JP7152153B2 - A method for purifying nucleic acids from specimens containing colloidal particles - Google Patents

Description

The present invention relates to methods for purifying nucleic acids from specimens containing colloidal particles.

Genetic testing is a method of testing specimens based on qualitative or quantitative analysis of nucleic acids. Genetic testing is an industrially important testing method that is applied in various fields such as medical care, agriculture, livestock and fisheries, and quality inspection, because it is highly sensitive and allows results to be determined in a short period of time. For this test, it is necessary to purify the nucleic acid from the specimen, and the most commonly used nucleic acid purification method is the bind-elute method, which utilizes the property that nucleic acids bind to a nucleic acid adsorption carrier in the presence of a chaotropic agent. (Patent Document 1). This purification method generally includes (a) a lysis step of dissolving the sample and eluting the nucleic acid, (b) an adsorption step of binding the nucleic acid and the nucleic acid adsorption carrier, and (c) washing the nucleic acid bound to the nucleic acid adsorption carrier. It consists of a washing step and (d) an elution step of eluting the nucleic acid bound to the nucleic acid-adsorptive carrier. In particular, the method using a spin column is an excellent method for easily purifying highly pure nucleic acids. However, it has been known that nucleic acid recovery efficiency is greatly reduced when purifying nucleic acid from a sample such as blood containing a large amount of colloidal particles. This is because the colloidal particles or aggregates thereof contained in the specimen physically block the binding of the nucleic acid to the nucleic acid-adsorptive carrier. In particular, in a technique using a spin column, aggregates of colloidal particles cause clogging of the column, resulting in a significant drop in nucleic acid recovery efficiency.

As a method to solve this problem, there is a method of separating and removing colloidal particles contained in the sample by high-speed centrifugation to suppress the decrease in nucleic acid recovery efficiency, but the high-speed centrifugation operation is complicated and efficiency is a problem. There is Also known is a method of adding an alcohol-based organic solvent, a polyoxyethylene fatty alcohol ether, and a chaotropic agent to a specimen to suppress a decrease in nucleic acid recovery efficiency without requiring high-speed centrifugation (Patent document 2) It is not suitable for a large amount of specimen, and nucleic acid recovery efficiency is insufficient in nucleic acid purification from specimens containing high concentrations of colloidal particles such as blood and milk.

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

An object of the present invention is to provide a highly efficient method for purifying nucleic acids from a sample 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 selected from the group consisting of (i) amide-type surfactants and/or (ii) sulfoxide-based and amine-based amphiphilic organic solvents. and (iii) a solubilizing agent containing a chaotropic agent to solubilize the colloidal particles contained in the sample, so that the nucleic acid in the sample can be released with high efficiency. and found that it can be purified, and completed the present invention.

Accordingly, the present invention includes the following aspects.
(1) A method for purifying nucleic acids from a sample containing colloidal particles, comprising:
(a) 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 to solubilize the colloidal particles in the sample by treating the sample with a solubilizing agent,
(b) a step of contacting a specimen treated with a solubilizing agent with a nucleic acid-adsorbing carrier; and (c) a step of eluting the nucleic acid from the nucleic acid-adsorbing carrier;
The above method, comprising
(2) A method for purifying nucleic acids from a sample containing colloidal particles, comprising:
(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 treating the specimen with a solubilizing agent comprising an agent to solubilize colloidal particles in the specimen;
(b) a step of contacting a specimen treated with a solubilizing agent with a nucleic acid-adsorbing carrier; and (c) a step of eluting the nucleic acid from the nucleic acid-adsorbing carrier;
The above method, comprising
(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 amphipathic 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 body fluid, whole blood, serum, lymph, urine, sweat, nasal fluid, spinal fluid, interstitial fluid, semen, vaginal fluid, amniotic fluid, tears, saliva, feces, selected from the group consisting of pus, sputum, casey tissue, cell suspension, cell-containing fluid, tissue fluid, milky sap, milky fruit juice, vegetable milk, dairy products, and cosmetics, pharmaceuticals and detergents containing colloidal particles , (1) to (5).
(7) colloidal particles from the group consisting of micelles, liposomes, fat globules, solid microparticles, foams, animal cells, plant cells, parasites, fungi, molds, 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 for purifying nucleic acids from a sample containing the colloidal particles, comprising (i) an amide surfactant, and (ii) a sulfoxide and an amine and (iii) a chaotropic agent.
(9) A solubilizer composition for solubilizing colloidal particles for purifying nucleic acids from a sample containing the colloidal particles, comprising (i) an amide surfactant, and (ii) a sulfoxide and an amine A solubilizer composition comprising one or more amphiphilic organic solvents selected from the group consisting of system amphiphilic organic solvents, and (iii) a chaotropic agent.
(10) The solubilizer composition according to (8) or (9), wherein the amide surfactant is a fatty acid alkanolamide.
(11) The solubilizer composition according to any one of (8) to (10), wherein the amphipathic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine, and pyridine. thing.
(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 (iii) a chaotropic agent, for solubilizing colloidal particles.
(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.
(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 agents.
(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) the 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 for solubilizing colloidal particles, comprising:
colloidal particles, (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, or both, as well as (iii) a method comprising the step of treating with a chaotropic agent.
(21) A method for solubilizing colloidal particles, comprising:
colloidal particles, (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 method comprising the step of treating with a chaotropic agent.
(22) In any one of (18) to (21) above, the colloidal particles are colloidal particles contained in the specimen. The specimen is preferably the specimen described in (6) above.
(23) In any one of (18) to (21) above, the colloidal particles are the colloidal particles described in (7) above.
(24) In any one of (18) to (21) above, the amide-type surfactant is the compound described in (3) above.
(25) In any one of (18) to (21) above, the amphipathic organic solvent is the compound described in (4) above.
(26) In any one of (18) to (21) above, the chaotropic agent is the compound described in (5) above.

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

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to purify nucleic acids from a sample containing colloidal particles with high efficiency and high purity.

1 is a diagram showing the results of measurement of changes in milk turbidity due to the addition of a surfactant (5% to 20% by weight) and an amphiphilic organic solvent (5% to 50% by volume) described in Example 1. be. FIG. 2 shows the results of real-time PCR analysis of nucleic acids purified from milk specimens using the solubilizing agent compositions A to L described in Example 2 and sterile distilled water M. FIG. Nucleic acids purified from milk samples using a solubilizer composition (15% by weight amizole CDE-G, 1.7 M guanidine thiocyanate, and 40% by volume DMSO) are amplified by PCR, and the results of electrophoresis are shown. It is a diagram. FIG. 2 shows the results of real-time PCR analysis of nucleic acids purified from blood samples using a solubilizer composition (15% by weight amizole CDE-G, 1.7 M guanidine thiocyanate, and 40% by volume DMSO).

In one aspect, the invention relates to a method of purifying nucleic acids from a sample containing colloidal particles.

As used herein, the term “colloidal particles” means that, when the shape of a substance is assumed to be a sphere, its radius is generally about 10 ⁻¹⁰ m (0.1 nm) to 10 ⁻⁵ m (10 μm), for example, about 10 ⁻⁹ The term "colloid" refers to a system in which ^colloidal particles are dispersed in a gas, liquid, or solid dispersion medium. As used herein, colloidal particles include not only particles but also droplets and oil droplets having the above particle sizes, film-encased droplets and oil droplets, bubbles, and mixtures thereof. Examples of colloidal particles include, but are not limited to, micelles, liposomes, fat globules, solid microparticles, foams, and cells (Oleoscience, Vol. 8, No. 2, pp.33-38, (2008)). , for example, animal cells, plant cells, parasites, fungi, molds, yeasts, bacteria, archaea, viruses, spores, spores, cysts, and the like.

Specimens containing colloidal particles include, but are not limited to, colloidal liquids; emulsions; animal milk, preferably milk, derived from mammals such as cows, goats, sheep, and pigs; milky body fluids, whole blood, serum , lymph, urine, sweat, nasal fluid, spinal fluid, interstitial fluid, semen, vaginal fluid, amniotic fluid, tears, saliva, feces, pus, sputum, and casey tissue; cell suspensions; cell-containing fluids milky sap; milky fruit juice; vegetable milks such as soy milk, coconut milk, and almond milk; dairy products such as yogurt, cheese, butter, and cream; goods are mentioned.

In the present specification, nucleic acids are broadly divided into natural nucleic acids and non-natural nucleic acids. "Natural nucleic acids" refer to nucleotides as the basic unit, with the 3'- and 5'-position carbons of the sugar between each nucleotide. Polynucleotides linked by phosphodiester bonds. Examples of natural nucleic acids include deoxyribonucleotide and ribonucleotide polymers such as DNA and RNA. The term "non-natural nucleic acid" refers to a nucleic acid containing non-natural nucleotides in place of or in addition to the above-mentioned natural nucleotides. Nucleotides, or nucleotide analogs that have similar properties to artificially made nucleotides, such as xanthosines and diaminopyrimidines. Nucleic acids of the present invention are derived from, for example, eukaryotes, bacteria, archaea, viruses, viroids, artificial synthetics, and the like.

As used herein, “purification” of nucleic acid means partially or completely separating and purifying nucleic acid contained in a sample from contaminants other than nucleic acid. As a measure of the degree of purification when partially purifying a nucleic acid, for example, using the purified nucleic acid, nucleic acid amplification methods such as PCR and isothermal nucleic acid amplification method, restriction enzyme treatment, cloning, base sequence analysis, Southern blotting, Northern blotting, hybrid capture analysis, line probe assay, microarray analysis, electrophoresis, mass spectrometry, fluorescent staining, dye staining, and molecular biological analysis using complementary binding between natural or non-natural nucleic acid strands It is preferable that the analysis can be performed without problems.

The method of purifying nucleic acid from a specimen containing colloidal particles of the present invention includes a solubilization step, a contacting step with a nucleic acid-adsorbing carrier, and an elution step from the nucleic acid-adsorbing carrier. In addition to these steps, the method of the present invention may optionally include a pretreatment step prior to the solubilization step. Each step constituting the present invention will be described in detail below.

<Pretreatment process>
In the method of the present invention, the “pretreatment step” is a step for pretreating the specimen to increase the solubilization efficiency of the subsequent solubilization step. Pretreatment steps include, for example, agitation with a stirrer, filtration by passing through pores such as a syringe or a filter, crushing with crushing beads, ultrasonic treatment, centrifugation, freeze-thaw, heat treatment, and reduction. treatment, alkali treatment, and enzymatic treatment with protease, lipase, lysozyme, and the like.

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

As used herein, "treating a sample with a solubilizing agent" means a step of allowing a solubilizing agent to act on a sample, for example, a step of mixing a solubilizing agent and a sample. This step can be performed, for example, by adding a solubilizing agent to the specimen and optionally stirring the mixture. If the mixture is agitated, the agitation may occur only during mixing, or may occur continuously or intermittently throughout the process. This step can be performed, for example, for 1 second to 30 minutes, preferably 5 seconds to 20 minutes, more preferably 10 seconds to 15 minutes.

In this step, when the sample is treated with the solubilizing agent, the 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 even more preferably 70°C or higher. The heat treatment time is preferably 1 second to 30 minutes, more preferably 5 seconds to 20 minutes, still more preferably 10 seconds to 15 minutes. Normally, when a sample such as blood or milk containing a high concentration of colloidal particles is heat-treated, the protein denatures and aggregates are formed, resulting in a decrease in nucleic acid purification efficiency. Since aggregates are not formed even when this is carried out, nucleic acids can be solubilized and purified with high efficiency.

As used herein, "solubilization" refers to reducing the particle size and/or molecular weight of colloidal particles without aggregating or aggregating them.

In the present invention, "solubilizer" refers to a substance used to solubilize colloidal particles, and may be a combination of two or more ingredients. 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 the form of a combination of individual independent components. "Treatment of the sample with a solubilizing agent" in the method of the present invention includes processing of the sample with any form of solubilizing agent. The solubilizers used in the present invention can solubilize not only colloidal particles, but also aggregates and aggregates of colloidal particles. Furthermore, the solubilizing agent used in the present invention disrupts the structure of colloidal particles when they are biological samples such as parasites, fungi, molds, yeasts, bacteria, archaea, spores, spores, cysts, or viruses. can be used to solubilize and disperse the nucleic acids they contain in solution.

Surfactants that can be used as solubilizers in the present invention are not particularly limited as long as they solubilize colloidal particles. Ionic surfactants can be mentioned. Anionic surfactants such as sodium dodecyl sulfate (SDS) and nonionic surfactants are preferred, and ester type, ether type, ester ether type, alkyl ether type, phenyl ether type and sorbitan are more preferred. Examples include derivative-type, alkanolamine-type, amide-type, ethylene glycol-type, and polyoxyethylene fatty alcohol ether-type nonionic surfactants. Specific examples of nonionic surfactants that can be used as solubilizers in the present invention include Tween 20 (trademark) (polysorbate 20, polyoxyethylene sorbitan monolaurate), Tween 80 (trademark) (polysorbate 80 , polyoxyethylene sorbitan monooleate), Triton X-100™ (poly(oxyethylene) octylphenyl ether), Nonidet P-40™ ((octylphenoxy) polyethoxyethanol), polyoxyethylene (20 ) cetyl ether, Amizol CDE-G™ (coconut fatty acid diethanolamide), and Amizol LDE-G™ (lauric acid diethanolamide).

Preferred surfactants that can be used as solubilizers in the present invention include, for example, amide-type surfactants such as fatty acid alkanolamides, polyoxyethylene fatty acid amides, and fatty acid amidopropylbetaines. As a surfactant that can be used as a solubilizing agent in the present invention, amide-type nonionic surfactants such as fatty acid alkanolamides and polyoxyethylene fatty acid amides are preferred, and fatty acid alkanolamides are particularly preferred. As used herein, fatty acid alkanolamides are preferably represented by the following general formula.

［式中、R¹は、炭素数5～21の直鎖若しくは分岐鎖のアルキル基若しくはアルケニル基、好ましくは炭素数7以上、9以上又は11以上であり、好ましくは炭素数19以下又は17以下であり、より好ましくは炭素数7～19、炭素数9～17、又は炭素数11～17の直鎖若しくは分岐鎖のアルキル基若しくはアルケニル基であり、
R²は、水素、又は炭素数1～3のヒドロキシアルキル基、好ましくは、水素又は炭素数2のヒドロキシアルキル基であり、
R³は、炭素数1～3のヒドロキシアルキル基、好ましくは、炭素数2のヒドロキシアルキル基である]

[In the formula, 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 carbon atoms, preferably 19 or less or 17 or less carbon atoms. and more preferably a linear or branched alkyl group 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 with 1 to 3 carbon atoms, preferably a hydroxyalkyl group with 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, and myristic acid monoethanolamide. Fatty acid monoethanolamides such as ethanolamide, palmitic acid monoethanolamide, stearic acid monoethanolamide, and oleic acid monoethanolamide, as well as coconut oil fatty acid diethanolamide, palm kernel oil fatty acid diethanolamide, lauric acid diethanolamide, myristic acid Fatty acid diethanolamides such as diethanolamide, palmitate diethanolamide, stearate diethanolamide, and oleate diethanolamide are included. Surfactants that can be used as solubilizers in the present invention are preferably fatty acid diethanolamides, particularly preferably coconut fatty acid diethanolamide or lauric fatty acid diethanolamide.

In the solubilizer used in the present invention, 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 the concentration is sufficient to solubilize the colloidal particles, and a person skilled in the art can easily determine the optimum value. can. If the concentration of the surfactant is too low, the colloidal particles will not be sufficiently solubilized. The concentration of the surfactant is the final concentration when mixed with the specimen. % 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 20% by weight or less, such as 0.05% to 90% by weight, 0.1% to 80% by weight, 0.5% to 70% by weight, 1% to 60% by weight, 2% to 50% by weight, It may be from 3% to 40%, from 4% to 30%, or from 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 solubilizes colloidal particles. , thiols, sulfoxides, alkanes, alkenes, alkynes, amines, imines, amides, and nitrile amphiphilic organic solvents, preferably alcohol, sulfoxide and amine amphiphilic organic solvents. Specific examples of alcohol-based amphiphilic organic solvents include methanol, ethanol, 1-propanol, and isopropanol. Specific examples of sulfoxide-based amphiphilic organic solvents include dimethyl sulfoxide (DMSO), diethyl sulfoxide, and ethyl sulfoxide. methyl sulfoxide and the like, and specific examples of amine-based amphipathic organic solvents include aminoalcohols 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 dimethylsulfoxide (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 sufficient to solubilize the colloidal particles, and a person skilled in the art can easily determine the optimum value. be able to. The concentration of the amphipathic 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, 95 wt% or less, 90 wt% or less, 80 wt% or less, 70 wt% or less, or 60 wt% or less, such as 1 wt% to 95 wt%, 5 wt% to 90 wt% , 10% to 80%, 20% to 70%, or 30% to 60% by weight.

The solubilizing agents used in the present invention contain chaotropic agents in addition to surfactants and/or amphiphilic organic solvents. The chaotropic agent contained in the solubilizer used in the present invention is not particularly limited as long as it has the effect of promoting the binding of the nucleic acid and the nucleic acid-adsorptive carrier. 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 phosphate, sodium perchlorate, sodium trichloroacetate, and sodium trifluoroacetate, preferably guanidine salts. 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 solubilizing agent may be sufficient for binding the nucleic acid to the nucleic acid-adsorptive carrier, and the optimum value can be easily determined by those skilled in the art. The final concentration of the chaotropic agent when mixed with the sample should be, 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, 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, such as 0.1% to 90% by weight, 0.5% by weight or less. % to 80%, 1% to 70%, 2% to 60%, 5% to 50%, or 10% to 40% by weight.

By combining a surfactant capable of solubilizing colloidal particles and an amphiphilic organic solvent, an additive or synergistic solubilizing effect is expected. Therefore, the solubilizer used in the present invention is preferably: Contains both surfactants and amphiphilic organic solvents. Preferably, the solubilizer used in the present invention contains an amide-type surfactant and an amphipathic 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, amphipathic organic solvent, chaotropic agent), (amide-type surfactant, alcoholic organic solvent, chaotropic agent), (amide surfactant, sulfoxide organic solvent, chaotropic agent), (amide surfactant, amine organic solvent, chaotropic agent), (surfactant, sulfoxide 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 (Amide surfactants, sulfoxide organic solvents, chaotropic agents) and (Amide surfactants, amine organic solvents, chaotropic agents), which have a high ability to solubilize colloidal particles. Further preferred examples of the combination contained in the solubilizer of the present invention include (coconut fatty acid diethanolamide or lauric fatty acid diethanolamide, DMSO, guanidinium salts such as guanidine thiocyanate).

The solubilization step of the present invention can be performed in the presence of a buffer. A buffer such as a phosphate buffer or Good's buffer is preferably used as the buffer. 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 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 any pH as long as the colloidal particles and the like in the sample do not coagulate when the solubilizing agent and the sample are mixed, and the pH at the time of mixing with the sample is preferably pH 2 or higher. , more preferably pH 3 or higher, and still more preferably pH 4 or higher.

The solubilization step of the present invention includes, for example, a reducing agent such as dithiothreitol (DTT), a chelating agent such as ethylenediaminetetraacetic acid (EDTA), an alkalinizing agent, and protease, lipase, and It can be carried out 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 an RNase in order to selectively purify DNA, or selectively purify RNA. Purification can also be performed in the presence of, for example, RNA protecting agents, RNase inhibitors, and/or DNases.

In the solubilization step of the present invention, each component constituting the solubilizer may be added separately or together when mixing with the sample. That is, the solubilization step of the present invention, for example, the surfactant and / or amphipathic organic solvent, and the chaotropic agent, may be carried out by adding separately, or by adding a mixture containing each component can be done by More preferably, in the solubilization step of the present invention, the solubilizer is added as the solubilizer composition described below.

<Step of Contacting with Nucleic Acid-Adsorbing Carrier>
As used herein, the “step of contacting with a nucleic acid-adsorbing carrier” refers to bringing the sample treated with a solubilizing agent in the “solubilization step” into contact with the nucleic acid-adsorbing carrier, thereby binding the nucleic acid in the sample to the nucleic acid-adsorbing carrier. means the step of allowing

This step is not limited as long as the nucleic acid contained in the sample can be adsorbed to the nucleic acid adsorption carrier. For example, when the nucleic acid-adsorbing carrier is in the form of a column, it can be carried out by passing the sample through this column. It can be done by mixing.

As used herein, the nucleic acid-adsorbing carrier is not particularly limited as long as it adsorbs nucleic acid in the presence of a chaotropic agent and/or an organic solvent. Examples include inorganic substances such as silica, resins, and insoluble polysaccharides. A nucleic acid adsorption carrier composed of can be used. These nucleic acid-adsorbing carriers may be in any shape, such as columnar, powdery, fibrous, bead-like, membrane-like, and porous, as long as they are capable of adsorbing nucleic acids. The nucleic acid-adsorbing carrier may be magnetic or modified.

<Elution process from nucleic acid adsorption carrier>
The step of eluting from the nucleic acid-adsorbing carrier in the present invention means a step of eluting the nucleic acid adsorbed to the nucleic acid-adsorbing carrier by the above-described “contacting step with the nucleic acid-adsorbing carrier” with an eluent. This step optionally includes a washing step with a washing solution prior to 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 is adsorbed and contaminants derived from the specimen. molecular solutions, and sugar aqueous solutions. Ethanol, isopropanol, acetone, and the like are examples of the solvent contained in the cleaning solution, and those skilled in the art can easily determine the optimum values for their concentrations. The concentration of the solvent in the cleaning liquid may be, for example, 20% by weight or more, 30% by weight or more, or 40% by weight or more, and may be, 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.

After washing with the washing solution, the nucleic acid-adsorbing carrier may be subjected to centrifugal separation, heat treatment, and air-drying to remove the washing solution.

After washing, the elution solution used for eluting the nucleic acid from the nucleic acid-adsorbing carrier is not particularly limited as long as it can elute the nucleic acid from the nucleic acid-adsorbing carrier, and examples thereof include water and buffer solutions.

<Solubilizer composition>
In one aspect, the invention relates to solubilizer compositions that can be used in the methods of the invention. The solubilizer composition of the present invention contains at least the above surfactant and/or amphiphilic organic solvent, and a chaotropic agent, preferably the surfactant, amphiphilic organic solvent, and chaotropic agent. The configurations of the surfactant, amphipathic organic solvent, and chaotropic agent that can be contained in the solubilizer composition of the present invention are as described in the <Solubilization step> above, and therefore descriptions thereof are 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% 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 20% by weight or less, such as 0.05% to 90% by weight, 0.1% to 80% by weight, 0.5% to 70% by weight, 1% to 60% by weight, 2% by weight % to 50% by weight, 3% to 40% by weight, 4% to 30% by weight, or 5% to 20% by weight. In addition, when the solubilizer composition of the present invention contains an amphipathic organic solvent, the concentration of the amphipathic organic solvent is, 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, such as 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, such as 1% to 95% by weight. % by weight, 5% to 90% by weight, 10% to 80% by weight, 20% to 70% by weight, 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% 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, 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, such as 0.1% to 90% by weight. , 0.5% to 80%, 1% to 70%, 2% to 60%, 5% to 50%, or 10% to 40% by weight. The final concentration of each component when the solubilizing agent composition of the present invention is mixed with the specimen is as described in the <Solubilization step> above, so the description is omitted here.

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

The pH of the solubilizer composition of the present invention may be any pH that does not coagulate colloidal particles and the like in the sample when mixed with the sample. It is preferably pH 3 or higher, more preferably pH 4 or higher.

For promoting solubilization, the solubilizer composition of the present invention contains, for example, a reducing agent such as dithiothreitol (DTT), a chelating agent such as ethylenediaminetetraacetic acid (EDTA), an alkalinizing agent, protease, and lipase. , and degrading enzymes such as lysozyme. Further, the solubilizer composition of the present invention may contain, for example, a DNA protectant, a DNase inhibitor, and/or an RNase to selectively purify DNA, and may also selectively purify RNA. For example, RNA protectants, RNase inhibitors, and/or DNases may be included for purification.

The solubilizer composition of the present invention can be used to solubilize colloidal particles in order to purify nucleic acids from specimens containing the 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 invention relates to a nucleic acid purification kit comprising a solubilizer composition of the invention. In addition to the solubilizer composition, the kit may contain, for example, a nucleic acid adsorption carrier, an adsorption solution, an elution solution, PCR primers, buffers, enzymes, and/or instructions for use.

In one aspect, the present invention separately contains (for example, contains 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 elution solution, PCR primers, buffers, enzymes, and/or instructions for use. The structures of the surfactant, amphipathic organic solvent, and chaotropic agent contained in the solubilizer used in the present invention are as described in the <Solubilization step> above, and therefore descriptions thereof are omitted here.

A person skilled in the art can appropriately determine the weight ratio of each component when the kit of the present invention contains each component separately. 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 per 100 parts by weight of the chaotropic agent, 1 part by weight or more, 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 to 10000 parts by weight, 1 to 1000 parts by weight, 5 to 500 parts by weight, 10 to 100 parts by weight, or 20 to 60 parts by weight good. When the kit of the present invention contains the amphipathic organic solvent and the chaotropic agent separately, the weight ratio of the amphipathic 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 parts by weight It may be parts by weight or less, more specifically 0.3 to 30000 parts by weight, 3 to 3000 parts by weight, 6 to 1500 parts by weight, 30 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 separately contains a surfactant, an amphipathic organic solvent, and a chaotropic agent, the kit of the present invention contains surfactants in the above weight ratios, for example, with respect to 100 parts by weight of the chaotropic agent. agent and amphiphilic organic solvent in the above weight ratio. The final concentration of each component when the surfactant and/or amphipathic organic solvent and the chaotropic agent contained in the kit of the present invention are mixed with the specimen is as described in <Solubilization step> above. 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, an automatic nucleic acid analysis device, and the like.

EXAMPLES The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these examples.
<Example 1: Evaluation 1 of ability to solubilize colloidal particles>
1) Overview In order to evaluate the ability of surfactants and amphipathic organic solvents to solubilize colloidal particles, milk is mixed with each surfactant or amphipathic organic solvent and the change in turbidity (OD ₆₆₀ ) is measured. The degree of solubilization of colloidal particles was evaluated by When the colloidal particles are solubilized without agglomeration, the colloidal particle diameter becomes small, so the light transmittance increases, which can be observed as a decrease in turbidity.

2) Method Mix 10 μl of commercially available milk (Megmilk Snow Brand) with 190 μl of surfactant or amphiphilic organic solvent, leave to stand for 10 minutes, collect 100 μl from the mixture and transfer to ASSAY PLATE 96WELL (IWAKI). Transferred and OD ₆₆₀ 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 Chemical), Amizol CDE-G (Kawaken Fine Chemicals), Amizol LDE-G (Kawaken Fine Chemicals) and SDS, and DMSO, ethanol, isopropanol, ethylenediamine, and pyridine as amphipathic organic solvents. The surfactant was diluted to 5% by weight to 20% by weight, and the amphiphilic organic solvent was diluted to 5% by volume to 50% by volume with sterilized distilled water for use in the test.

3) Results A decrease in turbidity was confirmed in all samples to which each surfactant or amphipathic organic solvent was added, and coagulation of milk components was not confirmed. In particular, the turbidity is greatly reduced in the samples added with the amide-type surfactant Amizol CDE-G or Amizol LDE-G, the sulfoxide organic solvent DMSO, or the amine organic solvent ethylenediamine or pyridine. It was confirmed that they have a particularly strong ability to solubilize colloidal particles (Fig. 1).

<Example 2: Evaluation 2 of ability to solubilize colloidal particles>
1) Method Solubilizer compositions A to L containing a surfactant and/or an amphipathic organic solvent and a chaotropic agent at the following concentrations in sterile distilled water were prepared (A: 10% by weight Amizol CDE-G and 3M (about 35 wt%) guanidine thiocyanate, B: 20 wt% Amizol CDE-G and 3M guanidine thiocyanate, C: 30 wt% (about 32 wt%) DMSO and 3M guanidine thiocyanate, D: 60 wt% (about 62% by weight) DMSO and 3M guanidine thiocyanate, E: 10% by weight Amisole CDE-G, 30% by volume DMSO, and 3M guanidine thiocyanate, F: 10% by weight Amisole CDE-G, 30% by volume (about 25% by volume) %) 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% by weight Nonidet P-40, 30% by volume DMSO, and 3M guanidine thiocyanate, 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 guanidine thiocyanate, L: 3M guanidine thiocyanate). As a control, M: sterilized distilled water was used. In order to evaluate the ability of each solubilizer composition to solubilize colloidal particles, milk was mixed with each solubilizer composition and the degree of solubilization of colloidal particles was evaluated by measuring the decrease in turbidity. That is, 100 μl of commercial milk (Megmilk Snow Brand) and 500 μl of each solubilizer composition were mixed and allowed to stand for 10 minutes. OD ₆₆₀ was measured by Benchmark Plus (Bio-RAD). In addition, it was visually evaluated whether or not the mixed liquid had coagulated. Furthermore, the mixture was heat-treated at 80° C. for 10 minutes, and similarly evaluated whether or not coagulation would occur.

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 samples to which the solubilizer compositions B, D, and E were added showed a large decrease in turbidity, confirming that they have a particularly strong ability to solubilize colloidal particles. In addition, no coagulation of milk components due to the addition of each solubilizer composition was observed before and after the heat treatment (Table 1). On the other hand, in the solubilizer compositions K and L, a slight decrease in turbidity was observed compared to the control solution M, but the decrease in turbidity was slight compared to the solubilizer compositions A to J. there were. Further, in the solubilizer compositions K to L and sterile distilled water M, coagulation of milk components was confirmed by heat treatment of the solution after mixing with milk (Table 1).

<Example 3>
1) Overview Nucleic acids were purified from milk containing microorganisms using the solubilizer compositions A to L or sterile distilled water M prepared in Example 2 and a silica column. Subsequently, the purified nucleic acid was used to perform real-time PCR targeting the microorganism-derived nucleic acid, and the appropriate purification of the nucleic acid and the nucleic acid recovery efficiency were evaluated.

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

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

Real-time PCR targeting S. thermophilus was performed using the purified nucleic acid. The composition of the PCR reaction solution is as follows: 10 µM forward primer STf (5'-GCTCCCACTACAAGATGGACCTGC-3' (sequence number 1)) 1.5 µl, 10 µM reverse primer STr (5'-TAGGAGTCTGGGCCGTGTCTCAG-3' (sequence number 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) 2.5 μl, purified DNA 2.5 μl, and sterilized distilled water 31.5 μl. The reaction was performed using the LightCycler 96 system (Roche), and after 1 minute at 98°C, 45 PCR cycles of 98°C 5 seconds → 60°C 5 seconds → 72°C 15 seconds were performed and the nucleic acid amplification reaction was monitored.

3) Results Nucleic acid amplification was confirmed from the samples purified using the solubilizer compositions A to J, confirming that the nucleic acids were properly purified. Samples purified using solubilizer compositions B, D, E, and G were found to have particularly low Cq values, confirming that nucleic acid recovery efficiency is high 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, but it was confirmed that the Cq values were higher and the nucleic acid recovery efficiency was lower than those of the solubilizer compositions A to J. (Fig. 2).

<Example 4>
1) Overview A solubilizer composition and silica-coated magnetic particles were used to purify nucleic acids from milk containing microorganisms. Using the purified nucleic acid, PCR targeting the microorganism-derived nucleic acid was performed to evaluate whether the nucleic acid was appropriately purified and the lower limit of detection.

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) in commercial milk (Megmilk Snow Brand) A positive bacterium, Streptococcus thermophilus, was added and suspended, and this was used as a milk sample.

As the solubilizer composition, a mixture of 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 solubilizing agent composition was added to 100 µl of the milk sample added to a 1.5 ml tube and incubated at 80°C for 10 minutes to solubilize the colloidal particles. 20 μl of silica-coated magnetic particles MagPrep (registered trademark) Silica Particles (Merck Millipore) were added to the solubilized milk sample and mixed well. 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 to separate the magnetic particles and remove the supernatant. After repeating 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 collected as purified nucleic acid. In this example, nucleic acid purification was possible in about 10 minutes without using a high-speed centrifuge or special equipment.

The composition of the PCR reaction solution is as follows: 10 µM forward primer STf (5'-GCTCCCACTACAAGATGGACCTGC-3' (sequence number 1)) 1.5 µl, 10 µM reverse primer STr (5'-TAGGAGTCTGGGCCGTGTCTCAG-3' (sequence number 2)) 1.5 µl, 0.5 µl of Kaneka high-speed amplification DNA Polymerase (Kaneka), 5.0 µl of 10x Kaneka high-speed amplification DNA Polymerase buffer, 5.0 µl of 2 mM dNTPs, 2.5 µl of purified DNA, and 34.0 µl of sterilized distilled water. The reaction was carried out 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, and a 130 bp amplified fragment was visualized.
3) Results An amplified fragment of 130 bp was confirmed by electrophoresis, confirming that the nucleic acid was properly purified from the model milk containing S. thermophilus at a concentration of 3.5 x 103 cfu/m or ^{3.5 x} 102 cfu/ml. (Fig. 3).

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

2) Method 10 ⁵ cfu/ml Gram-positive bacterium Streptococcus thermophilus was added to heparinized mouse blood and suspended, and this was used as model blood.

As the solubilizer composition, a mixture of 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 solubilizing agent composition was added to 100 μl of model blood in a 1.5 ml tube and incubated at 80° C. for 10 minutes to solubilize the colloidal particles. The sample after heat treatment was completely solubilized, and no aggregates due to protein or the like were confirmed. Thereafter, the same procedure as in Example 3 was used to recover the purified nucleic acid. The purified nucleic acid was then used to perform real-time PCR targeting S. thermophilus. The reaction and preparation of reagents 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 the nucleic acid was properly purified from the blood sample containing S. thermophilus (Fig. 4).

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

Claims (17)

A method for purifying nucleic acids contained in a sample from a sample containing colloidal particles, comprising:
(a) one or more amphiphilic organic solvents selected from the group consisting of (i) amide-type surfactants, (ii) chaotropic agents, and (iii) sulfoxide-based and amine-based amphiphilic organic solvents treating the sample with a solubilizing agent containing a solvent to solubilize the colloidal particles in the sample;
(b) a step of contacting a specimen treated with a solubilizing agent with a nucleic acid-adsorbing carrier; and (c) a step of eluting the nucleic acid from the nucleic acid-adsorbing carrier;
including
The amide type surfactant is 2% to 50% by weight of coconut oil fatty acid diethanolamide or lauric acid diethanolamide,
the chaotropic agent is 10% to 40% by weight of a guanidine salt;
5% to 70% by weight of the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine and pyridine;
The above method, wherein the colloidal particles are selected from the group consisting of micelles, liposomes, fat globules, and solid microparticles . A method for purifying nucleic acids contained in a sample from a sample containing colloidal particles, comprising:
(a) (i) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and (ii) a solubilizer containing a chaotropic agent. treating to solubilize the colloidal particles in the specimen;
(b) a step of contacting a specimen treated with a solubilizing agent with a nucleic acid-adsorbing carrier; and (c) a step of eluting the nucleic acid from the nucleic acid-adsorbing carrier;
including
the chaotropic agent is 10% to 40% by weight of a guanidine salt;
5% to 70% by weight of the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine and pyridine;
The above method, wherein the colloidal particles are selected from the group consisting of micelles, liposomes, fat globules, and solid microparticles . 3. The method of claim 2, wherein the solubilizing agent further comprises a nonionic surfactant. A method according to any one of claims 1 to 3, wherein the chaotropic agent is guanidine thiocyanate. Colloidal fluid, emulsion, animal milk, milky body fluid, whole blood, serum, lymphatic fluid, urine, sweat, nasal fluid, spinal fluid, interstitial fluid, semen, vaginal fluid, amniotic fluid, tears, saliva, feces, pus, sputum , casey tissue, cell suspension, cell-containing fluid, tissue fluid, milky sap, milky fruit juice, vegetable milk, dairy products, and cosmetics, pharmaceuticals, and detergents containing colloidal particles. The method according to any one of 1-4. 6. The method of claim 5, wherein the specimen is selected from the group consisting of animal milk, milky fluid, whole blood, serum, and lymph. A solubilizer composition for solubilizing colloidal particles for purifying nucleic acids contained in a specimen from a specimen containing colloidal particles, comprising (i) an amide-type surfactant, (ii) a chaotropic agent, and (iii) containing one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents,
The amide type surfactant is 2% to 50% by weight of coconut oil fatty acid diethanolamide or lauric acid diethanolamide,
the chaotropic agent is 10% to 40% by weight of a guanidine salt;
5% to 70% by weight of the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine and pyridine;
A solubilizer composition, wherein the colloidal particles are selected from the group consisting of micelles, liposomes, fat globules, and solid microparticles . A solubilizer composition for solubilizing colloidal particles in order to purify nucleic acid contained in a specimen from a specimen containing colloidal particles, comprising (i) a group consisting of sulfoxide-based and amine-based amphipathic organic solvents one or more amphiphilic organic solvents selected from and (ii) a chaotropic agent,
the chaotropic agent is 10% to 40% by weight of a guanidine salt;
5% to 70% by weight of the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine and pyridine;
A solubilizer composition, wherein the colloidal particles are selected from the group consisting of micelles, liposomes, fat globules, and solid microparticles . 9. The solubilizer composition of claim 8, further comprising a nonionic surfactant. The solubilizer composition according to any one of claims 7 to 9, wherein the chaotropic agent is guanidine thiocyanate. The solubilizer composition according to any one of claims 7 to 10, wherein the specimen is selected from the group consisting of animal milk, milky body fluid, whole blood, serum and lymph. 12. A nucleic acid purification kit for purifying a nucleic acid contained in a specimen from a specimen containing colloidal particles, comprising the solubilizer composition according to any one of claims 7 to 11. A solubilizer kit for solubilizing colloidal particles, which is selected from the group consisting of (i) an amide-type surfactant, (ii) a chaotropic agent, and (iii) a sulfoxide-based and amine-based amphiphilic organic solvent. containing one or more amphiphilic organic solvents,
The amide type surfactant is 2% to 50% by weight of coconut oil fatty acid diethanolamide or lauric acid diethanolamide,
the chaotropic agent is 10% to 40% by weight of a guanidine salt;
5% to 70% by weight of the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine and pyridine;
the colloidal particles are selected from the group consisting of micelles, liposomes, fat globules, and solid microparticles ;
Solubilizer Kit. A solubilizer kit for solubilizing colloidal particles, comprising (i) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents; (ii) contains a chaotropic agent;
the chaotropic agent is 10% to 40% by weight of a guanidine salt;
5% to 70% by weight of the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine and pyridine;
the colloidal particles are selected from the group consisting of micelles, liposomes, fat globules, and solid microparticles ;
Solubilizer kit. A nucleic acid purification kit for purifying nucleic acids contained in a specimen from a specimen containing colloidal particles,
(i) an amide-type surfactant, (ii) a chaotropic agent, and (iii) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents. ,
The amide type surfactant is 2% to 50% by weight of coconut oil fatty acid diethanolamide or lauric acid diethanolamide,
the chaotropic agent is 10% to 40% by weight of a guanidine salt;
5% to 70% by weight of the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine and pyridine ;
wherein the amide-type surfactant, chaotropic agent, and amphiphilic organic solvent are used to solubilize the colloidal particles ;
Nucleic acid purification kit. A nucleic acid purification kit for purifying nucleic acids contained in a specimen from a specimen containing colloidal particles,
(i) one or more amphiphilic organic solvents selected from the group consisting of sulfoxide-based and amine-based amphiphilic organic solvents, and (ii) a chaotropic agent,
the chaotropic agent is 10% to 40% by weight of a guanidine salt;
5% to 70% by weight of the amphiphilic organic solvent is one or a combination of two or more selected from the group consisting of DMSO, ethylenediamine and pyridine ;
wherein the amphiphilic organic solvent and chaotropic agent are used to solubilize the colloidal particles ;
Nucleic acid purification kit. 17. The solubilizer kit of claim 14, or the nucleic acid purification kit of claim 16, further comprising a nonionic detergent.

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