Method for extracting a target substance and method for detecting a target substance

Abstract

The present invention aims to provide a method for suppressing the contamination of the eluent with ethanol. [Solution] The present invention's method for extracting a target substance 20 comprises: (a) contacting a magnetic carrier 10 holding the target substance 20 with an ethanol-containing washing solution; (b) after step (a), contacting the magnetic carrier 10 with a liquid organic compound 200; and (c) after step (b), contacting the magnetic carrier 10 with an elution solution 300 that is immiscible with the organic compound 200 to elute the target substance 20 into the elution solution 300, wherein the organic compound 200 is miscible with ethanol and miscible with water.

Description

Technical Field

[0001] The present invention relates to a method for extracting a target substance and a method for detecting a target substance.

Background Art

[0002] In recent years, with the development of gene utilization technology, gene-based medical applications such as genetic diagnosis and gene therapy have attracted attention. In addition, many gene-based techniques for cultivar identification and cultivar improvement have been developed in the field of agriculture and livestock. As a technique for utilizing genes, techniques such as the PCR (Polymerase Chain Reaction) method are widely widespread. Today, the PCR method has become an indispensable technique for elucidating the information of biological materials.

[0003] The PCR method is a technique for amplifying a target nucleic acid by subjecting a solution (reaction solution) containing the nucleic acid to be amplified (target nucleic acid) and reagents to thermal cycling. Thermal cycling is a treatment in which two or more temperature stages are periodically applied to a reaction solution. In PCR methods, methods involving two-step or three-step thermal cycling are common.

[0004] For example, Patent Document 1 discloses a nucleic acid-binding solid support comprising magnetic particles of amorphous metal containing Fe, Cr, Si, and B, and a silicon oxide film provided on the surface of the magnetic particles. According to the nucleic acid-binding solid support described in Patent Document 1, it is stated that nucleic acids can be extracted efficiently.

Prior Art Literature

Patent Literature

[0005]

Patent Document 1

Summary of the Invention

Problem to be Solved by the Invention

[0006] Ethanol is commonly used to clean magnetic carriers that hold target substances. After cleaning the magnetic carriers with ethanol, if the carriers are brought into contact with an eluent, ethanol may become mixed into the eluent. This ethanol mixed into the eluent can inhibit the amplification reaction of the target substance.

[0007] Therefore, the present invention aims to provide a method for suppressing the contamination of the eluent with ethanol. [Means for solving the problem]

[0008] The inventors, after diligent research, discovered that the above problems could be solved by the following means, and thus completed the present invention. That is, the present invention is as follows.

[0009] (Aspect 1) (a) Contacting the magnetic carrier holding the target substance with an ethanol-containing cleaning solution, (b) After step (a), the magnetic carrier is brought into contact with a liquid organic compound, and (c) After step (b), the magnetic carrier is brought into contact with an eluate that is incompatible with the organic compound, and the target substance is eluted into the eluate. Includes, The above organic compound is miscible with ethanol and miscible with water. A method for extracting target substances. (Aspect 2) The method according to embodiment 1, wherein the above organic compound comprises at least one compound selected from aliphatic alcohols, linear alkanes, linear fatty acids, and cyclic fatty acids. (Aspect 3) The method according to embodiment 2, wherein the organic compound comprises at least one compound selected from (A) to (D) below: (A) Aliphatic alcohols with 6 to 13 carbon atoms; (B) Straight-chain alkanes with 6 to 19 carbon atoms; (C) Straight-chain fatty acids with 6 to 11 carbon atoms; (D) A cyclic fatty acid with 10 carbon atoms. (Aspect 4) The method according to any one of embodiments 1 to 3, wherein the eluent is an aqueous solution containing a component selected from the group consisting of a metal salt, a pH buffer, and a surfactant, or pure water. (Aspect 5) The method according to any one of embodiments 1 to 4, further comprising removing at least a portion of the ethanol-containing cleaning solution from the magnetic carrier after step (a) and before step (b). (Aspect 6) The method according to any one of embodiments 1 to 5, wherein the ethanol-containing cleaning solution is not air-dried after step (a) and before step (b). (Aspect 7) The method according to any one of embodiments 1 to 6, further comprising removing at least a portion of the organic compound from the magnetic support after step (b) and before step (c). (Pattern 8) The method according to any one of embodiments 1 to 7, wherein the magnetic carrier has an inorganic oxide surface. (Aspect 9) The method according to embodiment 8, wherein the inorganic oxide surface is silicon oxide. (Aspect 10) The method according to embodiment 8 or 9, wherein the magnetic carrier is composed of iron-based metal particles and the inorganic oxide surface. (Aspect 11) Extracting the target substance by the method described in any one of the embodiments 1 to 10, and To detect the extracted target substance. A method for detecting a target substance, including the detection of a target substance. (Aspect 12) The method according to embodiment 11, comprising performing an enzymatic reaction on the extracted target substance, and then detecting the purified and enzymatically reacted target substance. (Aspect 13) The method according to embodiment 12, wherein the above-mentioned enzymatic reaction includes a nucleic acid amplification reaction. [Effects of the Invention]

[0010] According to the method of the present invention, mixing of ethanol into the eluate can be suppressed. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] [Figure 1] FIG. 1 is a schematic diagram for explaining step (a) of the present invention. [Figure 2] FIG. 2 is a schematic diagram for explaining step (b) of the present invention. [Figure 3] FIG. 3 is a schematic diagram for explaining step (c) of the present invention. MODE FOR CARRYING OUT THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail. The present invention is not limited to the following embodiments, and can be implemented with various modifications within the scope of the gist of the present invention.

[0013] <<Method for Extracting Target Substance>> The method of the present invention for extracting a target substance comprises the following steps: (a) contacting a magnetic carrier holding a target substance with an ethanol-containing washing solution, (b) after step (a), contacting the magnetic carrier with a liquid organic compound, and (c) after step (b), contacting the magnetic carrier with an eluate immiscible with the organic compound to elute the target substance into the eluate, the organic compound is compatible with ethanol and incompatible with water.

[0014] In the present invention, a magnetic carrier holding a target substance is contacted with an ethanol-containing washing solution, and then the magnetic carrier is contacted with a liquid organic compound. Ethanol is miscible with such a liquid organic compound.

[0015] Subsequently, the magnetic carrier, which has been in contact with the liquid organic compound, is brought into contact with an eluent that is immiscible with the liquid organic compound, and the target substance is eluted into the eluent. At this time, since the liquid organic compound is immiscible with the eluent, it separates into two layers: one mainly composed of the liquid organic compound and the other mainly composed of the eluent. As described above, ethanol is miscible with the liquid organic compound, so during this layer separation, ethanol enters the layer mainly composed of the liquid organic compound, and therefore the inclusion of ethanol in the eluent can be suppressed.

[0016] The process may further include removing at least a portion of the ethanol-containing cleaning solution from the magnetic carrier after step (a) and before step (b). For example, at least a portion of the ethanol-containing cleaning solution may be removed from the magnetic carrier by applying an external magnetic field to the magnetic carrier using a magnetic field source to form an aggregate of magnetic carriers and separate it from the ethanol-containing cleaning solution.

[0017] Furthermore, in the present invention, the removal of at least a portion of the ethanol-containing cleaning solution from the magnetic carrier may be repeated one or more times after step (a) and before step (b).

[0018] In the present invention, the process may or may not include air drying of the ethanol-containing cleaning solution after step (a) and before step (b), but air drying of the ethanol-containing cleaning solution can be omitted to simplify the process.

[0019] In the present invention, the process may further include removing at least a portion of the liquid organic compound from the magnetic carrier after step (b) and before step (c). For example, at least a portion of the liquid organic compound may be removed from the magnetic carrier by applying an external magnetic field to the magnetic carrier using a magnetic field source to form an aggregate of magnetic carriers and separate it from the liquid organic compound.

[0020] <Step of bringing the magnetic carrier into contact with the cleaning solution (Step (a))> In the present invention's method for extracting a target substance, first, the magnetic carrier holding the target substance is brought into contact with an ethanol-containing washing solution. This makes it possible to remove at least a portion of the impurities adsorbed on the magnetic carrier.

[0021] As shown in Figure 1, the magnetic carrier 10 holding the target substance 20 is brought into contact with the ethanol-containing cleaning solution 100 (Figure 1(a)). For example, as shown in Figures 1(b) and (c), before step (b), the magnetic carrier 10 may be magnetized using a magnet 50 to remove a portion of the ethanol-containing cleaning solution 100. At this time, some of the ethanol-containing cleaning solution 100 may remain between the magnetized magnetic carriers 10.

[0022] In the present invention, step (a) may be performed two or more times.

[0023] (Target substance) In this invention, the target substance is held on a magnetic carrier.

[0024] In the present invention, the target substance is not particularly limited, but examples include nucleic acids such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), proteins, vesicles, extracellular vesicles, viruses, cells, cell aggregates, protein aggregates, and the like.

[0025] (Magnetic carrier) In this invention, the magnetic carrier holds the target substance.

[0026] In the present invention, the magnetic carrier is not particularly limited, as long as it has residual magnetization and is capable of adsorbing a target substance. For example, the magnetic carrier includes ferrite fine particles and magnetic metal particles.

[0027] In the present invention, to increase the resistance of the liquid to organic compounds, the magnetic carrier may have, for example, an inorganic oxide surface. When the magnetic carrier has an inorganic oxide surface, examples of inorganic oxide surfaces include silicon oxide, magnesium oxide, calcium oxide, aluminum oxide, titanium oxide, zirconium oxide, boron oxide, yttrium oxide, etc., and may be one or a mixture of two or more of these.

[0028] In the present invention, the inorganic oxide surface is preferably silicon oxide. Silicon oxide can specifically adsorb the target substance in the adsorption solution, thereby enabling efficient extraction and recovery of the target substance. Furthermore, because silicon oxide is chemically stable, it can particularly suppress oxidation of the magnetic carrier and corrosion by the organic compound of the present invention.

[0029] Furthermore, because of its high specific gravity, the magnetic carrier may be iron-based metal particles, or it may be a particle composed of a combination of iron-based metal particles and an inorganic oxide surface.

[0030] In the present invention, the saturation magnetization of the magnetic carrier may be 5 emu / g or more, 10 emu / g or more, 20 emu / g or more, 40 emu / g or more, 50 emu / g or more, or 60 emu / g or more, and may be 300 emu / g or less, 250 emu / g or less, 200 emu / g or less, 150 emu / g or less, 120 emu / g or less, 110 emu / g or less, or 100 emu / g or less.

[0031] The saturation magnetization of a magnetic carrier can be measured using a vibrating sample magnetometer (VSM), for example. An example of a vibrating sample magnetometer is the TM-VSM1230-MHHL manufactured by Tamagawa Seisakusho Co., Ltd. The maximum applied magnetic field when measuring saturation magnetization is, for example, 0.5 T or higher.

[0032] In the present invention, the particle size D of the magnetic carrier 50The particle size may be 0.1 μm or larger, 0.2 μm or larger, or 0.3 μm or larger, and may be 30 μm or smaller, 20 μm or smaller, or 10 μm or smaller.

[0033] Note that the particle size D of the magnetic carrier 50 This can be determined by measuring the volume-based particle size distribution using laser diffraction-dispersion and then obtaining the integrated distribution curve from this particle size distribution. Specifically, in the integrated distribution curve, the particle size D is the one where the cumulative value from the smallest diameter side reaches 50%. 50 (Median diameter) is the particle size D of the magnetic carrier. 50 Examples of devices that measure particle size distribution using laser diffraction and dispersion include the MT3300 series manufactured by Microtrac-Bell. Note that methods other than laser diffraction and dispersion, such as image analysis, may also be used.

[0034] (Ethanol-containing cleaning solution) In the present invention, the ethanol-containing cleaning solution comes into contact with the magnetic carrier in step (a).

[0035] In the present invention, the ethanol-containing cleaning solution is not particularly limited as long as it is a liquid containing ethanol. The ethanol-containing cleaning solution may further contain components that do not promote the elution of target substances and do not promote the binding of impurities to the magnetic carrier. These components are not particularly limited, but may further contain, for example, organic solvents such as isopropyl alcohol, phenol, and chloroform, water, or a low-salt aqueous solution. Examples of low-salt aqueous solutions include buffer solutions. The salt concentration of the low-salt aqueous solution is preferably 0.1 mM to 100 mM, and more preferably 1 mM to 50 mM. The salt used to make the buffer solution is not particularly limited, but salts such as TRIS (tris(hydroxymethyl)aminomethane), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), PIPES (Piperazine-1,4-bis (2-ethanesulfonic acid)), and phosphoric acid are preferably used.

[0036] In the present invention, the ethanol-containing cleaning solution may contain surfactants such as Triton®, Tween®, or sodium dodecyl sulfate (SDS). Furthermore, the ethanol-containing cleaning solution may contain chaotropic substances such as guanidine hydrochloride. The pH of the ethanol-containing cleaning solution is not particularly limited.

[0037] In the present invention, when step (a) is performed two or more times, the concentration of ethanol in the ethanol-containing cleaning solution may be the same or different in each of the multiple steps (a).

[0038] <Step (b)) of bringing the magnetic carrier into contact with a liquid organic compound> In the present invention's method for extracting a target substance, the magnetic carrier is then brought into contact with a liquid organic compound. This allows at least a portion of the ethanol-containing cleaning solution surrounding the magnetic carrier to dissolve in the liquid organic compound.

[0039] As shown in Figure 2, the magnetic carrier 10 holding the target substance 20 is brought into contact with the liquid organic compound 200 (Figure 2(a)). As a result, the remaining portion of the ethanol-containing cleaning solution 100 (not shown) used in step (a) around the magnetic carrier is mixed with the liquid organic compound 200. For example, as shown in Figures 2(b) and (c), the magnetic carrier 10 may be magnetized using a magnet 50 before step (c) to remove some of the liquid organic compound 200. At this time, some of the liquid organic compound 200 may remain between the magnetized magnetic carriers 10.

[0040] (organic compound) In the present invention, the organic compound (hereinafter also referred to as "oil cleaning solution") comes into contact with a magnetic carrier. The organic compound of the present invention is miscible with ethanol but miscible with water. In the present invention, the organic compound is immiscible with the eluate. In the present invention, "immiscible" means a state in which the organic compound and the eluate are separated into two layers, and the organic compound may be dissolved in the eluate at room temperature (20°C) in an amount of 0.0% by mass or more, 0.2% by mass or more, or 0.4% by mass or more, or in an amount of 10.0% by mass or less, 5.0% by mass or less, 3.0% by mass or less, 1.0% by mass or less, 0.8% by mass or less, or 0.6% by mass or less.

[0041] In the present invention, the organic compound is not particularly limited, but examples include alcohols, carboxylic acids, hydrocarbons, etc. The organic compound may be an aliphatic organic compound or an aromatic organic compound. The aliphatic organic compound may have a linear structure, a branched structure, or a cyclic structure. The organic compound may contain at least one selected from aliphatic alcohols, aliphatic carboxylic acids, and aliphatic hydrocarbons, or it may contain at least one selected from aliphatic alcohols, linear alkanes, linear fatty acids, and cyclic fatty acids. Furthermore, the organic compound of the present invention may be a mixture containing any of the above substances.

[0042] In the present invention, the aliphatic alcohol is not particularly limited, but examples include aliphatic alcohols having 6 to 13 carbon atoms, particularly 10 to 12 carbon atoms, and particularly 11 carbon atoms. As a result, the aliphatic alcohol is not miscible with the eluate. Specifically, the aliphatic alcohol may be hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, and tridecanol, and particularly 1-undecanol.

[0043] Aliphatic carboxylic acids include straight-chain fatty acids, branched fatty acids, and cyclic fatty acids. In the present invention, the aliphatic carboxylic acid is not particularly limited, but examples include aliphatic carboxylic acids having 6 to 11 carbon atoms, specifically hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, naphthenic acid, and the like.

[0044] In the present invention, the aliphatic hydrocarbon is not particularly limited, but examples include aliphatic hydrocarbons having 6 to 19 carbon atoms, specifically hexane, heptane, octane, nonane, decane, undecane, tetradecane, pentadecane, hexadecane, heptadecane, nonadecane, etc. The aliphatic hydrocarbon may also be a straight-chain alkane.

[0045] The organic compound may be at least one selected from (A) to (D) below: (A) Aliphatic alcohols with 6 to 13 carbon atoms; (B) Straight-chain alkanes with 6 to 19 carbon atoms; (C) Straight-chain fatty acids with 6 to 11 carbon atoms; (D) A cyclic fatty acid with 10 carbon atoms.

[0046] The oil cleaning solution may be a mixture of the above-mentioned organic compounds in any proportion.

[0047] If the organic compound of the present invention is solid or semi-solid at room temperature (20°C), or has high viscosity, it may be liquefied and / or its viscosity reduced by heating before use. The heating temperature is not particularly limited.

[0048] <Elution step (step (c))> In the present invention's method for extracting target substances, the magnetic carrier is then brought into contact with an eluent that is immiscible with organic compounds, thereby eluting the target substance into the eluent. This separates the organic compounds present around the magnetic carrier from the eluent, thus reducing the amount of impurities in the eluent, particularly ethanol-containing washing solutions.

[0049] As shown in Figure 3, the magnetic carrier 10 holding the target substance 20 is brought into contact with the eluent 300. This causes the liquid organic compound 200, which is mixed with the ethanol-containing washing solution 100 (not shown), to separate from the eluent. This suppresses the inclusion of the ethanol-containing washing solution in the eluent, and therefore reduces the ethanol content in the eluent.

[0050] (Eluate) In the present invention, the eluent comes into contact with the magnetic carrier in step (c). In the present invention, the eluent is a liquid that elutes the target substance and is immiscible with the organic compound in the present invention.

[0051] The eluate is a liquid that separates the target substance from the magnetic carrier on which it is adsorbed, and then elutes the nucleic acid into the eluate.

[0052] In the present invention, the eluate may be an aqueous solution containing components selected from the group consisting of metal salts, pH buffers, and surfactants. Alternatively, the eluate may be, for example, pure water.

[0053] In the present invention, the eluent may be, but is not limited to, a solution containing trishydroxymethylaminomethane (Tris) and ethylenediaminetetraacetic acid (EDTA), i.e., a TE buffer.

[0054] ≪Method for detecting target substances≫ The present invention's method for detecting a target substance is: Extracting the target substance by the method of the present invention, and To detect the extracted target substance. Includes.

[0055] The present invention may include performing an enzymatic reaction on the extracted target substance, and then detecting the purified and enzymatically reacted target substance. In this case, the enzymatic reaction may include nucleic acid amplification reactions or the like.

[0056] The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples. [Examples]

[0057] ≪Comparative Example 1≫ 600 μL of adsorbent solution was mixed with 200 μL of nucleic acid-containing solution. The resulting adsorbent solution was mixed with 4 mg of magnetic carrier and stirred at room temperature for 3 minutes to adsorb nucleic acid onto the magnetic carrier. Then, a magnet was brought close to the bottom of the container from outside to magnetize the magnetic carrier inside the container and remove the adsorbent solution.

[0058] As the first washing solution, 400 μL of an 80% ethanol aqueous solution was prepared. 400 μL of the first washing solution was added to the container, and the magnetic carrier was redispersed in the washing solution. A magnet was brought close to the bottom of the container from outside to magnetize the magnetic carrier and remove the first washing solution. This operation was repeated several times.

[0059] As a second washing solution, 600 μL of an 80% ethanol aqueous solution was prepared. 600 μL of the second washing solution was added to the container, and the magnetic carrier was redispersed in the second washing solution. A magnet was brought close to the bottom of the container from outside to magnetize the magnetic carrier, and the second washing solution was removed.

[0060] 100 μL of TE buffer (10 mmol / L Tris-HCl (pH 8.0), 1 mmol / L EDTA-2Na (pH 8.0)) was prepared as the eluate. 100 μL of the eluate was added to the container, and the magnetic carrier was redispersed within the eluate. A magnet was brought close to the bottom of the container from outside to magnetize the magnetic carrier, and the eluate was collected with a pipette. The amount of ethanol in the collected eluate was measured. The ethanol content in the eluate was as shown in Table 1 below.

[0061] ≪Comparative Example 2≫ Comparative Example 2 was carried out in the same manner as Comparative Example 1, except that mineral oil was used as the second cleaning solution. The ethanol content in the eluate is shown in Table 1 below.

[0062] Example 1 Example 1 was carried out in the same manner as Comparative Example 1, except that 1-undecanol was used as the second washing solution. Here, 1-undecanol used as the second washing solution was miscible with ethanol but not with the eluate. The ethanol content in the eluate is shown in Table 1 below.

[0063] ≪Rating≫ The ethanol content in the eluate for Comparative Examples 1 and 2, and Example 1, was as shown in Table 1 below. The ethanol content in the eluate of Example 1, which used 1-undecanol as the second washing solution, was lower than that of Comparative Examples 1 and 2, which used an 80% ethanol aqueous solution and mineral oil as the second washing solution. Therefore, by using 1-undecanol, a liquid organic compound, as the second washing solution, the ethanol content in the eluate could be effectively reduced.

[0064] [Table 1]

[0065] ≪Comparative Example 3≫ In Example 3, the ethanol content in the eluate was confirmed when ethanol was used as the first and second washing solutions, and an organic compound was used as the third washing solution.

[0066] <Comparative Example 3-1> Magnetic support 1 was prepared using the following method: Fe 92 A magnetic metal powder consisting of Si4Cr4 (composition formula is atomic %) was prepared by high-pressure water atomization. Subsequently, a magnetic support with a predetermined particle size was obtained using a wet classifier. Furthermore, a magnetic support 1 was obtained by depositing a silicon oxide (SiO2) film with a thickness of 50-100 nm using the Stöber method.

[0067] In addition to magnetic carrier 1, the following magnetic carriers 2-4 were prepared: Magnetic carrier 2: Thermo Fisher Scientific Inc. Dynabeads® MyOne® Silane Magnetic carrier 3: Precision System Science Co., Ltd. MagDEA(registered trademark) Dx SV Magnetic carrier 4: Ferrotec Ferrobeads FB-30SH

[0068] 600 μL of adsorbent solution was mixed with 200 μL of nucleic acid-containing solution. The resulting adsorbent mixture was mixed with magnetic carrier 1 (10 mg) and stirred at room temperature for 3 minutes to adsorb nucleic acid onto the magnetic carrier. Then, a magnet was brought close to the bottom of the container from outside to magnetize the magnetic carrier inside the container and remove the adsorbent solution.

[0069] As the first washing solution, 600 μL of an 80 vol% ethanol aqueous solution was prepared. 600 μL of the first washing solution was added to the container, and the magnetic carrier was redispersed in the washing solution. A magnet was brought close to the bottom of the container from outside to magnetize the magnetic carrier, and the first washing solution was removed.

[0070] As a second washing solution, 600 μL of a 99.5% vol. ethanol aqueous solution was prepared. 600 μL of the second washing solution was added to the container, and the magnetic carrier was redispersed in the second washing solution. A magnet was brought close to the bottom of the container from outside to magnetize the magnetic carrier, and the second washing solution was removed.

[0071] As a third cleaning solution, 1000 μL of mineral oil was prepared. 1000 μL of the third cleaning solution was added to the container, and the magnetic carrier was redispersed in the third cleaning solution. A magnet was brought close to the bottom of the container from outside to magnetize the magnetic carrier, and the third cleaning solution was removed.

[0072] 100 μL of TE buffer (10 mmol / L Tris-HCl (pH 8.0), 1 mmol / L EDTA-2Na (pH 8.0)) was prepared as the eluate. 100 μL of the eluate was added to the container, and the magnetic carrier was redispersed within the eluate. After magnetizing the magnetic carrier by bringing a magnet close to the bottom of the container from outside, only the eluate, which had separated into layers and contained mineral oil, was collected using a pipette. The amount of ethanol in the collected eluate was measured. The ethanol content in the eluate was as shown in Table 2 below.

[0073] Subsequently, the same procedure was performed when magnetic carrier 1 (10 mg) was replaced with magnetic carrier 2 (2 mg), magnetic carrier 3 (4 mg), or magnetic carrier 4 (4 mg), and the ethanol content in the eluate was confirmed. The amount of magnetic carrier added was set considering the true specific gravity of each magnetic carrier so that the volume of added particles was equivalent.

[0074] <Comparative Examples 3-2 and 3-3> Comparative Examples 3-2 and 3-3 were carried out in the same manner as Comparative Example 3-1, except that the compounds shown in Table 2 were used as the third washing solution. The ethanol content in the eluate is as shown in Table 2 below.

[0075] [Table 2]

[0076] Example 3 In Example 3, multiple linear alkanes were used as the third washing solution, and the ethanol content in the eluate was confirmed.

[0077] <Examples 3-1 to 3-8> Examples 3-1 to 3-8 were carried out in the same manner as Comparative Example 3-1, except that the compounds shown in Table 3 were used as the third washing solution. The ethanol content in the eluate is as shown in Table 3 below. In Examples 3-7 and 3-8, the third washing solution and the eluate were heated to 50°C.

[0078] <Rating> The conditions and results for Example 3 are shown in Table 3. By comparing with Comparative Examples 3-1 to 3-3 in Table 2, it was confirmed that using a linear alkane as the third washing solution effectively reduces the ethanol content in the eluate.

[0079] [Table 3]

[0080] Example 4 In Example 4, multiple aliphatic alcohols were used as the third washing solution, and the ethanol content in the eluate was confirmed.

[0081] <Examples 4-1 to 4-8> Examples 4-1 to 4-8 were carried out in the same manner as Comparative Example 3-1, except that the compounds shown in Table 4 were used as the third washing solution. The ethanol content in the eluate is as shown in Table 4 below. In Examples 4-7 and 4-8, the third washing solution and the eluate were heated to 50°C.

[0082] <Rating> The conditions and results for Example 4 are shown in Table 4. By comparing with Comparative Examples 3-1 to 3-3 in Table 2, it was confirmed that using an aliphatic alcohol as the third washing solution effectively reduces the ethanol content in the eluate.

[0083] [Table 4]

[0084] Example 5 In Example 5, multiple linear fatty acids were used as the third washing solution, and the ethanol content in the eluate was confirmed.

[0085] <Examples 5-1 to 5-6> Examples 5-1 to 5-6 were carried out in the same manner as Comparative Example 3-1, except that the compounds shown in Table 5 were used as the third washing solution. The ethanol content in the eluate is as shown in Table 5 below. In Examples 5-1 to 5-3 and Examples 5-5 and 5-6, the cases using magnetic carriers 2 to 4 were not performed, and in Examples 5-5 and 5-6, the third washing solution and eluate were heated to 50°C.

[0086] <Rating> The conditions and results for Example 5 are shown in Table 5. By comparing with Comparative Examples 3-1 to 3-3 in Table 2, it was confirmed that using linear fatty acids as the third washing solution effectively reduces the ethanol content in the eluate.

[0087] [Table 5]

[0088] Example 6 In Example 6, a cyclic fatty acid was used as the third washing solution, and the ethanol content in the eluate was confirmed.

[0089] <Example 6-1> Example 6-1 was carried out in the same manner as Comparative Example 3-1, except that the compound shown in Table 6 was used as the third washing solution. The ethanol content in the eluate is as shown in Table 6 below.

[0090] <Rating> The conditions and results for Example 6 are shown in Table 6. By comparing with Comparative Examples 3-1 to 3-3 in Table 2, it was confirmed that using a cyclic fatty acid as the third washing solution effectively reduces the ethanol content in the eluate.

[0091] [Table 6] [Explanation of Symbols]

[0092] 10 Magnetic carrier 20 Target substance 50 magnets 100% Ethanol-containing cleaning solution 200 Organic compounds 300 eluate

Claims

1. (a) Contacting the magnetic carrier holding the target substance with an ethanol-containing cleaning solution, (b) After step (a), the magnetic carrier is brought into contact with a liquid organic compound, and (c) After step (b), the magnetic carrier is brought into contact with an eluent that is incompatible with the organic compound, thereby eluting the target substance into the eluent. Includes, The aforementioned organic compound is compatible with ethanol and not compatible with water. A method for extracting target substances.

2. The method according to claim 1, wherein the organic compound comprises at least one compound selected from aliphatic alcohols, linear alkanes, linear fatty acids, and cyclic fatty acids.

3. The method according to claim 2, wherein the organic compound comprises at least one compound selected from (A) to (D) below: (A) Aliphatic alcohols having 6 to 13 carbon atoms; (B) Straight-chain alkanes with 6 to 19 carbon atoms; (C) Straight-chain fatty acids with 6 to 11 carbon atoms; (D) A cyclic fatty acid with 10 carbon atoms.

4. The method according to claim 1, wherein the eluent is an aqueous solution or pure water containing a component selected from the group consisting of metal salts, pH buffers, and surfactants.

5. The method according to claim 1, further comprising removing at least a portion of the ethanol-containing cleaning solution from the magnetic carrier after step (a) and before step (b).

6. The method according to claim 1, wherein the ethanol-containing cleaning solution is not air-dried after step (a) and before step (b).

7. The method according to claim 1, further comprising removing at least a portion of the organic compound from the magnetic carrier after step (b) and before step (c).

8. The method according to claim 1, wherein the magnetic carrier has an inorganic oxide surface.

9. The method according to claim 8, wherein the inorganic oxide surface is silicon oxide.

10. The method according to claim 8, wherein the magnetic carrier is composed of iron-based metal particles and the inorganic oxide surface.

11. Extracting the target substance by the method described in claim 1, and To detect the extracted target substance. A method for detecting a target substance, including the following.

12. The method according to claim 11, comprising performing an enzymatic reaction on the extracted target substance, and then detecting the purified and enzymatically reacted target substance.

13. The method according to claim 12, wherein the enzymatic reaction includes a nucleic acid amplification reaction.

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