-accepting group other than the N atom in general formula (1), the compound of general formula (1) can be a proton acceptor. Examples of such compounds include imidazole. R １ and R ２ And, R ２ and R ３ and, or R １ and R ３ For example, R １ , R ２ or R ３ The ring is closed by bonding via a single bond or a double bond between two carbon atoms, a single bond between a carbon atom and an oxygen atom or a sulfur atom, or a single bond between a carbon atom and a nitrogen atom, each of which is derived from the above. Examples of the 5- to 7-membered cyclic structure include an imidazole ring, a pyrrolidine ring, a piperazine ring, a piperidine ring, a thiomorpholine ring, and a morpholine ring. Specific examples of the compound include 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, and morpholine or a derivative thereof.

[0030] In the general formula (1), the total number of primary, secondary or tertiary amino groups is -COOH and -SO ３ The number of H is more than the total number of H, for example, it may be more than one, more than two, or more than three. Primary, secondary, or tertiary amino groups are proton-accepting groups, while -COOH and -SO ３ Since H is a proton donating group, the total number of primary, secondary or tertiary amino groups is -COOH and -SO ３ If the total number of H is greater than the total number of H, the compound as a whole is a proton acceptor.

[0031] As described above, the compound represented by the general formula (1) is a proton acceptor, and in terms of chemical structure, includes some amines or derivatives thereof, some amino acids or derivatives thereof, etc. The amines may be, for example, primary amines, secondary amines, or tertiary amines, and may be aliphatic amines, aromatic amines, or heterocyclic amines.

[0032] Examples of the aliphatic amine, which is a compound represented by the general formula (1), include trishydroxymethylaminomethane, tert-butylamine, ethylamine, isopropylamine, ethanolamine, 1,3-bis[tris(hydroxymethyl)methylamino]propane, diethylamine, trimethylamine, triethylamine, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (bis-tris), arginine, and lysine.

[0033] Examples of the heterocyclic amine, which is a compound represented by the above general formula (1), include 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 histidine.

[0034] Examples of imidazole derivatives include 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, and 1,2,4,5-tetramethylimidazole.

[0035] Examples of piperazine derivatives include 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.

[0036] Examples of piperidine derivatives include 1-methylpiperidine, 2-methylpiperidine, 3-methylpiperidine, 4-methylpiperidine, 1-aminopiperidine, 4-aminopiperidine, 3-hydroxypiperidine, 4-hydroxypiperidine, 2-ethylpiperidine, 2,6-dimethylpiperidine, ethyl 3-piperidinecarboxylate, and ethyl 2-piperidinecarboxylate.

[0037] Examples of pyrrolidine derivatives include 1-methylpyrrolidine, 2-methylpyrrolidine, 3-aminopyrrolidine, 3-hydroxypyrrolidine, 1-ethylpyrrolidine, 3-hydroxy-1-methylpyrrolidine, 3-methoxypyrrolidine, (S)-(+)-2-(hydroxymethyl)pyrrolidine, 3-hydroxymethylpyrrolidine, 3-(dimethylamino)pyrrolidine, and 1-ethyl-3-pyrrolidinol.

[0038] Examples of the thiomorpholine derivatives include thiomorpholine 1,1-dioxide, 4-aminothiomorpholine 1,1-dioxide, and the like.

[0039] Examples of morpholine derivatives include 4-methylmorpholine, 2-methylmorpholine, 4-aminomorpholine (N-aminomorpholine), 4-ethylmorpholine, 2,6-dimethylmorpholine, 4-(2-aminoethyl)morpholine, 4-(2-hydroxyethyl)morpholine, 2-morpholinoethanesulfonic acid, and 3-morpholinepropanesulfonic acid.

[0040] <Contacting Step> In the contacting step, a sample containing nucleic acid is contacted with zeolite and a proton acceptor to adsorb substances other than nucleic acid. Therefore, in the purification method according to this embodiment, nucleic acid can be purified from a sample containing nucleic acid by the contacting step. In this specification, "purification" means increasing the purity of nucleic acid from a sample containing nucleic acid.

[0041] In the contacting step, "contacting" may mean mixing a sample containing nucleic acid with zeolite and a proton acceptor; for example, the sample containing nucleic acid may be added to a container containing zeolite, or zeolite may be added to the sample containing nucleic acid; the order in which the sample containing nucleic acid, zeolite, and proton acceptor are contacted is not particularly limited.

[0042] The amount of zeolite in the contacting step may 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 mL of the sample containing nucleic acid. The amount of zeolite in the contacting step may 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 mL of the sample containing nucleic acid.

[0043] The amount of proton acceptor in the contacting step may vary depending on the molar equivalent of the compound that can accept a proton, and for example, in the case of trishydroxymethylaminomethane (Tris), it may be 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 relative to the total amount of the sample containing nucleic acids. The content of Tris in the contacting step may 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 relative to the total amount of the sample containing nucleic acids.

[0044] The contact time in the contacting step is not particularly limited as long as it allows sufficient contact between the nucleic acid-containing sample, the zeolite, and the proton acceptor, and may be, for example, 10 seconds to 10 minutes, 20 seconds to 10 minutes, or 30 seconds to 5 minutes.

[0045] The contacting step adsorbs at least a portion of substances other than nucleic acids in a sample containing nucleic acids to the zeolite. "Adsorption" refers to a state in which substances are bound or encapsulated on the surface or in the pores of the zeolite, and the adsorbed substances are removed together with the zeolite when the zeolite is removed. The contacting step adsorbs 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 substances other than nucleic acids in a sample containing nucleic acids to the zeolite.

[0046] <Extraction Step> The purification method according to this embodiment may include a step (extraction step) of obtaining a sample containing the nucleic acid by mixing the specimen with a nucleic acid extraction reagent containing a surfactant and / or by heat-treating the specimen before the contact step. Cell membranes, viral envelopes, etc. in the specimen are destroyed by the surfactant or heat treatment, allowing the nucleic acid to be dissolved or suspended in the nucleic acid-containing sample. When both mixing the specimen with the nucleic acid extraction reagent containing a surfactant and heat-treating the specimen are performed, the order of these steps is not particularly limited. When the purification method according to this embodiment includes an extraction step, the purification method according to this embodiment can also be considered an extraction method.

[0047] The surfactant is not particularly limited and may be an ionic surfactant (anionic surfactant, cationic surfactant), a nonionic surfactant, or an amphoteric surfactant, but an ionic surfactant is preferred, and an anionic surfactant is more preferred, from the viewpoint of easier destruction of cell membranes, viral envelopes, etc. The nucleic acid extraction reagent may contain one type of surfactant or two or more types of surfactants.

[0048] Examples of anionic surfactants include higher fatty acid salts, linear alkylbenzene sulfonates, α-sulfofatty acid methyl ester salts, α-olefin sulfonates, alkyl sulfate salts, and polyoxyethylene alkyl sulfate salts. Alkyl sulfate salts are preferred, and lithium dodecyl sulfate (LDS), sodium dodecyl sulfate (SDS), or sodium cholate are more preferred.

[0049] Examples of cationic surfactants include alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkyldimethylbenzylammonium salts, and benzyltrimethylammonium salts, and may also be benzalkonium chloride.

[0050] Examples of nonionic surfactants include 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, and may also include TRITON (registered trademark)-X100, Tween (registered trademark) 40, and the like.

[0051] Examples of amphoteric surfactants include alkylamino fatty acid salts, alkylbetaines, and alkylamine oxides, and may include 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).

[0052] The surfactant content in the nucleic acid extraction reagent may be a normal content sufficient for extracting nucleic acids, and may vary depending on the type of surfactant. For example, based on the total amount of the nucleic acid extraction reagent, the surfactant content may be, for example, 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 surfactant content in the nucleic acid extraction reagent may be, for example, 5 w / v% or less, 4 w / v% or less, or 3 w / v% or less. Note that when multiple types of surfactants are contained in the nucleic acid extraction reagent, the surfactant content in the nucleic acid extraction reagent is the total content of the multiple types of surfactants.

[0053] The nucleic acid extraction reagent may contain an alkali in addition to the surfactant. Examples of alkali include sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, and aqueous ammonia, with sodium hydroxide being preferred. The nucleic acid extraction reagent may contain one type of alkali or two or more types.

[0054] When the alkali is sodium hydroxide, the content of alkali in the nucleic acid extraction reagent may be, for example, 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. When the alkali is sodium hydroxide, the content of alkali in the nucleic acid-containing reagent may be, for example, 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.

[0055] The pH of the nucleic acid extraction reagent is not particularly limited as long as it is a normal pH that does not decompose nucleic acids, but may be, for example, 5-9, 5-7, or 6-7.

[0056] The heat treatment is not particularly limited as long as it can extract nucleic acids from a specimen and dissolve or suspend the nucleic acids in a sample containing the nucleic acids, and may be, for example, heat treatment of the specimen using a heat block, etc. The temperature for the heat treatment may be, for example, 50°C to 200°C, 60°C to 150°C, or 70°C to 100°C. The time for the heat treatment may be, for example, 1 minute to 30 minutes, 5 minutes to 30 minutes, or 15 minutes to 30 minutes.

[0057] <Removal Step> The purification method according to this embodiment may include a step of removing the zeolite (removal step) after the contact step. In the removal step, the zeolite is removed to obtain a sample containing purified nucleic acids.

[0058] The zeolite can be removed, for example, by subjecting the mixture of the nucleic acid-containing sample, zeolite, and proton acceptor after the contact step to centrifugation, filtration, or a combination thereof. Removal by centrifugation can be easily carried out, for example, using a commercially available spin column.

[0059] The sample containing the purified nucleic acid obtained in the removal step can be used, for example, in nucleic acid amplification analysis.

[0060] [Nucleic Acid Amplification Method] The nucleic acid amplification method according to this embodiment comprises a step of purifying nucleic acid by carrying out the purification method according to this embodiment, and a step of performing a nucleic acid amplification reaction on the purified nucleic acid (nucleic acid amplification step).

[0061] <Nucleic Acid Amplification Step> The nucleic acid amplification step can be carried out according to a conventional method. More specifically, the nucleic acid amplification step can be carried out by incubating a reaction solution containing a purified nucleic acid, a primer set containing a base sequence specific to the nucleic acid, a nucleic acid synthesizing enzyme such as DNA polymerase, deoxynucleoside triphosphates (dNTPs: dATP, dTTP, dCTP, and dGTP), if necessary, a reverse transcriptase, a labeled probe for detecting the amplification product, and the like.

[0062] When the nucleic acid is DNA, the nucleic acid amplification reaction may 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 concerted (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, or an isothermal chimeric primer-initiated amplification (ICAN) reaction; when the nucleic acid is RNA, the nucleic acid amplification reaction may be, for example, a reverse transcription PCR (RT-PCR), a reverse transcription LAMP (RT-LAMP) reaction, a transcription-mediated amplification (TMA) reaction, or a nucleic acid sequence-based amplification (NASBA) reaction.

[0063] [Kit for purifying nucleic acid] A kit for purifying nucleic acid according to this embodiment (hereinafter also referred to as "purification kit according to this embodiment") includes a zeolite and a proton acceptor. By using the purification kit according to this embodiment, nucleic acid can be purified easily and with high purity from a sample containing nucleic acid. The sample containing nucleic acid, zeolite, and proton acceptor are as described above.

[0064] When the nucleic acid-containing sample used in the purification kit according to this embodiment is derived from a specimen, the kit may include a nucleic acid extraction reagent containing a surfactant. The nucleic acid extraction reagent containing a surfactant is as described above.

[0065] The purification kit according to this embodiment may include, in addition to the nucleic acid extraction reagent containing zeolite, a proton acceptor, and a surfactant, a buffer solution, a nuclease inhibitor, a device used for purifying nucleic acids (e.g., a spin column, etc.), and the like.

[0066] [Kit for amplifying nucleic acid] The kit for amplifying nucleic acid according to this embodiment (hereinafter also referred to as "nucleic acid amplification kit according to this embodiment") comprises a zeolite, a proton acceptor, a DNA polymerase, and deoxynucleoside triphosphates.

[0067] The nucleic acid amplification kit according to this embodiment may contain known reagents for carrying out various nucleic acid amplification reactions in addition to those contained in the purification kit according to this embodiment. When the target nucleic acid is RNA, the nucleic acid amplification kit according to this embodiment may further contain a reverse transcriptase. Examples of other known reagents for carrying out various nucleic acid amplification reactions include a buffer solution that provides conditions suitable for the enzymatic reaction, an enzyme such as dithiothreitol (DTT), a protective agent that stabilizes the target nucleic acid sequence, a labeled probe for detecting the amplification product, an intercalator, etc. The nucleic acid amplification kit according to this embodiment may also contain a device used to detect nucleic acid amplification.

[0068] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples.

[0069] Test Example 1: Evaluation of the Adsorption Effect of Zeolite on Substances Other Than Nucleic Acids 1. Ferrierite-NH4 zeolite (Tosoh Corporation, Model No. 720NHA) was used as the zeolite. Two types of nucleic acid extraction reagents were used: 0.5% sodium dodecyl sulfate (SDS) or 0.5% SDS + 50 mM trishydroxymethylaminomethane buffer (Tris-HCl) (pH 7.0). SARS-CoV-2 transcript RNA (250 copies / test) was used as the target nucleic acid solution.

[0070] 200 μL of saliva sample was added to 800 μL of nucleic acid extraction reagent and mixed. 240 mg of zeolite (720NHA) was added and further mixed. The mixture was then passed through a 0.45 μm filter to remove the zeolite and any adsorbed substances. The resulting filtrate was designated zeolite-treated sample 1-3 (SDS only) and 1-6 (SDS + Tris-HCl). 0.5 μL of target nucleic acid solution was added to 9.5 μL of the zeolite-treated sample, and the mixture was incubated at 62.5°C for 35 minutes using the Loopamp® Novel Coronavirus 2019 (SARS-CoV-2) Detection Reagent Kit (manufactured by Eiken Chemical Co., Ltd.) to amplify the target nucleic acid. The turbidity increase accompanying the amplification of the target nucleic acid (cDNA of SARS-CoV-2 transcript RNA) was measured in real time using a Loopamp EXIA amplification unit (manufactured by Eiken Chemical Co., Ltd.).

[0071] As positive controls, samples 1-1 (SDS only) and 1-4 (SDS + Tris-HCl) were used, which were prepared by adding distilled water (DW) to the nucleic acid extraction reagent instead of the saliva sample. Furthermore, as negative controls, zeolite-untreated samples 1-2 (SDS only) and 1-5 (SDS + Tris-HCl), obtained without adding zeolite, were used. Target nucleic acids were also amplified by the LAMP method in the same manner as above for these samples. The results are shown in Table 1. In Table 1, samples that could not be detected within the measurement time (35 minutes) are indicated as "N.D."

[0072]

[0073] In samples not containing saliva (positive controls), amplification of target nucleic acids was confirmed regardless of the presence or absence of buffer, and the detection times were similar (Samples 1-1 and 1-4). This confirmed that the presence or absence of buffer does not affect the detection time of target nucleic acids. On the other hand, in samples not treated with zeolite (negative controls) containing saliva specimens, amplification of target nucleic acids was not confirmed regardless of the presence or absence of buffer (Samples 1-2 and 1-5). This suggested that nucleic acids cannot be purified using buffer alone without zeolite. Furthermore, in zeolite-treated samples containing saliva specimens (Samples 1-3 and 1-6), amplification of target nucleic acids was confirmed when a nucleic acid extraction reagent containing Tris-HCl (Sample 1-6) was used, and the detection time was similar to that of the positive control. However, amplification of target nucleic acids was not confirmed when a nucleic acid extraction reagent not containing Tris-HCl (Sample 1-3) was used. These results suggest that zeolite adsorbs and removes substances other than nucleic acids in the sample in the presence of Tris-HCl. That is, it was suggested that nucleic acids can be purified to high purity from a sample containing nucleic acids simply and quickly by contacting the sample with zeolite and Tris-HCl.

[0074] Test Example 2: Evaluation of the Adsorption Effect of Zeolite on Substances Other Than Nucleic Acids (2) Various samples were prepared in the same manner as in Test Example 1, except that three types of nucleic acid extraction reagents were used: 0.5% SDS without buffer, 0.5% SDS + 50 mM Tris-HCl (pH 7.0), or 0.5% SDS + 50 mM Tricine (pH 7.0). The obtained samples were reacted at 62.5°C for 35 minutes using the Loopamp (registered trademark) Novel Coronavirus 2019 (SARS-CoV-2) Detection Reagent Kit (manufactured by Eiken Chemical Co., Ltd.) to amplify the target nucleic acid (cDNA of SARS-CoV-2 transcribed RNA), and the turbidity value that increased with the amplification of the target nucleic acid was measured in real time using a Loopamp EXIA Amplification Unit (manufactured by Eiken Chemical Co., Ltd.). The results are shown in Table 2. In Table 2, those that could not be detected within the measurement time (35 minutes) are indicated as "ND."

[0075]

[0076] In samples not containing saliva, amplification of target nucleic acids was confirmed even without zeolite treatment (Samples 2-1, 2-2, and 2-3). Nucleic acid amplification was hardly affected by the presence or type of buffer. On the other hand, in zeolite-untreated samples containing saliva specimens, amplification of target nucleic acids was not confirmed regardless of the presence or absence of buffer (Samples 2-4, 2-5, and 2-6). Furthermore, in zeolite-treated samples containing saliva specimens (Samples 2-7, 2-8, and 2-9), amplification of target nucleic acids was confirmed when a nucleic acid extraction reagent containing Tris-HCl (Sample 2-8) was used, and the detection time was faster than that of Sample 2-7 without buffer and comparable to that of Sample 2-2 without saliva specimens. On the other hand, when a nucleic acid extraction reagent containing tricine (Sample 2-9) was used, amplification of target nucleic acids was confirmed, but was significantly slower than that of Sample 2-3 without saliva specimens and comparable to that of Sample 2-7 without buffer. These results suggest that the adsorption effect of zeolite on substances other than nucleic acids in samples is enhanced by the presence of Tris-HCl, but this enhancement effect was not observed with tricine, which has a buffering action (pH adjusting action) similar to that of Tris-HCl. This suggests that the enhancement effect of Tris-HCl is not simply due to its pH adjusting action.

[0077] Test Example 3: Effect of each compound on adsorption of substances other than nucleic acids by zeolite As nucleic acid extraction reagents, 0.5% SDS and 0.5% SDS to which the compounds shown in Table 3 below had been added to make 50 mM were used, and the pH was adjusted to 7.0. 200 μL of saliva sample was added to 800 μL of each nucleic acid extraction reagent and mixed, and then ferrierite-NH ４240 mg of type zeolite (Tosoh Corporation, model number: 720NHA) was added and further mixed, and then the mixture was passed through a 0.45 μm filter to remove the zeolite and any substances adsorbed thereto. The resulting filtrate was designated as zeolite-treated samples 3-3 to 3-40. Similarly, as positive controls, samples 3-1 and 3-2 were prepared by adding distilled water instead of the saliva specimen, and as negative controls, zeolite-untreated samples 3-41 to 3-42 were also prepared without adding zeolite. As in Test Example 1, 0.5 μL of the target nucleic acid solution, SARS-CoV-2 transcript RNA (250 copies / test), was added to 9.5 μL of each obtained sample, and the mixture was reacted at 62.5°C for 35 minutes using a Loopamp (registered trademark) Novel Coronavirus 2019 (SARS-CoV-2) Detection Reagent Kit (manufactured by Eiken Chemical Co., Ltd.) to amplify the target nucleic acid. The turbidity value, which increases with the amplification of the target nucleic acid (cDNA of SARS-CoV-2 transcript RNA), 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, samples that could not be detected within the measurement time (35 minutes) are indicated as "N.D."

[0078] In the samples 3-1 and 3-2 that did not contain saliva, amplification of the target nucleic acid was confirmed even without zeolite. Furthermore, when Tris was used alone without zeolite treatment, there was almost no difference in detection time compared to when Tris was not used.

[0079] It was also confirmed that the compounds did not promote or delay the reaction in untreated zeolite samples using any type of nucleic acid extraction reagent (partial data not shown).Neither nucleic acid extraction reagent was used in untreated zeolite samples containing saliva samples, and no nucleic acid amplification (turbidity) was detected within the measurement time (partial data not shown).

[0080]

[0081] When a nucleic acid extraction reagent not containing the test compound was used (Sample 3-3), the detection time was 22.1 minutes. Therefore, in samples with a detection time shorter than 22.1 minutes, which is the detection time of Sample 3-3, it is suggested that the test compound contained therein improves the effect of zeolite on adsorption of substances other than nucleic acids. Such compounds were the compounds contained in Samples 3-4 to 3-13, 3-15 to 3-21, and 3-23 to 3-29. All of these compounds are 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, and 3-30 to 3-40 did not improve the above effect. These compounds were neither proton acceptors nor compounds represented by the above general formula (1).

[0082] Tert-butylamine has a partial structure other than the three hydroxyl groups of Tris. Furthermore, 1,1,1-tris(hydroxymethyl)ethane has a partial structure other than the amino group of Tris. Among samples containing tert-butylamine and 1,1,1-tris(hydroxymethyl)ethane (Samples 3-5 and 3-36), Sample 3-5 improved the effect of zeolite on adsorption of substances other than nucleic acids, while Sample 3-36 did not. From this, it was thought that the amino group possessed by Tris is related to the reason why samples containing Tris improve the above-mentioned effect. The amino group is a functional group containing a nitrogen atom, and since the nitrogen atom has a lone electron pair, a proton forms a coordinate bond. Considering these properties, it is thought that one of the reasons for the improvement in the above-mentioned effect is that Tris acts as a proton acceptor.

[0083] In fact, the compound that improved the above effect was a proton acceptor. Furthermore, aniline (see Sample 3-14), a compound that did not improve the above effect, has a nitrogen atom and an aromatic hydrocarbon group bonded to the carbon atom closest to the nitrogen atom, and therefore is not a compound represented by the above general formula (1). Furthermore, pyrrole (see Sample 3-22) has a nitrogen atom and forms a cyclic structure with a double bond, but does not contain any nitrogen atoms other than the nitrogen atom, and therefore is not a compound represented by the above general formula (1). Since the lone electron pair on the nitrogen atom is delocalized due to the resonance effect, aniline and pyrrole are unlikely to form coordinate bonds with protons, and therefore cannot be considered proton acceptors. Furthermore, cysteine, glycine, proline, and serine (see Samples 3-30 to 3-33), which do not improve the above effect, have a greater number of moles of carboxylic acid groups that donate protons than the number of moles of nitrogen atoms that accept protons, and therefore are not compounds represented by the above general formula (1) and therefore cannot be considered proton acceptors. Thus, whether or not a substance is a proton acceptor is considered to be important in determining whether or not the effect of zeolite on adsorption of substances other than nucleic acids is improved.

[0084] Test Example 4: Confirmation of RNA adsorption to various zeolites In order to remove substances that inhibit nucleic acid amplification reactions (hereinafter referred to as nucleic acid amplification inhibitors) contained in nucleic acid extracts prepared from biological specimens using zeolites, it is necessary that the target nucleic acid to be detected does not adsorb to zeolites. Therefore, tests were conducted to determine whether the target nucleic acid adsorbs to zeolites.

[0085] To 1,000 μL of a solution prepared by adding SARS-CoV-2 transcript RNA to a nucleic acid extraction reagent (0.5% SDS, 50 mM Tris-HCl (pH 7.0)), 480 mg of the zeolite described below was added and mixed by inversion. The zeolite used was Tosoh Corporation, model number 930NHA (beta-NH ４ type), 940NHA (beta-NH ４ type), 840NHA (ZSM-5-NH ４ type), 720NHA (ferrierite-NH ４The zeolites were zeolite type), 720KOA (ferrierite-K type), or 320NAA (Y-Na type). The suspension was mixed by inversion and passed through a 0.45 μm filter to remove the zeolite and any substances adsorbed thereto, and the resulting filtrates were designated zeolite-treated samples 4-1 to 4-6. Using 5 μL of the zeolite-treated sample and 20 μL of quantitative PCR reaction reagent, amplification of the target nucleic acid was performed by quantitative PCR. As a control, sample 4-7 was also prepared in which SARS-CoV-2 transcript RNA was added to the nucleic acid extraction reagent but no zeolite was added, and amplification of the target nucleic acid by quantitative PCR was similarly performed.

[0086] Amplification of the target nucleic acid by quantitative PCR was carried out using the primer set shown in Table 5 in a quantitative PCR reaction reagent solution having the composition shown in Table 6, at the temperature and time shown in Table 7. The fluorescence intensity, which increased with the amplification of the target nucleic acid, was measured in real time using a Thermal Cycler Dice® Real Time System III. The probe shown in Table 5 was labeled with FAM at the 5' end and BHQ1 at the 3' end of the base sequence shown in SEQ ID NO: 3. The results of confirmation of amplification by quantitative PCR are shown in Table 8. In Table 8, the "copy number ratio" indicates the ratio when the copy number for "without zeolite treatment" is set to 100%.

[0087]

[0088]

[0089]

[0090]

[0091] Amplification of the target nucleic acid was confirmed in zeolite-treated samples 4-1 to 4-6, and the amplification (copy number) was similar to that of the control sample 4-7, suggesting that almost no RNA is adsorbed to zeolite in the presence of Tris-HCl.

[0092] Test Example 5: Confirmation of DNA Adsorption to Various Zeolites Zeolite-treated samples 5-1 to 5-6 were obtained in the same manner as in Test Example 4, except that Saccharomyces cerevisiae (hereinafter also referred to as "S. cerevisiae") DNA was added instead of SARS-CoV-2 transcript RNA. Additionally, as a control, sample 5-7 was prepared in which S. cerevisiae DNA was added to the nucleic acid extraction reagent without adding zeolite. These samples were subjected to quantitative PCR amplification of the target nucleic acid (S. cerevisiae DNA) in the same manner as in Test Example 4, except that the primer and probe sets shown in Table 9, the quantitative PCR reaction reagent solutions shown in Table 10, and the temperatures and times shown in Table 11 were used for the reactions. The fluorescence intensity, which increased with the amplification of the target nucleic acid, was measured in real time. The probe shown in Table 9 was labeled with FAM at the 5' end and BHQ1 at the 3' end of the base sequence shown in SEQ ID NO: 6. The results of confirmation of amplification by quantitative PCR are shown in Table 12. In Table 12, the "copy number ratio" indicates the ratio when the copy number in the "no zeolite treatment" case is set to 100%.

[0093]

[0094]

[0095]

[0096]

[0097] Similar results were obtained with DNA as with RNA. Amplification of the target nucleic acid was confirmed in zeolite-treated samples 5-1 to 5-6, and amplification (copy number) was observed to be comparable to that of the control sample 5-7. This suggests that almost no DNA is adsorbed to zeolite in the presence of Tris-HCl.

[0098] Test Example 6: Evaluation of RNA extraction and purification using various zeolites It was investigated whether the enhancement of the adsorption effect by Tris-HCl observed with zeolite 720NHA could also be obtained with other types of zeolites.

[0099] Zeolite-treated samples 6-5 to 6-16 were obtained in the same manner as in Test Example 1, except that in addition to 720NHA, Tosoh Corporation's model numbers 690HOA, 642NAA, 840NHA, 722HOA, or 720KOA were used as the zeolite. Furthermore, as positive controls, samples 6-1 to 6-2 were prepared by adding and mixing distilled water (DW) instead of the saliva sample. Furthermore, as negative controls, zeolite-untreated samples 6-3 to 6-4 were also prepared by mixing without adding zeolite. For the obtained samples, target nucleic acid amplification was performed for SARS-CoV-2 transcript RNA using the Loopamp (registered trademark) Novel Coronavirus 2019 (SARS-CoV-2) Detection Reagent Kit (manufactured by Eiken Chemical Co., Ltd.) in the same manner 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 are shown in Table 13. In Table 13, those that could not be detected within the measurement time (90 minutes) are indicated as "ND."

[0100]

[0101] In samples not containing saliva, amplification of target nucleic acids was confirmed even without zeolite treatment (Samples 6-1 and 6-2). Furthermore, nucleic acid amplification was hardly affected by the presence or absence of buffer. On the other hand, in zeolite-untreated samples containing saliva specimens, amplification of target nucleic acids was not confirmed, regardless of the presence or absence of buffer (Samples 6-3 and 6-4). Furthermore, in all of the zeolite-treated samples containing saliva specimens (6-5 to 6-16), the detection time for target nucleic acids was significantly shorter in the presence of Tris-HCl than in the absence of Tris-HCl. These results suggest that contacting a sample containing nucleic acids with zeolite and Tris-HCl improves the adsorption effect of zeolite on substances other than nucleic acids, enabling nucleic acids to be extracted and purified easily and with high purity in a short period of time.

[0102] Test Example 7: Evaluation of DNA extraction and purification using various zeolites Zeolite-treated samples 7-5 to 7-8 were obtained in the same manner as Test Example 6, except that instead of SARS-CoV-2 transcript RNA, Bordetella pertussis (hereinafter also referred to as "B. pertussis") DNA was used as the target nucleic acid, and 720NHA or 840NHA was used as the zeolite. Furthermore, as positive controls, samples 7-1 and 7-2 were prepared by adding and mixing distilled water (DW) instead of the saliva sample. Furthermore, as negative controls, untreated samples 7-3 and 7-4 were also prepared by mixing without adding zeolite. To 11.5 μL of the resulting sample, 11.5 μL of distilled water and 2 μL of target nucleic acid solution (2,000 copies / test) were added, and the mixture was reacted at 64°C for 50 minutes using a Simprova® Respiratory Infection Panel (manufactured by Eiken Chemical Co., Ltd.) to amplify the target nucleic acid. The fluorescence intensity, which increased with amplification of the target nucleic acid, was measured using a Thermal Cycler Dice® Real Time System III. The results are shown in Table 14. In Table 14, samples that were not detected within the measurement time (50 minutes) are indicated as "N.D."

[0103]

[0104] In samples not containing saliva, amplification of target nucleic acids was confirmed even without zeolite treatment (Samples 7-1 and 7-2). Furthermore, nucleic acid amplification was hardly affected by the presence or absence of buffer. On the other hand, in zeolite-untreated samples containing saliva specimens, amplification of target nucleic acids was not confirmed, regardless of the presence or absence of buffer (Samples 7-3 and 7-4). Furthermore, in all of the zeolite-treated samples containing saliva specimens (7-5 ​​to 7-8), the detection time for target nucleic acids was significantly shorter in the presence of Tris-HCl than in the absence of Tris-HCl. These results suggest that contacting a sample containing nucleic acids with zeolite and Tris-HCl improves the adsorption effect of zeolite on substances other than nucleic acids, enabling nucleic acids to be extracted and purified easily and with high purity in a short period of time.

[0105] Test Example 8: Application of Zeolite-Purified RNA to PCR. 200 μL of a solution obtained by adding influenza A virus to a 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). Next, 240 mg of zeolite (Model No. 720NHA, manufactured by Tosoh Corporation) was added and mixed by inversion. The suspension mixed by inversion was passed through a 0.45 μm filter to obtain zeolite-treated samples 8-6 and 8-8. Additionally, as positive controls, samples 8-1 to 8-4 were prepared by adding physiological saline instead of the saliva sample. Furthermore, as negative controls, zeolite-untreated samples 8-5 and 8-7 were also prepared by mixing without adding zeolite. 5 μL of each of the obtained samples and 20 μL of quantitative PCR reaction reagent were used to amplify target nucleic acids by quantitative PCR.

[0106] The target nucleic acid was amplified by quantitative PCR using the primer set shown in Table 15, in the quantitative PCR reaction reagent solution shown in Table 6, at the temperature and time shown in Table 7. The fluorescence intensity, which increased with the amplification of the target nucleic acid, was measured in real time using a LightCycler (registered trademark) 480 System. The probe consisting of the base sequence shown in SEQ ID NO: 9 in Table 15 was labeled with FAM at the 5' end and MGB at the 3' end. The results are shown in Table 16. In Table 16, the "copy number ratio" indicates the ratio when the copy number of sample 8-1 is set to 100%.

[0107]

[0108]

[0109] In samples containing saline, but not saliva, amplification of target nucleic acids was confirmed (Samples 8-1 to 8-4), regardless of the presence or absence of zeolite treatment and Tris-HCl. In the negative control samples (Samples 8-5 and 8-7) that were not treated with zeolite, amplification was only about half that of the positive control, regardless of the presence or absence of Tris-HCl. These results suggest that nucleic acid amplification is inhibited by saliva. Furthermore, in a sample containing only influenza A virus and saliva (Sample 8-6), target nucleic acids were detected at only about half the copy number of samples containing saline, but not saliva (Samples 8-1 to 8-4). That is, target nucleic acids were detected in Sample 8-6 only at a copy number similar to that of the negative control (Sample 8-5). On the other hand, in a sample containing influenza A virus, saliva, and Tris-HCl (Sample 8-8), target nucleic acids were detected at copy numbers similar to those of samples containing saline, but not saliva (Samples 8-1 to 8-4). These results also suggest that contacting a sample containing nucleic acids with zeolite and Tris-HCl improves the adsorption effect of zeolite on substances other than nucleic acids, enabling nucleic acids to be extracted and purified simply, quickly, and with high purity.

[0110] Test Example 9: Application of DNA purified by zeolite to PCR. 200 μL of a solution obtained by adding Streptococcus pneumoniae (hereinafter also referred to as "S. pneumoniae") to a 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 (model number 720NHA) manufactured by Tosoh Corporation was added and mixed by inversion. The suspension mixed by inversion was passed through a 0.45 μm filter to obtain zeolite-treated samples 9-6 and 9-8. Additionally, as positive controls, samples 9-1 to 9-4 were prepared by adding and mixing physiological saline instead of the saliva sample. Furthermore, as negative controls, zeolite-untreated samples 9-5 and 9-7 were also prepared. Using 5 μL of each of the obtained samples and 20 μL of quantitative PCR reaction reagent, amplification of the target nucleic acid was carried out by quantitative PCR.

[0111] Amplification of the target nucleic acid by quantitative PCR was carried out using the primer and probe set shown in Table 17 in a quantitative PCR reaction reagent solution having the composition shown in Table 18, at the temperature and time shown in Table 19. The fluorescence intensity, which increased with the amplification of the target nucleic acid, was measured in real time using a LightCycler® 480 System. The probe shown in Table 17 was labeled with FAM at the 5' end and TAMRA at the 3' end of the base sequence shown in SEQ ID NO: 12. The results of confirmation of amplification by quantitative PCR are shown in Table 20. In Table 20, the "copy number ratio" indicates the ratio when the copy number of sample 9-1 is set to 100%.

[0112]

[0113]

[0114]

[0115]

[0116] In samples containing saline, rather than saliva, amplification of the target nucleic acid was confirmed (Samples 9-1 to 9-4), regardless of the presence or absence of zeolite treatment and Tris-HCl. The negative control samples (Samples 9-5 and 9-7) that were not treated with zeolite had a lower amplification (copy number) rate than the positive control, regardless of the presence or absence of Tris-HCl. These results suggest that nucleic acid amplification is inhibited by saliva. Furthermore, in a sample (Sample 9-6) containing only S. pneumoniae and a saliva sample in the nucleic acid extraction reagent, target nucleic acid was detected at a lower copy number than the positive control samples (Samples 9-1 to 9-4) containing saline rather than saliva. On the other hand, in a sample (Sample 9-8) containing S. pneumoniae, a saliva sample, and Tris-HCl, target nucleic acid was detected at a copy number similar to that of the samples (Samples 9-1 to 9-4) containing saline rather than saliva. These results also suggest that contacting a sample containing nucleic acids with zeolite and Tris-HCl improves the adsorption effect of zeolite on substances other than nucleic acids, enabling nucleic acids to be extracted and purified simply, quickly, and with high purity.

[0117] [Test Example 10: Application of RNA purification by zeolite to PCR in other samples] As the zeolite, mordenite-H type zeolite or ferrierite-NH ４Zeolite-treated samples were obtained in the same manner as in Test Example 1, except that a type zeolite (Tosoh Corporation, model number: 690HOA or 720NHA) was used and various specimens (serum, urine, sputum, nasal swab, cerebrospinal fluid, feces, whole blood) were mixed with the nucleic acid extraction reagent. A positive control sample was prepared by adding distilled water (DW) instead of the saliva specimen. Furthermore, a negative control sample was prepared by mixing without adding zeolite. For the obtained sample, target nucleic acid amplification was performed on SARS-CoV-2 transcript RNA (250 copies / test) using the Loopamp® Novel Coronavirus 2019 (SARS-CoV-2) Detection Reagent Kit (manufactured by Eiken Chemical Co., Ltd.) in the same manner 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 are shown in Tables 21 to 26. In Tables 21 to 26, those that were not detected within the measurement time (35 minutes) are indicated as "ND."

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] Regardless of the specimen used, the detection time of the target nucleic acid was shortened in the zeolite-treated sample containing Tris-HCl.

Claims

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

2. The proton acceptor is represented by the general formula (1) (In the formula, R １ , R ２ and R ３ are each independently a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may have a substituent, or a chain aliphatic hydrocarbon group having 2 to 10 carbon atoms which may have a substituent and which has a nitrogen atom, a sulfur atom or an oxygen atom interposed between the carbon atoms, said 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 guanidyl group, an aromatic hydrocarbon group having 6 to 12 carbon atoms, 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-constituting atom and which may have a substituent which is an aliphatic hydrocarbon group having 1 to 6 carbon atoms, When the optionally substituted aliphatic hydrocarbon group having 1 to 6 carbon atoms has a substituent which is an aromatic hydrocarbon group having 6 to 12 carbon atoms, 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), １ and R ２ And, R ２ and R ３ And, or R １ and R ３ and may be bonded to form a cyclic structure, in which case the cyclic structure is a 5- to 7-membered ring and may further contain a nitrogen atom, a sulfur atom or an oxygen atom, and when the cyclic structure has a double bond, it contains a nitrogen atom other than the N atom in general formula (1), and in general formula (1), the total number of primary, secondary or tertiary amino groups is -COOH and -SO ３ The method of claim 1, wherein the compound is a compound represented by the formula:

3. The method of claim 1, wherein the proton acceptor is trishydroxymethylaminomethane 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 specimen selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal discharge, swabs, amniotic fluid, and gargle.

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

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

7. The zeolite is ferrierite-NH ４ type zeolite, ferrierite-K type zeolite, ZSM-5-NH ４ type zeolite, Y-Na type zeolite, beta-NH ４ 4. The method according to claim 1, wherein the zeolite is at least one selected from the group consisting of mordenite-H type zeolite, mordenite-Na type zeolite, and ferrierite-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 a zeolite and a proton acceptor.

10. The proton acceptor is represented by the general formula (1) (In the formula, R １ , R ２ and R ３ are each independently a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms which may have a substituent, or a chain aliphatic hydrocarbon group having 2 to 10 carbon atoms which may have a substituent and which has a nitrogen atom, a sulfur atom or an oxygen atom interposed between the carbon atoms, said 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 guanidyl group, an aromatic hydrocarbon group having 6 to 12 carbon atoms, 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-constituting atom and which may have a substituent which is an aliphatic hydrocarbon group having 1 to 6 carbon atoms, When the optionally substituted aliphatic hydrocarbon group having 1 to 6 carbon atoms has a substituent which is an aromatic hydrocarbon group having 6 to 12 carbon atoms, 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), １ and R ２ And, R ２ and R ３ And, or R １ and R ３ and may be bonded to form a cyclic structure, in which case the cyclic structure is a 5- to 7-membered ring and may further contain a nitrogen atom, a sulfur atom or an oxygen atom, and when the cyclic structure has a double bond, it contains a nitrogen atom other than the N atom in general formula (1), and in general formula (1), the total number of primary, secondary or tertiary amino groups is -COOH and -SO ３ The kit according to claim 9, wherein the compound is represented by the formula:

11. The kit of claim 9, wherein the proton acceptor is trishydroxymethylaminomethane and / or 2-morpholinoethanesulfonic acid.

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

13. The zeolite is ferrierite-NH ４ type zeolite, ferrierite-K type zeolite, ZSM-5-NH ４ type zeolite, Y-Na type zeolite, beta-NH ４ The kit according to any one of claims 9 to 11, wherein the zeolite is at least one selected from the group consisting of mordenite-H type zeolite, mordenite-Na type zeolite, and ferrierite-H type zeolite.

14. The kit according to any one of claims 9 to 11, which is a kit for purifying nucleic acid derived from at least one specimen selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal discharge, swabs, amniotic fluid, and gargle, and further comprises 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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