WO2016175239A1 - Method for purifying nucleic acid from specimen including colloidal particles - Google Patents

Abstract

The present invention addresses the problem of providing a method for purifying a nucleic acid from a specimen including colloidal particles in a highly-efficient manner, and an agent therefor. The present invention solves said problem by means of: a solubilizer including either or both of (i) an amide type surfactant and (ii) one or more amphipathic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphipathic organic solvents, and (iii) a chaotropic agent; and a method for purifying a nucleic acid using the solubilizing reagent.

Description

Nucleic acid purification method from specimens containing colloidal particles

The present invention relates to a method for purifying nucleic acid from a specimen containing colloidal particles.

Genetic testing is a method for testing specimens based on qualitative or quantitative analysis of nucleic acids. Genetic testing is an industrially important testing method applied in various fields such as medical care, agriculture, livestock and fisheries, and quality testing because it can determine results with high sensitivity and in a short time. For this test, it is necessary to purify the nucleic acid from the specimen, and the most widely used nucleic acid purification method is the bind-elut method, which applies the property that the nucleic acid binds to the nucleic acid adsorption carrier in the presence of a chaotropic agent. (Patent Document 1). This purification method generally includes (a) a lysis step for dissolving a sample and elution of a nucleic acid, (b) an adsorption step for binding a nucleic acid to a nucleic acid adsorption carrier, and (c) washing a nucleic acid bound to the nucleic acid adsorption carrier. A washing step, and (d) an elution step for elution of nucleic acid bound to the nucleic acid adsorption carrier. In particular, a method using a spin column is an excellent method by which high-purity nucleic acid can be easily purified. However, it has been known that when nucleic acid is purified from a specimen containing a large amount of colloidal particles such as blood, the nucleic acid recovery efficiency is greatly reduced. This is because colloidal particles or aggregates contained in the specimen physically block the binding between the nucleic acid and the nucleic acid adsorption carrier. In particular, in the method using a spin column, the aggregate of colloidal particles causes clogging of the column, and the nucleic acid recovery efficiency is significantly reduced.

As a technique to solve this problem, there is a technique that separates and removes colloidal particles contained in the specimen by high-speed centrifugation to suppress the decrease in nucleic acid recovery efficiency. However, the high-speed centrifugation operation is complicated, and there is a problem in efficiency. There is. In addition, a technique is known in which an alcohol-based organic solvent, polyoxyethylene fatty alcohol ether, and a chaotropic agent are added to a specimen to suppress a decrease in nucleic acid recovery efficiency without requiring high-speed centrifugation (Patent Document) 2) Not suitable for a large amount of specimen, and the efficiency of nucleic acid recovery was insufficient in the purification of nucleic acid from specimens containing high concentrations of colloidal particles such as blood and milk.

Japanese Patent Laid-Open No. 2001-78790 Special Table 2014-526255

An object of the present invention is to provide a method for highly efficiently purifying nucleic acid from a specimen containing colloidal particles and a drug for use in the method.

As a result of intensive studies to solve the above problems, the present inventors have selected from the group consisting of (i) an amide type surfactant and / or (ii) a sulfoxide-based and amine-based amphiphilic organic solvent. Highly efficient nucleic acid in a sample by solubilizing colloidal particles contained in the sample using one or more amphiphilic organic solvents and (iii) a solubilizing agent containing a chaotropic agent. As a result, the present invention was completed.

Accordingly, the present invention includes the following aspects.
(1) A method for purifying nucleic acid from a specimen containing colloidal particles,
One or both of (a) (i) an amide-type surfactant, and (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents And (iii) treating the specimen with a solubilizing agent containing a chaotropic agent to solubilize colloidal particles in the specimen,
(B) a step of contacting a specimen treated with a solubilizing agent with a nucleic acid adsorption carrier; and (c) a step of eluting nucleic acid from the nucleic acid adsorption carrier.
Said method.
(2) A method for purifying nucleic acid from a specimen containing colloidal particles,
(A) (i) an amide type surfactant, (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and (iii) chaotropic A step of solubilizing colloidal particles in the sample by treating the sample with a solubilizing agent containing the agent,
(B) a step of contacting a specimen treated with a solubilizing agent with a nucleic acid adsorption carrier; and (c) a step of eluting nucleic acid from the nucleic acid adsorption carrier.
Said method.
(3) The method according to (1) or (2), wherein the amide type surfactant is a fatty acid alkanolamide.
(4) The method according to any one of (1) to (3), wherein the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine, and pyridine.
(5) The method according to any one of (1) to (4), wherein the chaotropic agent is a guanidine salt.
(6) The specimen is colloidal fluid, emulsion, animal milk, milky fluid, whole blood, serum, lymph fluid, urine, sweat, nasal fluid, spinal fluid, tissue fluid, semen, vaginal fluid, amniotic fluid, tears, saliva, feces, Selected from the group consisting of pus, sputum, dried tissue, cell suspension, cell-containing fluid, tissue fluid, milky sap, milky juice, vegetable milk, dairy products, and cosmetics, pharmaceuticals, and detergents containing colloidal particles The method according to any one of (1) to (5).
(7) Colloidal particles are from the group consisting of micelles, liposomes, fat globules, solid particulates, bubbles, animal cells, plant cells, parasites, fungi, fungi, yeasts, bacteria, archaea, viruses, spores, spores, and cysts. The method according to any one of (1) to (6), which is selected.
(8) A solubilizer composition for solubilizing colloidal particles to purify nucleic acid from a specimen containing colloidal particles, comprising (i) an amide type surfactant, and (ii) a sulfoxide system and an amine A solubilizer composition comprising one or both of one or more amphiphilic organic solvents selected from the group consisting of a system amphiphilic organic solvent, and (iii) a chaotropic agent.
(9) A solubilizer composition for solubilizing colloidal particles to purify nucleic acid from a specimen containing colloidal particles, comprising (i) an amide type surfactant, and (ii) a sulfoxide system and an amine A solubilizer composition comprising one or more amphiphilic organic solvents selected from the group consisting of a systemic amphiphilic organic solvent, and (iii) a chaotropic agent.
(10) The solubilizer composition according to (8) or (9), wherein the amide type surfactant is a fatty acid alkanolamide.
(11) The solubilizer composition according to any one of (8) to (10), wherein the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine, and pyridine. object.
(12) The solubilizer composition according to any one of (8) to (11), wherein the chaotropic agent is a guanidine salt.
(13) A nucleic acid purification kit comprising the solubilizer composition according to any one of (8) to (12).
(14) one or both of (i) an amide type surfactant and (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents And a solubilizer kit for solubilizing colloidal particles, comprising (iii) a chaotropic agent.
(15) (i) an amide type surfactant, (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and (iii) chaotropic A solubilizer kit for solubilizing colloidal particles, comprising an agent.
(16) One or both of (i) an amide-type surfactant, and (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents And (iii) a chaotropic agent, a nucleic acid purification kit.
(17) (i) an amide type surfactant, (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and (iii) chaotropic A nucleic acid purification kit containing an agent.
(18) one or both of (i) an amide-type surfactant, and (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents And (iii) use of chaotropic agents to solubilize colloidal particles.
(19) (i) an amide type surfactant, and (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and (iii) Use of chaotropic agents to solubilize colloidal particles.
(20) A method of solubilizing colloidal particles,
One or two or more amphiphilic organic solvents selected from the group consisting of (i) an amide-type surfactant and (ii) a sulfoxide-based and amine-based amphiphilic organic solvent, or A process comprising both, and (iii) treating with a chaotropic agent.
(21) A method of solubilizing colloidal particles,
Colloidal particles are made of (i) an amide type surfactant, and (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and (iii) ) A process comprising a step of treating with a chaotropic agent.
(22) In any one of the above (18) to (21), the colloidal particles are colloidal particles contained in a specimen. The sample is preferably the sample described in (6) above.
(23) In any one of the above (18) to (21), the colloidal particles are the colloidal particles described in the above (7).
(24) In any one of the above (18) to (21), the amide type surfactant is the compound described in the above (3).
(25) In any one of the above (18) to (21), the amphiphilic organic solvent is the compound described in the above (4).
(26) In any one of the above (18) to (21), the chaotropic agent is the compound described in the above (5).

This specification includes the disclosure of Japanese Patent Application No. 2015-090596, which is the basis of the priority of the present application.

According to the present invention, nucleic acid can be purified with high efficiency and high purity from a specimen containing colloidal particles.

The figure which shows the result of having measured the change of the turbidity of milk by addition of surfactant (5 weight%-20 weight%) and an amphiphilic organic solvent (5 volume%-50 volume%) as described in Example 1. is there. FIG. 3 shows the results of real-time PCR analysis of nucleic acids purified from milk samples using solubilizer compositions A to L and sterilized distilled water M described in Example 2. The results of electrophoresis and amplification of nucleic acid purified from a milk sample using a solubilizer composition (15% by weight Amizole CDE-G, 1.7M guanidine thiocyanate, and 40% by volume DMSO) are shown. FIG. It is a figure which shows the result of having analyzed the nucleic acid refine | purified from the blood sample using the solubilizer composition (15 weight% amizole CDE-G, 1.7M guanidine thiocyanate, and 40 volume% DMSO) by real-time PCR.

In one aspect, the present invention relates to a method for purifying nucleic acid from a specimen containing colloidal particles.

In the present specification, the term “colloidal particles” means that the radius is generally about 10 ⁻¹⁰ m (0.1 nm) to 10 ⁻⁵ m (10 μm), for example, about 10 ⁻⁹ , assuming that the substance is a sphere. Particles of m (1 nm) to 10 ⁻⁶ m (1 μm) are referred to. “Colloid” refers to a system in which colloidal particles are dispersed in a dispersion medium of gas, liquid, or solid. In the present specification, colloidal particles include not only particles but also droplets and oil droplets having the above particle diameters, droplets and oil droplets wrapped in a film, bubbles, and mixtures thereof. Examples of colloidal particles include, but are not limited to, micelles, liposomes, fat globules, solid microparticles, and bubbles, and cells (Oreoscience, Vol. 8, No. 2, pp. 33-38, (2008)). Examples include animal cells, plant cells, parasites, fungi, molds, yeasts, bacteria, archaea, viruses, spores, spores, cysts, and the like.

Examples of the specimen containing colloidal particles include, but are not limited to, colloidal liquid; emulsion; animal milk derived from mammals such as cows, goats, sheep, and pigs, preferably milk; milky fluid, whole blood, serum Animal-derived samples such as lymph, urine, sweat, nasal fluid, spinal fluid, tissue fluid, semen, vaginal fluid, amniotic fluid, tears, saliva, feces, pus, sputum, and dried tissue; cell suspension; cell-containing fluid Tissue fluid; milky sap; milk juice; vegetable milk such as soy milk, coconut milk, and almond milk; dairy products such as yogurt, cheese, butter, and cream; and processing of cosmetics, pharmaceuticals, and detergents containing colloidal particles Goods.

In the present specification, nucleic acids are roughly divided into natural nucleic acids and non-natural nucleic acids. “Natural nucleic acids” have nucleotides as a basic unit, and each nucleotide has a 3′-position and a 5′-position carbon. A polynucleotide linked by a phosphodiester bond. Natural nucleic acids include polymers of deoxyribonucleotides and ribonucleotides such as DNA and RNA. “Non-natural nucleic acid” refers to a nucleic acid containing a non-natural nucleotide in place of or in addition to the above-mentioned natural nucleotide. The non-natural nucleotide is an artificial modification of the base part of the nucleotide. Nucleotides or nucleotide analogs having properties similar to artificially produced nucleotides, such as xanthosines and diaminopyrimidines. The nucleic acid of the present invention is derived from, for example, eukaryotes, bacteria, archaea, viruses, viroids, and artificial compounds.

In the present specification, “purification” of nucleic acid means to partially or completely separate and purify nucleic acid contained in a sample from impurities other than nucleic acid. As a standard of the degree of purification when partially purifying nucleic acid, for example, using purified nucleic acid, nucleic acid amplification methods such as PCR and isothermal nucleic acid amplification method, restriction enzyme treatment, cloning, nucleotide sequence analysis, Southern blot, Molecular biology such as Northern blot, hybrid capture analysis, line probe assay, microarray analysis, electrophoresis, mass spectrometry, fluorescent staining, dye staining, and analysis using complementary binding of natural or non-natural nucleic acid strands It is preferable that the analysis can be performed without any problem.

The method for purifying nucleic acid from a specimen containing colloidal particles of the present invention includes a solubilization step, a contact step with a nucleic acid adsorption carrier, and an elution step from the nucleic acid adsorption carrier. In addition to these steps, the method of the present invention may optionally include a pretreatment step before the solubilization step. Each process which comprises this invention is demonstrated in detail below.

<Pretreatment process>
In the method of the present invention, the “pretreatment step” is a step for increasing the solubilization efficiency of the next solubilization step by pretreating the specimen. Examples of the pretreatment step include stirring by a stirrer, filtration by passing through a pore or filter such as a syringe, crushing by crushing beads, ultrasonic treatment, centrifugation, freezing and thawing, heat treatment, reduction Examples include treatment, alkali treatment, and enzyme treatment with protease, lipase, lysozyme, and the like.

<Solubilization process>
In the method of the present invention, the solubilization step is a step of solubilizing colloidal particles in the sample by treating the sample with a solubilizing agent.

In the present specification, “treating a sample with a solubilizer” means a step of allowing the sample to act on the sample, for example, a step of mixing the solubilizer and the sample. This step can be performed, for example, by adding a solubilizer to the specimen and optionally stirring the mixture. When the mixture is stirred, stirring may be performed only during mixing, or may be performed continuously or intermittently throughout this step. This step can be performed, for example, for 1 second to 30 minutes, preferably 5 seconds to 20 minutes, and more preferably 10 seconds to 15 minutes.

In this step, when the specimen is treated with the solubilizing agent, solubilization can be further promoted by adding other treatment such as heat treatment. The heat treatment temperature is preferably 40 ° C. or higher, more preferably 60 ° C. or higher, and further preferably 70 ° C. or higher. The heat treatment time is preferably 1 second to 30 minutes, more preferably 5 seconds to 20 minutes, and even more preferably 10 seconds to 15 minutes. Usually, heat treatment of specimens such as blood and milk containing high concentrations of colloidal particles will denature proteins and form aggregates and reduce nucleic acid purification efficiency. However, specimens solubilized with the following solubilizers are heat treated. Even if aggregates are not formed, the nucleic acid can be solubilized and purified with high efficiency.

In the present specification, “solubilization” refers to reduction in particle diameter and / or reduction in molecular weight without coagulation or aggregation of colloidal particles.

In the present invention, “solubilizing agent” refers to a substance used to solubilize colloidal particles, and may be a combination of two or more components. The solubilizer used in the present invention contains at least a surfactant and / or an amphiphilic organic solvent, and a chaotropic agent. Each component contained in the solubilizer used in the present invention may be used in the form of a mixture in which two or more of them are mixed, or may be used in a form in which individual independent components are used in combination. In the method of the present invention, “treating a specimen with a solubilizing agent” includes treating the specimen with either form of the solubilizing agent. The solubilizer used in the present invention can solubilize not only colloidal particles but also aggregates and aggregates of colloidal particles. Furthermore, the solubilizer used in the present invention destroys the structure when the colloidal particles are biological samples such as parasites, fungi, molds, yeasts, bacteria, archaea, spores, spores, cysts, or viruses. Thus, the solubilized nucleic acids can be dispersed in the solution.

The surfactant that can be used as a solubilizer in the present invention is not particularly limited as long as it can solubilize colloidal particles, and examples thereof include anionic, cationic, zwitterionic, and non-ionic. An ionic surfactant is mentioned. Preferred examples include anionic surfactants and nonionic surfactants such as Sodium Dodecyl Sulfate (SDS), and more preferred are ester types, ether types, ester ether types, alkyl ether types, phenyl ether types, sorbitans. Derivative type, alkanolamine type, amide type, ethylene glycol type, polyoxyethylene fatty alcohol ether type nonionic surfactants may be mentioned. Specific examples of the nonionic surfactant that can be used as a solubilizer in the present invention include, for example, Tween® 20 ™ (polysorbate 20, polyoxyethylene sorbitan monolaurate), Tween® 80 ™ (polysorbate 80). , Polyoxyethylene sorbitan monooleate), Triton X-100 ™ (poly (oxyethylene) octylphenyl ether), Nonidet P-40 ™ ((octylphenoxy) polyethoxyethanol), polyoxyethylene (20 ) Cetyl ether, Amizole CDE-G ™ (coconut oil fatty acid diethanolamide), and Amizole LDE-G ™ (lauric acid diethanolamide).

Preferred surfactants that can be used as a solubilizer in the present invention include amide type surfactants such as fatty acid alkanolamides, polyoxyethylene fatty acid amides, and fatty acid amidopropylbetaines. As the surfactant that can be used as a solubilizer in the present invention, amide type nonionic surfactants such as fatty acid alkanolamides and polyoxyethylene fatty acid amides are preferable, and fatty acid alkanolamides are particularly preferable. In the present specification, the fatty acid alkanolamide is preferably represented by the following general formula.

[Wherein R ¹ is a linear or branched alkyl or alkenyl group having 5 to 21 carbon atoms, preferably 7 or more, 9 or more, or 11 or more, preferably 19 or less or 17 or less carbon atoms. And more preferably a linear or branched alkyl or alkenyl group having 7 to 19 carbon atoms, 9 to 17 carbon atoms, or 11 to 17 carbon atoms,
R ² is hydrogen or a hydroxyalkyl group having 1 to 3 carbon atoms, preferably hydrogen or a hydroxyalkyl group having 2 carbon atoms;
R ³ is a hydroxyalkyl group having 1 to 3 carbon atoms, preferably a hydroxyalkyl group having 2 carbon atoms]

Examples of fatty acid alkanolamides that can be used as solubilizers in the present invention include, for example, coconut oil fatty acid monoethanolamide, palm kernel oil fatty acid monoethanolamide, lauric acid monoethanolamide, lauric acid monoisopropanolamide, myristic acid monoester. Fatty acid monoethanolamides such as ethanolamide, palmitic acid monoethanolamide, stearic acid monoethanolamide, and oleic acid monoethanolamide, and palm oil fatty acid diethanolamide, palm kernel oil fatty acid diethanolamide, lauric acid diethanolamide, myristic acid And fatty acid diethanolamides such as diethanolamide, palmitic acid diethanolamide, stearic acid diethanolamide, and oleic acid diethanolamide. That. The surfactant that can be used as a solubilizer in the present invention is preferably fatty acid diethanolamide, particularly preferably coconut oil fatty acid diethanolamide or lauric acid fatty acid diethanolamide.

In the solubilizer used in the present invention, the surfactants may be used alone or in combination.

The concentration of the surfactant in the solubilizer used in the present invention is not particularly limited as long as it is a concentration sufficient to solubilize the colloidal particles, and those skilled in the art can easily determine the optimum value. it can. If the surfactant concentration is too low, solubilization of the colloidal particles will be insufficient, and if it is too high, the viscosity of the solution will increase, making it difficult to handle. The concentration of the surfactant is the final concentration when mixed with the specimen, for example, 0.05% by weight or more, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, 2% by weight or more, 3% by weight or more, 4% % By weight or more or 5% by weight or more, for example, 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, 40% by weight or less, 30% by weight or less or May be up to 20% by weight, for example 0.05% to 90%, 0.1% to 80%, 0.5% to 70%, 1% to 60%, 2% to 50%, It may be 3% to 40%, 4% to 30%, or 5% to 20% by weight.

The amphiphilic organic solvent that can be used as a solubilizer in the present invention is not particularly limited as long as it can solubilize colloidal particles. For example, alcohol, aldehyde, carboxylic acid, ketone, ester, ether , Thiol, sulfoxide, alkane, alkene, alkyne, amine, imine, amide, and nitrile amphiphilic organic solvent are preferable, and alcohol-based, sulfoxide-based, and amine-based amphiphilic organic solvents are preferable. Specific examples of the alcohol-based amphiphilic organic solvent include methanol, ethanol, 1-propanol, and isopropanol. Specific examples of the sulfoxide-based amphiphilic organic solvent include dimethyl sulfoxide (DMSO), diethyl sulfoxide, and ethyl. Examples of amine-based amphiphilic organic solvents include amino alcohols such as monoethanolamine, diethanolamine, and triethanolamine; methylamine, dimethylamine, ethylamine, ethylenediamine, diethylamine, dimethylformamide, dimethyl Aliphatic amines such as acetamide, cyclohexylamine, and N-methylpyrrolidone; and aromatic amines such as pyridine. Amphiphilic organic solvents that can be used as solubilizers in the present invention are preferably dimethyl sulfoxide (DMSO), ethylenediamine, and pyridine. These amphiphilic organic solvents may be used alone or in combination.

The concentration of the amphiphilic organic solvent that can be used as the solubilizer is not particularly limited as long as it is a concentration sufficient to solubilize the colloidal particles, and those skilled in the art can easily determine the optimum value. be able to. The concentration of the amphiphilic organic solvent is the final concentration when mixed with the specimen, and may be, for example, 1% by weight or more, 5% by weight or more, 10% by weight or more, 20% by weight or more, or 30% by weight or more. For example, it may be 95% by weight or less, 90% by weight or less, 80% by weight or less, 70% by weight or less, or 60% by weight or less, for example, 1% by weight to 95% by weight, 5% by weight to 90% by weight 10 wt% to 80 wt%, 20 wt% to 70 wt%, or 30 wt% to 60 wt%.

The solubilizer used in the present invention contains a chaotropic agent in addition to a surfactant and / or an amphiphilic organic solvent. The chaotropic agent contained in the solubilizer used in the present invention is not particularly limited as long as it has an action of promoting the binding between the nucleic acid and the nucleic acid adsorption carrier. For example, guanidine isothiocyanate, guanidine thiocyanate, sulfuric acid Guanidine and guanidine salts such as guanidine hydrochloride, urea, sodium iodide, potassium iodide, sodium bromide, potassium bromide, calcium bromide, ammonium bromide, sodium perchlorate, sodium cyanate, potassium cyanate, thiocyanate Examples thereof include sodium acid salt, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, and the like, and preferably a guanidine salt. These chaotropic agents may be used alone or in combination. In addition, the chaotropic agent not only binds the nucleic acid and the nucleic acid adsorption carrier, but also acts as a protein denaturant and can contribute to solubilization of the colloidal particles. The concentration of the chaotropic agent contained in the solubilizer may be an amount sufficient to bind the nucleic acid and the nucleic acid adsorption carrier, and those skilled in the art can easily determine the optimum value. The concentration of the chaotropic agent is the final concentration when mixed with the specimen, for example, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, 2% by weight or more, 5% by weight or more, or 10% by weight or more. For example, it may be 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, or 40% by weight or less, for example, 0.1% by weight to 90% by weight, 0.5% by weight, It may be from wt% to 80 wt%, 1 wt% to 70 wt%, 2 wt% to 60 wt%, 5 wt% to 50 wt%, or 10 wt% to 40 wt%.

Since an additive or synergistic solubilizing effect is expected by combining a surfactant having the ability to solubilize colloidal particles and an amphiphilic organic solvent, the solubilizer used in the present invention is preferably Contains both surfactant and amphiphilic organic solvent. Preferably, the solubilizer used in the present invention contains an amide type surfactant and an amphiphilic organic solvent, or contains a surfactant and a sulfoxide-based or amine-based organic solvent. Exemplary combinations of solubilizers used in the present invention include (amide type surfactant, amphiphilic organic solvent, chaotropic agent), (amide type surfactant, alcohol-based organic solvent, chaotropic agent), (amide) Type surfactant, sulfoxide type organic solvent, chaotropic agent), (amide type surfactant, amine type organic solvent, chaotropic agent), (surfactant, sulfoxide type organic solvent, chaotropic agent), (surfactant, amine) Organic solvent, chaotropic agent), (nonionic surfactant, sulfoxide organic solvent, chaotropic agent), and (nonionic surfactant, amine organic solvent, chaotropic agent), more preferably High solubilizing ability of colloidal particles (amide type surfactant, sulfoxide organic solvent, chaotropic agent) And (amide type surfactants, amine-based organic solvents, chaotropic agents) and the like. More preferable examples of the combination contained in the solubilizer of the present invention include (guanidium salts such as coconut oil fatty acid diethanolamide or lauric acid fatty acid diethanolamide, DMSO, and guanidine thiocyanate).

The solubilization step of the present invention can be performed in the presence of a buffer. As the buffer, a buffer such as a phosphate buffer or a Good buffer is preferably used. Among these buffers, MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Tris, Bicine, TAPS, CHES, CAPSO And Good buffers such as CAPS are particularly preferred. These buffering agents may be used alone or in combination.

The pH in the solubilization step of the present invention may be a pH at which colloidal particles and the like in the sample do not coagulate when the solubilizer and the sample are mixed, and the pH at the time of mixing with the specimen is preferably pH 2 or more. More preferably, the pH is 3 or more, and still more preferably 4 or more.

In the solubilization process of the present invention, for the purpose of promoting solubilization, for example, a reducing agent such as dithiothreitol (DTT), a chelating agent such as ethylenediaminetetraacetic acid (EDTA), an alkalizing agent, and a protease, lipase, and It can be performed in the presence of a degrading enzyme such as lysozyme. Furthermore, the solubilization step of the present invention can be performed in the presence of, for example, a DNA protecting agent, a DNase inhibitor, and / or RNase in order to selectively purify DNA. For purification, for example, it can be performed in the presence of an RNA protecting agent, an RNase inhibitor, and / or DNase.

In the solubilization step of the present invention, each component constituting the solubilizing agent may be added separately when mixing with the specimen, or may be added together. That is, the solubilization step of the present invention may be performed, for example, by separately adding the surfactant and / or the amphiphilic organic solvent and the chaotropic agent, or adding a mixture containing each component. May be performed. More preferably, in the solubilization step of the present invention, the solubilizer is added as the following solubilizer composition.

<Contacting step with nucleic acid adsorption carrier>
In this specification, the “contact step with the nucleic acid adsorption carrier” means that the sample treated with the solubilizing agent in the “solubilization step” is brought into contact with the nucleic acid adsorption carrier, and the nucleic acid in the sample is bound to the nucleic acid adsorption carrier. Means the step of

This step is not limited as long as the nucleic acid contained in the sample can be adsorbed on the nucleic acid adsorption carrier. For example, when the nucleic acid adsorption carrier is in the form of a column, it can be carried out by passing the sample through this column. When the nucleic acid adsorption carrier is in the form of a solid such as a bead, the nucleic acid adsorption carrier and the sample are This can be done by mixing.

In the present specification, the nucleic acid adsorption carrier is not particularly limited as long as it is a carrier that adsorbs nucleic acid in the presence of a chaotropic agent and / or an organic solvent. For example, inorganic substances such as silica, resins, insoluble polysaccharides, and the like The nucleic acid adsorption carrier comprised by these can be used. These nucleic acid adsorption carriers may have any shape such as a column shape, a powder shape, a fiber shape, a bead shape, a membrane shape, and a porous shape as long as nucleic acid can be adsorbed. The nucleic acid adsorption carrier may be magnetized or may be modified.

<Elution process from nucleic acid adsorption carrier>
The elution step from the nucleic acid adsorbing carrier in the present invention means a step of eluting the nucleic acid adsorbed on the nucleic acid adsorbing carrier by the “contacting step with the nucleic acid adsorbing carrier” with an eluent. This step optionally includes a washing step with a washing solution before elution.

The washing solution in this step is not limited as long as it can wash the solubilizing agent remaining on the nucleic acid adsorption carrier to which the nucleic acid has been adsorbed and impurities derived from the specimen. Examples include a molecular solution and an aqueous sugar solution. Examples of the solvent contained in the cleaning liquid include ethanol, isopropanol, acetone and the like, and those concentrations can be easily determined by those skilled in the art. The concentration of the solvent in the cleaning solution may be, for example, 20% by weight or more, 30% by weight or more, or 40% by weight or more, for example, 100% by weight or less, 90% by weight or less, or 80% by weight or less. For example, 20 to 100% by weight, preferably 30 to 90% by weight, more preferably 40 to 80% by weight.

The nucleic acid adsorption carrier after washing with the washing liquid may be subjected to a centrifugal separation process and a drying process by heat treatment and air drying in order to remove the washing liquid.

The eluate used for eluting the nucleic acid from the nucleic acid adsorption carrier after washing is not particularly limited as long as the nucleic acid is eluted from the nucleic acid adsorption carrier, and examples thereof include water and a buffer solution.

<Solubilizer composition>
In one aspect, the invention relates to a solubilizer composition that can be used in the methods of the invention. The solubilizer composition of the present invention contains at least the above-mentioned surfactant and / or amphiphilic organic solvent and a chaotropic agent, and preferably contains a surfactant, an amphiphilic organic solvent, and a chaotropic agent. Since the composition of the surfactant, the amphiphilic organic solvent, and the chaotropic agent that can be included in the solubilizer composition of the present invention is as described in the above <Solubilization step>, description thereof is omitted here. .

When the solubilizer composition of the present invention contains a surfactant, the concentration of the surfactant is, for example, 0.05% by weight or more, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, 2% by weight or more, 3 wt% or more, 4 wt% or more or 5 wt% or more, for example, 90 wt% or less, 80 wt% or less, 70 wt% or less, 60 wt% or less, 50 wt% or less, 40 wt% or less 30 wt% or less or 20 wt% or less, for example 0.05 wt% to 90 wt%, 0.1 wt% to 80 wt%, 0.5 wt% to 70 wt%, 1 wt% to 60 wt%, 2 wt % To 50%, 3% to 40%, 4% to 30%, or 5% to 20% by weight. Further, when the solubilizer composition of the present invention contains an amphiphilic organic solvent, the concentration of the amphiphilic organic solvent is, for example, 1 wt% or more, 5 wt% or more, 10 wt% or more, 20 wt% or more. Or 95% or less, 90% or less, 80% or less, 70% or less, or 60% or less, such as 1% to 95%. The weight may be 5%, 5% to 90%, 10% to 80%, 20% to 70%, or 30% to 60% by weight. In the solubilizer composition of the present invention, the concentration of the chaotropic agent is, for example, 0.1 wt% or more, 0.5 wt% or more, 1 wt% or more, 2 wt% or more, 5 wt% or more, or 10 wt% or more. For example, it may be 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, or 40% by weight or less, for example, 0.1% by weight to 90% by weight. 0.5 wt% to 80 wt%, 1 wt% to 70 wt%, 2 wt% to 60 wt%, 5 wt% to 50 wt%, or 10 wt% to 40 wt%. Since the final concentration of each component when the solubilizer composition of the present invention is mixed with the specimen is as described in the above <Solubilization step>, description thereof is omitted here.

The solubilizer composition of the present invention may contain water as a solvent other than the above components, but may contain one or more of the above buffering agents in order to stabilize the pH of the solubilizer composition.

The pH of the solubilizer composition of the present invention may be a pH at which colloidal particles and the like in the sample do not coagulate when mixed with the sample, and the pH at the time of mixing with the specimen is preferably pH 2 or more. The pH is preferably 3 or more, more preferably 4 or more.

In order to promote solubilization, the solubilizer composition of the present invention can be used, for example, by reducing agents such as dithiothreitol (DTT), chelating agents such as ethylenediaminetetraacetic acid (EDTA), alkalizing agents, proteases, and lipases. And a degrading enzyme such as lysozyme. Further, the solubilizer composition of the present invention may contain, for example, a DNA protecting agent, a DNase inhibitor, and / or an RNase in order to selectively purify DNA, and selectively select RNA. For purification, for example, an RNA protecting agent, RNase inhibitor, and / or DNase may be included.

The solubilizer composition of the present invention can be used to solubilize colloidal particles in order to purify nucleic acids from a specimen containing colloidal particles.

The solubilizer composition of the present invention may be incorporated into, for example, a nucleic acid preparation device, a nucleic acid amplification device, or an automatic nucleic acid analysis device.

<Kit>
In one aspect, the present invention relates to a nucleic acid purification kit comprising the solubilizer composition of the present invention. In addition to the solubilizing agent composition, the kit may contain, for example, a nucleic acid adsorption carrier, an adsorption solution, an eluate, a PCR primer, a buffer, an enzyme, and / or instructions for use.

In one embodiment, the present invention separately includes each component contained in the solubilizer used in the present invention, that is, a surfactant and / or an amphiphilic organic solvent, and a chaotropic agent (for example, included in separate containers). ), A solubilizer kit or a nucleic acid purification kit for solubilizing colloidal particles. In addition to the solubilizer, the kit may contain, for example, a nucleic acid adsorption carrier, an adsorption solution, an eluate, a PCR primer, a buffer, an enzyme, and / or instructions for use. Since the composition of the surfactant, the amphiphilic organic solvent, and the chaotropic agent contained in the solubilizer used in the present invention is as described in the above <Solubilization step>, description thereof is omitted here.

The weight ratio of each component when the kit of the present invention includes each component separately can be determined as appropriate by those skilled in the art. When the kit of the present invention contains the surfactant and the chaotropic agent separately, the weight ratio of the surfactant in the kit of the present invention is, for example, 0.1 parts by weight or more with respect to 100 parts by weight of the chaotropic agent, 1 part by weight, 5 parts by weight or more, 10 parts by weight or more, or 20 parts by weight or more, for example, 10000 parts by weight or less, 1000 parts by weight or less, 500 parts by weight or less, 100 parts by weight or less, or 60 parts by weight or less Well, more specifically, 0.1 parts by weight to 10,000 parts by weight, 1 part by weight to 1000 parts by weight, 5 parts by weight to 500 parts by weight, 10 parts by weight to 100 parts by weight, or 20 parts by weight to 60 parts by weight. Good. Further, when the kit of the present invention separately contains an amphiphilic organic solvent and a chaotropic agent, the weight ratio of the amphiphilic organic solvent in the kit of the present invention is, for example, 100 parts by weight of the chaotropic agent. For example, 0.3 parts by weight or more, 3 parts by weight or more, 6 parts by weight or more, 30 parts by weight or more, or 60 parts by weight or more, for example, 30000 parts by weight or less, 3000 parts by weight or less, 1500 parts by weight or less, 300 parts by weight or less, or 200 More specifically, it may be 0.3 parts by weight to 30000 parts by weight, 3 parts by weight to 3000 parts by weight, 6 parts by weight to 1500 parts by weight, 30 parts by weight to 300 parts by weight, or 60 parts by weight to It may be 200 parts by weight. When the kit of the present invention includes a surfactant, an amphiphilic organic solvent, and a chaotropic agent separately, the kit of the present invention is, for example, a surfactant having the above weight ratio with respect to 100 parts by weight of the chaotropic agent. And an amphiphilic organic solvent in the above weight ratio. Since the final concentration of each component when the surfactant and / or amphiphilic organic solvent contained in the kit of the present invention and the chaotropic agent are mixed with the specimen is as described in the above <Solubilization step> The description is omitted here.

The kit of the present invention may be incorporated into, for example, a nucleic acid preparation device, a nucleic acid amplification device, a nucleic acid automatic analysis device, or the like.

Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.
<Example 1: Evaluation 1 of solubilizing ability of colloidal particles>
1) Outline In order to evaluate the solubilizing ability of surfactants and amphiphilic organic solvents to colloidal particles, each surfactant or amphiphilic organic solvent and milk are mixed and the change in turbidity (OD ₆₆₀ ) is measured. Thus, the solubilization degree of the colloidal particles was evaluated. When the colloidal particles are solubilized without coagulation, the diameter of the colloidal particles is reduced, so that the light transmittance is increased and it can be observed as a decrease in turbidity.

2) Method Mix 10 μl of commercially available milk (Snow Brand Megmilk) with 190 μl of surfactant or amphiphilic organic solvent, let stand for 10 minutes, then collect 100 μl from the mixture and put it in ASSAY PLATE 96WELL (IWAKI). The OD ₆₆₀ was measured by Microplate Spectrophotometer Benchmark Plus (Bio-RAD). Surfactants include Tween 20, Tween 80, Triton X-100, Nonidet P-40, polyoxyethylene (20) cetyl ether (Wako Pure Chemicals), Amizole CDE-G (Kawaken Fine Chemical), Amizole LDE-G (Kawaken Fine Chemical) and SDS were used as the amphiphilic organic solvent, DMSO, ethanol, isopropanol, ethylenediamine, and pyridine. The surfactant was diluted to 5 to 20% by weight, and the amphiphilic organic solvent was diluted to 5 to 50% by volume with sterile distilled water and used for the test.

3) Results In all samples to which each surfactant or amphiphilic organic solvent was added, a decrease in turbidity was confirmed, and coagulation of the milk component was not confirmed. In particular, the turbidity is greatly reduced in the sample to which amide type surfactant Amido CDE-G or Amizole LDE-G, sulfoxide organic solvent DMSO, or amine organic solvent ethylenediamine or pyridine is added, It was confirmed that these have particularly strong colloid particle solubilizing ability (FIG. 1).

<Example 2: Evaluation 2 of colloidal particle solubilization ability>
1) Method Solubilizer compositions A to L containing a surfactant and / or an amphiphilic organic solvent and a chaotropic agent in sterile distilled water at the following concentrations were prepared (A: 10% by weight Amisole CDE-G) And 3M (about 35% by weight) guanidine thiocyanate, B: 20% by weight Amisole CDE-G and 3M guanidine thiocyanate, C: 30% by volume (about 32% by weight) DMSO and 3M guanidine thiocyanate, D: 60% by volume (About 62% by weight) DMSO and 3M guanidine thiocyanate, E: 10% by weight Amizole CDE-G, 30% by volume DMSO, and 3M guanidine thiocyanate, F: 10% by weight Amisol CDE-G, 30% by volume (about 25% % By weight) Ethanol, and 3M guanidine thiocyanate, G: 10% by weight Tween 20, 30% by volume DMSO, and 3M guanidine thiocyanate, H: 10% by weight Triton X-100, 30% by volume DMSO, 3M guanidine thiocyanate, I: 10 wt% Nonidet P-40, 30 vol% DMSO, and 3M Thioshi Guanidine acid, J: 10% by weight polyoxyethylene (20) cetyl ether, 30% by volume DMSO, 3M guanidine thiocyanate, K: 10% by weight polyoxyethylene (20) cetyl ether, 30% by volume ethanol, and 3M thiocyanate Guanidine acid, L: 3M guanidine thiocyanate). As a control, M: sterile distilled water was used. In order to evaluate the solubilizing ability of colloidal particles by each solubilizer composition, each solubilizer composition and milk were mixed, and the degree of solubilization of the colloidal particles was evaluated by measuring the decrease in turbidity. Specifically, 100 μl of commercially available milk (Snow Brand Megmilk) and 500 μl of each solubilizer composition were mixed, allowed to stand for 10 minutes, 100 μl was collected from the mixture, transferred to ASSAY PLATE 96WELL (IWAKI), and Microplate Spectrophotometer OD ₆₆₀ was measured by Benchmark Plus (Bio-RAD). Further, it was visually evaluated whether or not the mixed solution was coagulated. Furthermore, the mixed solution was heat-treated at 80 ° C. for 10 minutes, and whether or not coagulation occurred was evaluated in the same manner.

2) Results As a result of the evaluation, a decrease in turbidity was confirmed in the samples to which the solubilizer compositions A to J were added. In particular, the turbidity was greatly reduced in the sample to which the solubilizer composition B, D, or E was added, and it was confirmed that this has particularly strong colloid particle solubilizing ability. Moreover, coagulation | solidification of the milk component by addition of each solubilizer composition was not confirmed before and after heat processing (Table 1). In contrast, the solubilizer compositions K and L showed a slight decrease in turbidity compared to the control solution M, but the turbidity decrease was weaker than that of the solubilizer compositions A to J. there were. In the solubilizer compositions K to L and sterilized distilled water M, coagulation of milk components was confirmed by heat treatment of the solution after mixing the milk (Table 1).

<Example 3>
1) Outline Using the solubilizer compositions A to L or the sterilized distilled water M and the silica column prepared in Example 2, nucleic acid was purified from milk containing microorganisms. Subsequently, real-time PCR targeting the microorganism-derived nucleic acid was performed using the purified nucleic acid, and the nucleic acid was appropriately purified and the nucleic acid recovery efficiency was evaluated.

2) Method 10 ⁴ cfu / ml of Gram-positive bacteria Streptococcus thermophilus was added to commercially available milk (Snow Brand Megmilk) and suspended, and this was used as a milk sample.

As the solubilizer, the solubilizer compositions A to L and sterilized distilled water M described in Example 2 were used. 500 μl of each solubilizer composition or sterilized distilled water was added to 100 μl of a milk sample added to a 1.5 ml tube. Furthermore, after heat treatment at 80 ° C. for 10 minutes, the solubilized milk is applied to a silica column (FAVORGEN), centrifuged at 10000 μg for 1 minute, and then added with 500 μl of 70% ethanol to the column and centrifuged at 10000 μg for 1 minute. And washed. After washing with 70% ethanol again, the column was centrifuged at 10,000 g for 3 minutes and dried. 30 μl of sterilized distilled water was added to the column and centrifuged at 10,000 g for 1 minute, and then the eluate was recovered as purified nucleic acid. The time required for nucleic acid purification was about 20 minutes.

Real-time PCR targeting S. thermophilus was performed using the purified nucleic acid. The composition of the PCR reaction solution was 10 μM forward primer STf (5′-GCTCCACTACAAGATGGACCTGC-3 ′ (SEQ ID NO: 1)) 1.5 μl, 10 μM reverse primer STr (5′- TAGGAGTCTGGGCCGTGTCTCAG-3 ′ (SEQ ID NO: 2)) 1.5 μl, Kaneka high-speed amplification DNA Polymerase (Kaneka) 0.5 μl, 10x Kaneka high-speed amplification DNA Polymerase buffer 5.0 μl, 2 mM dNTPs 、 5.0 μl, 1000-fold diluted SYBR Green I (Lonza) 2.5μl, sterile DNA 2.5 μl 31.5 μl. The reaction was performed using a LightCycler 96 system (Roche), and after 98 minutes at 98 ° C., 45 cycles of 98 ° C. 5 seconds → 60 ° C. 5 seconds → 72 ° C. 15 seconds were performed to monitor the nucleic acid amplification reaction.

3) Results Nucleic acid amplification was confirmed from the sample purified using the solubilizer compositions A to J, and it was confirmed that the nucleic acid was appropriately purified. Samples purified using solubilizer compositions B, D, E, and G confirmed particularly low Cq values, confirming high nucleic acid recovery efficiency in nucleic acid purification methods using these (Fig. 2). Nucleic acid amplification was confirmed in the samples purified using the solubilizer compositions K and L. However, the Cq value was higher and the nucleic acid recovery efficiency was lower than in the solubilizer compositions A to J. (FIG. 2).

<Example 4>
1) Overview Nucleic acid was purified from milk containing microorganisms using a solubilizer composition and silica-coated magnetic particles. PCR was performed using the purified nucleic acid as a target for microorganism-derived nucleic acid, and the nucleic acid was appropriately purified and the lower detection limit was evaluated.

2) Method Grams of each cfu / ml (3.5 × 10 ³ cfu / ml, 3.5 × 10 ² cfu / ml, 3.5 × 10 ¹ cfu / ml, or 3.5 × 10 ⁰ cfu / ml) to commercial milk (Snow Brand Megmilk) A positive bacterium Streptococcus thermophilus was added and suspended, and this was used as a milk sample.

As a solubilizer composition, a mixed solution containing 15% by weight Amizole CDE-G, 1.7M (about 20% by weight) guanidine thiocyanate, and 40% by volume (about 42% by weight) DMSO in sterile distilled water was used. 500 μl of the solubilizer composition was added to 100 μl of the milk sample added to the 1.5 ml tube and incubated at 80 ° C. for 10 minutes to solubilize the colloidal particles. Add 20 μl of silica-coated magnetic particles MagPrep (registered trademark) Silica Particles (Merck Millipore) to the solubilized milk sample, mix well, set the tube on a magnetic stand, separate the magnetic particles, and remove the supernatant. Removed. After removing the tube from the magnetic stand, 500 μl of 70% ethanol was added, mixed well, and washed. Then, the tube was set on the magnetic stand, the magnetic particles were separated, and the supernatant was removed. After repeating the washing with 70% ethanol again, the magnetic particles in the tube were dried. After drying, 30 μl of sterilized distilled water was added, the tube was set on a magnetic stand, and the supernatant was recovered as purified nucleic acid. In this example, nucleic acid purification was possible in about 10 minutes without using a high-speed centrifuge or a special apparatus.

The composition of the PCR reaction solution was 10 μM forward primer STf (5′-GCTCCACTACAAGATGGACCTGC-3 ′ (SEQ ID NO: 1)) 1.5 μl, 10 μM reverse primer STr (5′-TAGGAGTCTGGGCCGTGTCTCAGAG-3 ′ (SEQ ID NO: 2)) 1.5 μl, Kaneka High-Speed Amplification DNA Polymerase (Kaneka) 0.5 μl, 10x Kaneka High-Speed Amplification DNA Polymerase Buffer 5.0 μl, 2 mM dNTPs 5.0 μl, Purified DNA 2.5 μl, and Sterile Distilled Water 34.0 μl. The reaction was performed at 98 ° C. for 1 minute, followed by 40 PCR cycles of 98 ° C., 5 seconds → 60 ° C., 5 seconds → 72 ° C., 15 seconds. Next, the PCR product was subjected to electrophoresis based on a conventional method to visualize an amplified fragment of 130 bp.
3) Results A 130 bp amplified fragment was confirmed by electrophoresis, and it was confirmed that the nucleic acid could be properly purified from model milk containing S. thermophilus at a concentration of 3.5 x 10 ³ cfu / m or 3.5 x 10 ² cfu / ml. (FIG. 3).

<Example 5>
1) Overview Nucleic acid was purified from blood containing microorganisms using a solubilizer composition and a silica column. Using the purified nucleic acid, real-time PCR targeting a nucleic acid derived from microorganisms was performed, and it was evaluated that the nucleic acid was appropriately purified.

2) Method 10 ⁵ cfu / ml of Gram positive bacterium Streptococcus thermophilus was added to and suspended in mouse blood to which heparin had been added, and this was used as model blood.

As a solubilizer composition, a mixed solution containing 15% by weight Amizole CDE-G, 1.7M (about 20% by weight) guanidine thiocyanate, and 40% by volume (about 42% by weight) DMSO in sterile distilled water was used. 500 μl of the solubilizer composition was added to 100 μl of model blood added to a 1.5 ml tube and incubated at 80 ° C. for 10 minutes to solubilize the colloidal particles. The sample after the heat treatment was completely solubilized, and aggregates due to proteins and the like were not confirmed. Thereafter, the purified nucleic acid was recovered by the same method as in Example 3. Subsequently, real-time PCR targeting S. thermophilus was performed using the purified nucleic acid. Reaction and reagent preparation were carried out under the same conditions as in Example 3.

3) Results As a result of real-time PCR, nucleic acid amplification was confirmed, and it was confirmed that nucleic acids were appropriately purified from blood samples containing S. thermophilus (FIG. 4).

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims (17)

1. A method for purifying nucleic acid from a specimen containing colloidal particles,
   One or both of (a) (i) an amide-type surfactant, and (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents And (iii) treating the specimen with a solubilizing agent containing a chaotropic agent to solubilize colloidal particles in the specimen,
   (B) a step of contacting a specimen treated with a solubilizing agent with a nucleic acid adsorption carrier; and (c) a step of eluting nucleic acid from the nucleic acid adsorption carrier.
   Said method.
2. A method for purifying nucleic acid from a specimen containing colloidal particles,
   (A) (i) an amide type surfactant, (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and (iii) chaotropic A step of solubilizing colloidal particles in the sample by treating the sample with a solubilizing agent containing the agent,
   (B) a step of contacting a specimen treated with a solubilizing agent with a nucleic acid adsorption carrier; and (c) a step of eluting nucleic acid from the nucleic acid adsorption carrier.
   Said method.
3. 3. The method according to claim 1, wherein the amide type surfactant is a fatty acid alkanolamide.
4. The method according to any one of claims 1 to 3, wherein the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine, and pyridine.
5. The method according to any one of claims 1 to 4, wherein the chaotropic agent is a guanidine salt.
6. Sample is colloidal fluid, emulsion, animal milk, milky fluid, whole blood, serum, lymph, urine, sweat, nasal fluid, spinal fluid, tissue fluid, semen, vaginal fluid, amniotic fluid, tears, saliva, feces, pus, sputum Claims selected from the group consisting of: dairy tissue, cell suspension, cell-containing fluid, tissue fluid, milky sap, milk juice, vegetable milk, dairy products, and cosmetics, pharmaceuticals, and detergents containing colloidal particles. The method according to any one of 1 to 5.
7. The colloidal particles are selected from the group consisting of micelles, liposomes, fat globules, solid particulates, bubbles, animal cells, plant cells, parasites, fungi, molds, yeasts, bacteria, archaea, viruses, spores, spores, and cysts The method according to any one of claims 1 to 6.
8. A solubilizer composition for solubilizing colloidal particles to purify nucleic acid from a specimen containing colloidal particles, comprising (i) an amide type surfactant, and (ii) a sulfoxide-based and amine-based amphiphile A solubilizer composition comprising one or both of one or two or more amphiphilic organic solvents selected from the group consisting of organic organic solvents, and (iii) a chaotropic agent.
9. A solubilizer composition for solubilizing colloidal particles to purify nucleic acid from a specimen containing colloidal particles, comprising (i) an amide type surfactant, and (ii) a sulfoxide-based and amine-based amphiphile A solubilizer composition comprising one or more amphiphilic organic solvents selected from the group consisting of volatile organic solvents, and (iii) a chaotropic agent.
10. 10. The solubilizer composition according to claim 8 or 9, wherein the amide type surfactant is a fatty acid alkanolamide.
11. The solubilizer composition according to any one of claims 8 to 10, wherein the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine, and pyridine.
12. The solubilizer composition according to any one of claims 8 to 11, wherein the chaotropic agent is a guanidine salt.
13. A nucleic acid purification kit comprising the solubilizer composition according to any one of claims 8 to 12.
14. One or both of (i) an amide type surfactant, and (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and ( iii) A solubilizer kit for solubilizing colloidal particles, comprising a chaotropic agent.
15. (I) an amide type surfactant, (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and (iii) a chaotropic agent. A solubilizer kit for solubilizing colloidal particles.
16. One or both of (i) an amide type surfactant, and (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and ( iii) a nucleic acid purification kit comprising a chaotropic agent.
17. (I) an amide type surfactant, (ii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and (iii) a chaotropic agent. Nucleic acid purification kit.
    

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