Nucleic acid purification method

Abstract

Provided is a method for purifying nucleic acid, the method being able to conveniently purify nucleic acid and efficiently recover same.　The method for purifying nucleic acid comprises a step for preparing a nucleic acid-containing extraction solution by bringing a nucleic acid-containing sample into contact with a metal cation-containing extraction solution; a step for bringing the nucleic acid-containing extraction solution into contact with an anionic adsorbent 4 in order to adsorb the nucleic acid to the anionic adsorbent 4; a step for bringing the anionic adsorbent 4 into contact with a wash solution having a pH of not more than 5.0, in order to wash the anionic adsorbent 4 to which the nucleic acid is adsorbed; and a step for isolating the nucleic acid from the anionic adsorbent 4 by bringing the anionic adsorbent 4 into contact with a recovery solution having a pH of at least 6.0.

Description

[Technical Field]

[0001] The present invention relates to a method for purifying nucleic acids. [Background technology]

[0002] Conventionally, a known method for purifying nucleic acids such as RNA and DNA contained in samples such as viruses, bacteria, fungi, or cells involves adding a precipitation reagent such as alcohol to an extract containing nucleic acids derived from the sample to precipitate the nucleic acids, adsorbing the precipitated nucleic acids onto a solid phase carrier, washing away impurities such as sample-derived proteins that have adhered to the solid phase carrier together with the nucleic acids, and then recovering the nucleic acids with an eluate. In this case, alcohols such as ethanol are typically used not only for the precipitation reagent but also for the extraction reagent and washing solution.

[0003] For example, Patent Document 1 below discloses a method for extracting nucleic acids by dissolving cellular material by contacting the cellular material with an extraction solution, in which an alcohol such as ethanol or butanol is used as the extraction solution.

[0004] Furthermore, Patent Document 2 below discloses a method for washing a solid phase bound to a nucleic acid by contacting the solid phase with a washing solution containing at least one of the components constituting a reaction solution applicable to an enzymatic reaction using nucleic acid. In the examples of Patent Document 2, alcohol is used as the second washing solution. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2018-134093 [Patent Document 2] Patent No. 4340298 Summary of the Invention [Problem to be solved by the invention]

[0006] However, when the extraction solution contains alcohol as in Patent Document 1, the coexistence of alcohol during nucleic acid extraction from a sample may prevent sufficient nucleic acid extraction, which necessitates a step of mixing alcohol to precipitate the nucleic acid after nucleic acid extraction from the sample, resulting in a problem of complicated steps.

[0007] Furthermore, when the cleaning solution contains alcohol, as in Patent Document 2, care must be taken to prevent the alcohol from volatilizing or catching fire when storing the cleaning solution, which presents a problem of poor handling. Furthermore, in Patent Document 2, three cleaning steps are required to prevent alcohol from being carried over to subsequent processes, making the process complicated.

[0008] If alcohol is not used, the nucleic acid recovery rate will decrease and the nucleic acids may not be recovered efficiently. In addition, substances other than alcohol used in nucleic acid purification may inhibit reactions in downstream testing and analysis.

[0009] An object of the present invention is to provide a method for purifying nucleic acids, which allows nucleic acids to be easily purified and efficiently recovered. [Means for solving the problem]

[0010] The method for purifying nucleic acids according to the present invention comprises the steps of: preparing an extraction solution containing nucleic acids by contacting a sample containing nucleic acids with an extraction solution containing metal cations; contacting the extraction solution containing nucleic acids with an anionic adsorbent to adsorb the nucleic acids; washing the anionic adsorbent to which the nucleic acids have been adsorbed by contacting the anionic adsorbent with a washing solution having a pH of 5.0 or less; and isolating the nucleic acids from the anionic adsorbent by contacting the anionic adsorbent with a recovery solution having a pH of 6.0 or more.

[0011] In a specific aspect of the nucleic acid purification method according to the present invention, the recovery liquid is a solution that does not inhibit a nucleic acid amplification reaction. The recovery liquid is preferably a buffer solution. The concentration of the buffer solution is more preferably 5 mmol / L or more and 100 mmol / L or less. The recovery liquid may also be a solution containing a nucleic acid amplification reagent.

[0012] In another specific aspect of the method for purifying nucleic acid according to the present invention, the wash solution having a pH of 5.0 or less is at least one selected from the group consisting of water, hydrochloric acid-potassium chloride buffer, glycine-hydrochloric acid buffer, citric acid-sodium citrate buffer, and citric acid-phosphate buffer.

[0013] In yet another specific aspect of the method for purifying nucleic acid according to the present invention, the concentration of metal cations in the extraction solution is 0.5 mol / L or more.

[0014] In yet another specific aspect of the method for purifying nucleic acid according to the present invention, the metal cation is an alkali metal ion, an alkaline earth metal ion, a transition metal ion, or a Group 12 metal ion.

[0015] In yet another particular aspect of the method for purifying nucleic acid according to the present invention, the valence of the metal cation is divalent or greater. [Effects of the Invention]

[0016] According to the present invention, a method for purifying nucleic acid can be provided, which allows nucleic acid to be easily purified and efficiently recovered. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram illustrating a state in which nucleic acids are adsorbed to an anionic adsorbent in a method for purifying nucleic acids according to one embodiment of the present invention. [Figure 2] FIG. 2 is a schematic plan view showing a chip used in a method for purifying nucleic acid according to one embodiment of the present invention. [Figure 3]FIG. 3 is a schematic cross-sectional view of a portion taken along line AA in FIG. [Figure 4] FIG. 4 is a graph showing the relationship between the pH of the washing solution and the proportion of RNA in the washing solution and in the recovery solution. [Figure 5] FIG. 5 is a graph showing the relationship between the pH of the washing solution and the recovery rate of RNA. [Figure 6] FIG. 6 is a graph showing the relationship between the pH of the washing solution when nasal mucus was added and the recovery rate of RNA. [Figure 7] FIG. 7 is a diagram showing the relationship between the number of PCR cycles and fluorescence intensity when a potassium chloride-hydrochloric acid buffer solution of pH 2 was used as the washing solution in Example 3. [Figure 8] FIG. 8 shows the relationship between the number of PCR cycles and fluorescence intensity when water of pH 2 was used as the washing liquid in Example 6. [Figure 9] FIG. 9 is a graph showing the relationship between the type of metal cation and the recovery rate of RNA. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be clarified below by describing specific embodiments of the present invention with reference to the drawings.

[0019] In the nucleic acid purification method according to the present invention, first, a sample containing nucleic acids is contacted with an extraction solution containing metal cations to prepare an extraction solution containing nucleic acids (extraction step). Next, the extraction solution containing nucleic acids is contacted with an anionic adsorbent to adsorb the nucleic acids to the anionic adsorbent (nucleic acid adsorption step). Next, a washing solution of pH 5.0 or less is contacted with the anionic adsorbent to wash the anionic adsorbent to which the nucleic acids are adsorbed (washing step). Next, a recovery solution of pH 6.0 or more is contacted with the anionic adsorbent to isolate the nucleic acids from the anionic adsorbent (nucleic acid recovery step).

[0020] According to the method for purifying nucleic acid of the present invention, nucleic acid can be easily purified and efficiently recovered, and the reason for this can be explained as follows.

[0021] In the present invention, the extraction solution used in the extraction step contains metal cations. Therefore, when a sample containing nucleic acids comes into contact with the extraction solution, the metal cations ionically bond to the negatively charged nucleic acids. When the extraction solution after nucleic acid extraction is brought into contact with an anionic adsorbent, the metal cations then ionically bond to the anionic adsorbent, as shown in Figure 1. This allows the nucleic acids to be adsorbed to the anionic adsorbent via the metal cations.

[0022] In this way, in the present invention, nucleic acids can be easily adsorbed to an anionic adsorbent via metal cations. Because metal cations do not inhibit nucleic acid extraction even when added to a nucleic acid extraction solution, there is no need to add an additional solution after nucleic acid extraction, as is the case with conventional methods that use alcohol. Therefore, no complicated steps are required.

[0023] Furthermore, the present invention is easy to handle because nucleic acids can be adsorbed onto anionic adsorbents without using alcohol. Furthermore, when a large amount of alcohol is added to precipitate nucleic acids, the volume of the extraction solution after nucleic acid extraction increases and the concentration of nucleic acids decreases. However, nucleic acid purification using metal cations in the present invention does not increase the volume of the extraction solution (the nucleic acid concentration does not decrease), so the recovery rate of nucleic acids can be increased. This allows a sufficient amount of nucleic acid to be obtained in a single purification process, thereby improving the sensitivity of analysis and testing in subsequent processes.

[0024] However, if a solution containing a large amount of metal cations is used in the subsequent nucleic acid amplification step, nucleic acid amplification may be inhibited. Therefore, it is necessary to wash away excess metal cations bound to the anionic adsorbent together with the nucleic acid in a washing step. However, if a solution containing no alcohol is used as the washing solution, the nucleic acid may be detached from the anionic adsorbent together with the metal cations.

[0025] In the present invention, by using a washing solution having a pH of 5.0 or less, excess metal cations can be washed away without releasing the nucleic acids bound to the anionic adsorbent. Therefore, nucleic acids can be recovered efficiently, and in subsequent steps, nucleic acid amplification is less likely to be inhibited by metal cations, thereby improving the accuracy of analysis and testing. Furthermore, since washing can be performed reliably in a single step without using alcohol, handling is excellent and nucleic acids can be easily purified. Furthermore, in the present invention, a recovery solution having a pH of 6.0 or higher is used, which also allows nucleic acids to be recovered efficiently, and in subsequent steps, nucleic acid amplification is less likely to be inhibited by metal cations, thereby improving the accuracy of analysis and testing.

[0026] Each step will be described in more detail below.

[0027] (extraction process) In the extraction step, a sample containing nucleic acids is brought into contact with an extraction solution, thereby extracting the nucleic acids from the sample and obtaining an extraction solution containing nucleic acids.

[0028] Examples of samples containing nucleic acids include biological samples containing nucleic acids such as DNA and RNA. Examples of such biological samples include cells, blood, tissue fluid, urine, and feces. Furthermore, the nucleic acid-containing sample may be, but is not limited to, a sample containing nucleic acids in the environment, such as soil, seawater, or river water.

[0029] The extraction solution may be, for example, a solution containing a protein denaturant, a metal cation, and a polar solvent. The polar solvent is preferably contained in an amount of 50% or more in the extraction solution.

[0030] Protein denaturants interact with proteins to disrupt their higher-order structure. Examples of protein denaturants that can be used include surfactants, reducing agents, guanidine derivatives, thiourea, urea, and salts thereof. Examples of surfactants that can be used include sodium dodecyl sulfate (SDS), polyoxyethylene sorbitan monolaurate (Tween 20), and the like. Examples of reducing agents that can be used include 2-mercaptoethanol, dithiothreitol (DTT), and the like. Examples of the salts that can be used include salts such as guanidine hydrochloride. These protein denaturants may be used alone or in combination.

[0031] The concentration of the protein denaturant in the extraction solution is not particularly limited, but is preferably 2 mol / L or more, more preferably 4 mol / L or more, even more preferably 8 mol / L or more, and preferably 10 mol / L or less. By setting the concentration of the protein denaturant within the above range, nucleic acids can be extracted more reliably. Note that when two or more types of protein denaturants are used, it is preferable that the total concentration is within the above range.

[0032] The metal cation is not particularly limited, but for example, an alkali metal ion, an alkaline earth metal ion, a transition metal ion, or a Group 12 metal ion can be used.

[0033] Examples of the alkali metal ion include a potassium ion, a sodium ion, a lithium ion, a rubidium ion, a cesium ion, and a francium ion.

[0034] Examples of alkaline earth metal ions include calcium ions, magnesium ions, beryllium ions, strontium ions, barium ions, and radium ions.

[0035] Examples of the transition metal ions include manganese ions, iron ions, cobalt ions, nickel ions, and copper ions.

[0036] Examples of the Group 12 metal ions include zinc ions, cadmium ions, and mercury ions.

[0037] These metal cations may be used alone or in combination of two or more.

[0038] The valence of the metal cation is preferably divalent or higher. Examples of divalent or higher metal cations include calcium ions and magnesium ions. When divalent or higher metal cations are used, nucleic acids can be recovered more efficiently.

[0039] The concentration of metal cations in the extraction solution is preferably 0.5 mol / L or more, more preferably 1 mol / L or more, and even more preferably 2 mol / L or more. When the concentration of metal cations is equal to or greater than the lower limit, nucleic acids can be recovered more efficiently. The upper limit of the concentration of metal cations is not particularly limited, but can be, for example, 6 mol / L.

[0040] The polar solvent is not particularly limited, and examples thereof include water, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethylacetamide (DMA), methoxypropanol, polyethylene glycol, pentanediol, propanediol, aminoethanol, and diethanolamine. These polar solvents may be used alone or in combination. The extraction solution may not contain a polar solvent. The extraction solution may also contain a solvent other than a polar solvent. However, from the perspective of more reliably extracting nucleic acids, it is preferable that the extraction solution contains a polar solvent such as water.

[0041] The extraction solution may further contain a coprecipitant, such as tRNA, polyadenine, acrylamide polymers, glycogen, etc. When the extraction solution further contains a coprecipitant, nucleic acids can be more efficiently adsorbed to the anionic adsorbent.

[0042] (Nucleic acid adsorption process) In the nucleic acid adsorption step, an extraction solution containing nucleic acids is brought into contact with an anionic adsorbent, thereby allowing the nucleic acids to be adsorbed onto the anionic adsorbent.

[0043] The anionic adsorbent is a support for supporting nucleic acids. The form of the anionic adsorbent is not particularly limited, and it can be used in the form of, for example, a membrane, a filter, a plate, a tube, or a fiber. The anionic adsorbent is preferably in the form of fiber, particles, or a porous material.

[0044] The anionic adsorbent is not particularly limited, and may be composed of, for example, silicon compounds, phosphate minerals, silicate minerals, or aluminosilicate minerals. Examples of silicon compounds include silica and glass. Examples of phosphate minerals include hydroxyapatite. Examples of silicate minerals include talc and montmorillonite. Examples of aluminosilicate minerals include zeolite. These may be used alone or in combination.

[0045] The anionic adsorbent is preferably a silica fiber or a glass fiber, and more preferably a silica fiber. In this embodiment, silica fiber is used as the anionic adsorbent. However, the anionic adsorbent may be a silica particle or a porous silica material.

[0046] (Cleaning process) In the washing step, the anionic adsorbent to which nucleic acids have been adsorbed in the nucleic acid adsorption step is brought into contact with a washing solution of pH 5.0 or less, thereby washing the anionic adsorbent to which nucleic acids have been adsorbed.

[0047] Examples of washing solutions having a pH of 5.0 or less include water, hydrochloric acid-potassium chloride buffer solution, glycine-hydrochloric acid buffer solution, citric acid-sodium citrate buffer solution, citric acid-phosphate buffer solution, etc. These may be used alone or in combination.

[0048] The pH of the washing solution is 5.0 or less, preferably 4.0 or less. When the pH of the washing solution is equal to or less than the upper limit, unnecessary metal ions can be more reliably removed without detaching nucleic acids bound to the anionic adsorbent, and the amplification of nucleic acids in subsequent steps can be more unlikely to be inhibited. Furthermore, since the washing solution does not contain alcohol, which inhibits nucleic acid amplification reactions, there is no need to remove alcohol before the nucleic acid amplification reaction, and nucleic acids can be purified more easily.

[0049] The lower limit of the pH of the cleaning liquid can be set to, for example, 2.0.

[0050] (Nucleic acid recovery process) Next, the anionic adsorbent washed in the washing step is contacted with a recovery solution to isolate and recover the nucleic acid from the anionic adsorbent.

[0051] The recovery liquid is preferably a solution that does not inhibit the nucleic acid amplification reaction. In this case, the accuracy of analysis and testing in subsequent steps can be further improved. The pH of the recovery liquid is 6.0 or higher, preferably 7.0 or higher, and more preferably 8.0 or higher. In this case, nucleic acids can be recovered more efficiently. The upper limit of the pH of the recovery liquid is not particularly limited, but can be, for example, 9.0.

[0052] The recovery liquid may be a buffer solution, a nucleic acid amplification reagent, or the like.

[0053] Examples of buffer solutions that can be used include trishydroxymethylaminomethane (Tris), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), tricine, PIPES, ACES, MOPSO, BES, MOPS, HEPES, TAPSO, POPSO, HEPSO, EPPS, Bicine, TAPS, and phosphate buffer solutions.

[0054] Examples of reagents that can be used for nucleic acid amplification include TaqPath 1-Step Multiplex MasterMix, TaqMan Gene Expression Master Mix, TaqMan Fast Advanced MasterMix Mix, TaqPath qPCR Master Mix, CG, and TaqMan Fast Virus 1-Step Master Mix (manufactured by ThermoFisher).

[0055] These may be used alone or in combination of two or more.

[0056] The pH of the buffer solution is 6.0 or higher, preferably 7.0 or higher, and more preferably 8.0 or higher. In this case, nucleic acids can be recovered more efficiently. The upper limit of the pH of the buffer solution is not particularly limited, but can be, for example, 9.0.

[0057] The buffer concentration is preferably 5 mmol / L or more, more preferably 10 mmol / L or more, and preferably 100 mmol / L or less, more preferably 50 mmol / L or less, and even more preferably 30 mmol / L or less. When the buffer concentration is within the above range, nucleic acids can be recovered more efficiently.

[0058] More specific embodiments will be exemplified below.

[0059] (First embodiment) Fig. 2 is a schematic plan view showing a chip used in the nucleic acid purification method according to the first embodiment of the present invention, and Fig. 3 is a schematic cross-sectional view of a portion taken along line AA in Fig. 2.

[0060] 2 and 3, the chip 1 has a flow path 2 through which a fluid is transported. A collection unit 3 is provided midway along the flow path 2. Therefore, the flow path 2 has an upstream flow path 2a provided upstream of the collection unit 3 and a downstream flow path 2b provided downstream of the collection unit 3. In addition, the collection unit 3 is provided with an anionic adsorbent 4 for adsorbing and purifying nucleic acids.

[0061] The chip 1 is a chip used for testing and analysis. Although the chip 1 is not particularly limited, in this embodiment, as shown in FIG. 3, the chip 1 has a plate-shaped substrate 5 and a cover member 6. The substrate 5 has a main surface 5a. A recess 5b is provided on the main surface 5a side of the substrate 5. The recess 5b is provided so as to open to the main surface 5a side of the substrate 5.

[0062] The material constituting the substrate 5 is not particularly limited, and for example, synthetic resin, rubber, metal, etc. can be used. The synthetic resin is not particularly limited, but a thermoplastic resin is preferable. Among them, for example, a cycloolefin polymer, a cycloolefin copolymer, polycarbonate, polymethyl methacrylate, or polypropylene can be used as the thermoplastic resin. These may be used alone or in combination.

[0063] The substrate 5 is preferably made of a molded body of the above-mentioned thermoplastic resin. The molding method is not particularly limited, and any known molding method can be used. Examples of molding methods include injection molding, injection compression molding, gas-assisted injection molding, extrusion molding, multi-layer extrusion molding, rotational molding, hot press molding, blow molding, and foam molding. Among these, injection molding is preferred.

[0064] The substrate 5 may be formed by laminating a plurality of synthetic resin sheets, and may be composed of a base sheet and a substrate body having through holes provided on the base sheet.

[0065] A cover member 6 is provided on the main surface 5a of the substrate 5. The cover member 6 is provided so as to close the recessed portion 5b of the substrate 5. The cover member 6 closes the recessed portion 5b of the substrate 5, thereby forming a recovery section 3. In this embodiment, the upstream flow path 2a and the downstream flow path 2b are also similarly formed by the cover members 6 closing the recessed portions 5b of the substrate 5.

[0066] The cover member 6 can be made of a flexible material such as a resin film, etc. Examples of the resin film that can be used include thermoplastic resins such as cycloolefin polymer, cycloolefin copolymer, polycarbonate, polymethyl methacrylate, and polypropylene.

[0067] Furthermore, the cover member 6 may be made of an elastic material. The elastic material is not particularly limited, but is preferably an elastomer. In the present invention, the substrate 5 and the cover member 6 may be integrally formed.

[0068] The substrate 5 is provided with the aforementioned flow channel 2 through which a fluid is transported. Here, the flow channel 2 is a micro-flow channel. The flow channel 2 may not be a micro-flow channel, but may be a flow channel with a larger cross-sectional area than a micro-flow channel. However, a micro-flow channel is preferable. This allows various analyses to be performed using a small amount of sample.

[0069] Microchannels are tiny channels that produce micro effects when transporting fluids. In these channels, liquids are strongly affected by surface tension and behave differently from liquids flowing through ordinary large channels.

[0070] The cross-sectional shape and size of the microchannel are not particularly limited as long as the micro-effect is generated. For example, when a pump or gravity is used to flow a fluid through the microchannel, in order to reduce flow resistance, if the cross-sectional shape of the microchannel is roughly rectangular (including square), the dimension of the smaller side is preferably 20 μm or more, more preferably 50 μm or more, and even more preferably 100 μm or more. In order to further reduce the size of a microfluidic device using chip 1, the dimension of the smaller side is preferably 5 mm or less, more preferably 1 mm or less, and even more preferably 500 μm or less.

[0071] Furthermore, when the cross-sectional shape of the microchannel is approximately circular, the diameter (minor axis in the case of an ellipse) is preferably 20 μm or more, more preferably 50 μm or more, and even more preferably 100 μm or more. From the viewpoint of further miniaturization of the microfluidic device, the diameter (minor axis in the case of an ellipse) is preferably 5 mm or less, more preferably 1 mm or less, and even more preferably 500 μm or less.

[0072] In this embodiment, such a chip 1 is used to carry out the nucleic acid purification method according to the present invention.

[0073] Specifically, the extraction solution after nucleic acid extraction in the nucleic acid extraction step is injected into the upstream flow path 2a from the injection port 7a in the chip 1 and sent to the recovery unit 3. This causes the nucleic acid to be adsorbed onto the anionic adsorbent 4 in the recovery unit 3.

[0074] Next, a washing liquid with a pH of 5.0 or less is sent to the recovery unit 3. This washes the anionic adsorbent 4 to which the nucleic acids have been adsorbed. Next, the recovery liquid is sent to the recovery unit 3, and while isolating the nucleic acids from the anionic adsorbent 4, it is sent to the downstream flow path 2b side, and the nucleic acids are recovered from the recovery port 7b.

[0075] In the nucleic acid purification method of this embodiment, nucleic acids can be easily adsorbed onto the anionic adsorbent 4 via metal cations. Because metal cations do not inhibit nucleic acid extraction even when added to the extraction solution before nucleic acid extraction, there is no need to add alcohol after extraction, as is the case with conventional methods that use alcohol. Therefore, no complicated steps are required.

[0076] Furthermore, the present invention is easy to handle because nucleic acids can be adsorbed to the anionic adsorbent 4 without using alcohol. Furthermore, when a large amount of alcohol is added to precipitate nucleic acids, the volume of the extraction solution after nucleic acid extraction increases and the concentration of nucleic acids decreases. However, nucleic acid purification using metal cations in the present invention does not involve an increase in the volume of the extraction solution (no decrease in nucleic acid concentration), and therefore the recovery rate of nucleic acids can be increased. This allows a sufficient amount of nucleic acid to be obtained in a single purification process, thereby improving the sensitivity of analyses and tests in subsequent processes.

[0077] However, if a solution containing a large amount of metal cations is used in the subsequent nucleic acid amplification step, nucleic acid amplification may be inhibited. Therefore, in the washing step, excess metal cations bound to the anionic adsorbent along with the nucleic acid must be washed away. However, if a solution containing no alcohol is used as the washing solution, the nucleic acid may be detached from the anionic adsorbent together with the metal cations.

[0078] In the present invention, by using a washing solution having a pH of 5.0 or less, excess metal cations can be washed away without releasing the nucleic acids bound to the anionic adsorbent. Therefore, in subsequent steps, nucleic acid amplification is less likely to be inhibited by metal cations, thereby improving the accuracy of analysis and testing. Furthermore, since washing can be performed reliably in one step without using alcohol, the solution is easy to handle and allows nucleic acids to be purified easily.

[0079] (Second embodiment) In the second embodiment, the above-described nucleic acid purification method according to the present invention is carried out using a container.

[0080] Specifically, an anionic adsorbent for adsorbing and recovering nucleic acids is placed in a plastic container such as polypropylene. The extraction solution obtained after nucleic acid extraction in the nucleic acid extraction step is poured into this container and stirred with the anionic adsorbent, allowing the nucleic acids to be adsorbed by the anionic adsorbent. The stirring method is not particularly limited, but can be performed using, for example, a vortex mixer, ultrasonic waves, or the like.

[0081] Next, the solution in the container is removed, and a washing solution is poured in and stirred. This washes the anionic adsorbent to which the nucleic acids have been adsorbed. Next, the washing solution is removed, and a recovery solution is poured in and stirred. This allows the nucleic acids to be isolated from the anionic adsorbent. Finally, the recovery solution is removed, and the isolated nucleic acids are recovered.

[0082] In the second embodiment, the extraction solution also contains metal cations. Therefore, when a sample containing nucleic acids is added to the extraction solution, the metal cations ionically bond to the negatively charged nucleic acids. When the mixture obtained after the nucleic acids to which the metal cations ionically bond are extracted is brought into contact with an anionic adsorbent, the metal cations ionically bond to the anionic adsorbent. This allows the nucleic acids to be adsorbed to the anionic adsorbent via the metal cations.

[0083] In this way, the purification method of this embodiment allows nucleic acids to be easily adsorbed to an anionic adsorbent via metal cations. Because metal cations do not inhibit nucleic acid extraction even when added to the extraction solution before nucleic acid extraction, there is no need to add alcohol after extraction, as is the case with conventional methods that use alcohol. Therefore, no complicated steps are required.

[0084] Furthermore, the present invention allows nucleic acids to be adsorbed onto anionic adsorbents without using alcohol, making it easy to handle. Furthermore, adding a large amount of alcohol to precipitate nucleic acids increases the volume of the extraction solution after nucleic acid extraction, resulting in a decrease in nucleic acid concentration. However, nucleic acid purification using metal cations in the present invention does not increase the volume of the extraction solution (no decrease in nucleic acid concentration), thereby increasing the nucleic acid recovery rate. This allows a sufficient amount of nucleic acid to be obtained in a single purification process, thereby improving the sensitivity of analyses and tests in subsequent processes.

[0085] However, if a solution containing a large amount of metal cations is used in the subsequent nucleic acid amplification step, nucleic acid amplification may be inhibited. Therefore, in the washing step, excess metal cations bound to the anionic adsorbent along with the nucleic acid must be washed away. However, if a solution containing no alcohol is used as the washing solution, the nucleic acid may be detached from the anionic adsorbent together with the metal cations.

[0086] In the present invention, by using a washing solution having a pH of 5.0 or less, excess metal cations can be washed away without releasing the nucleic acids bound to the anionic adsorbent. Therefore, in subsequent steps, nucleic acid amplification is less likely to be inhibited by metal cations, thereby improving the accuracy of analysis and testing. Furthermore, since washing can be performed reliably in one step without using alcohol, the solution is easy to handle and allows nucleic acids to be purified easily.

[0087] The present invention will be clarified below by showing specific examples and comparative examples of the present invention, but the present invention is not limited to the following examples.

[0088] (Examples 1 to 2 and Comparative Examples 1 to 4) In Examples 1 and 2 and Comparative Examples 1 to 4, the chip 1 shown in FIGS. 2 and 3 was fabricated as follows.

[0089] The substrate 5 was made of a cycloolefin polymer, which was injection molded to produce a substrate 5 having a recess 5b. A sealing tape was used as the cover member 6, and the recess 5b of the substrate 5 was closed with the sealing tape to produce a chip 1. Silica fiber (2 mm diameter, 0.8 mm thick) was placed in the collection section 3 as the anionic adsorbent 4. The width and depth of the flow channel 2 were 0.8 mm and 0.8 mm, respectively.

[0090] Using such a chip 1, the recovery rate was measured as follows.

[0091] First, 1 μL of a sample (virus; 30,000 copies) containing nucleic acid (RNA) was added to 150 μL of extraction solution (aqueous solution containing 4 mol / L urea, 4 mol / L guanidine hydrochloride, 2 mol / L calcium chloride, and Tris-HCl buffer (pH 7.0)) to extract the nucleic acid.

[0092] Next, the extracted solution after nucleic acid extraction was sent from the upstream flow path 2 a to the recovery unit 3 , whereby the nucleic acid was adsorbed onto the silica fiber of the recovery unit 3 .

[0093] Next, 400 μL of cleaning solution was sent to the collection unit 3 to wash the silica fiber carrying the nucleic acid, and the cleaning solution was collected after the solution was sent. 21 μL of recovery solution (10 mmol / L, Tris-HCl Buffer (pH 8.0)) was sent to the collection unit 3, and the nucleic acid (RNA) carried in the collection unit 3 was isolated and collected.

[0094] In Example 1, water with a pH of 4.0 was used as the cleaning solution. In Example 2, water with a pH of 5.0 was used. In Comparative Example 1, a buffer solution (Tris) with a concentration of 10 mmol / L and a pH of 6.0 was used. In Comparative Example 2, a buffer solution (Tris) with a concentration of 10 mmol / L and a pH of 7.0 was used. In Comparative Example 3, a buffer solution (Tris) with a concentration of 10 mmol / L and a pH of 8.0 was used. Furthermore, in Comparative Example 4, a buffer solution (Tris) with a concentration of 10 mmol / L and a pH of 9.0 was used.

[0095] Next, the proportion of RNA in the recovered wash solution and the recovered solution was evaluated. First, RNA in the wash solution after delivery was purified using a QIAamp 96 Virus QIAcube HT Kit (QIAGEN) and a QIAcube HT system (QIAGEN) (purified wash solution). Next, RT-PCR reaction solutions were prepared using 2 μL of the RNA solution purified from the wash solution or the recovered solution, primers, and TaqPath 1-Step Multiplex MasterMix (Thermofisher). Additionally, RT-PCR reaction solutions (nucleic acid concentrations of 50,000, 5,000, and 500 copies / μL) were prepared as standards, containing 2 μL of the same virus solution extracted and purified using the QIAamp Viral RNA Mini Kit (QIAGEN). Next, the RT-PCR reaction solutions prepared from the recovered solution and the standard RT-PCR reaction solutions were amplified using a thermal cycler "CFX96 (BIO RAD)." Amplification consisted of reverse transcription at 50°C for 30 seconds, followed by initial denaturation at 95°C for 20 seconds, followed by 45 PCR cycles at 95°C for 3 seconds and 60°C for 5 seconds. After amplification, the RNA recovery rate (nucleic acid recovery rate) was calculated using the following formula from the amount of nucleic acid automatically calculated by the CFX96 based on the standard. To ensure analytical accuracy in the subsequent nucleic acid amplification step, a nucleic acid recovery rate of 5% or higher is desirable. The results are shown in Figure 4.

[0096] Nucleic acid recovery rate (%) = {(amount of nucleic acid calculated by CFX96 × volume of recovered solution / 2) × 100} / 3,0000

[0097] 4, it can be seen that in Examples 1 and 2, where the pH is 5.0 or less, the outflow of RNA into the washing solution can be suppressed and nucleic acids can be recovered at a good recovery rate, compared to Comparative Examples 1 to 4, where the pH is higher than 5.0. Note that in Comparative Examples 1 to 4, taking into consideration the influence of metal cations, the pH was adjusted using a buffer solution rather than water, without adding metal cations such as sodium ions.

[0098] Examples 3 to 6 In Example 3, nucleic acids were recovered in the same manner as in Example 1, except that a hydrochloric acid-potassium chloride buffer solution with a concentration of 10 mmol / L and a pH of 2.0 was used as the washing solution. In Example 4, nucleic acids were recovered in the same manner as in Example 1, except that a glycine-hydrochloric acid buffer solution with a concentration of 10 mmol / L and a pH of 2.0 and a pH of 3.0 was used as the washing solution. In Example 5, nucleic acids were recovered in the same manner as in Example 1, except that a citric acid-sodium citrate buffer solution with a concentration of 10 mmol / L and a pH of 3.0 and a pH of 4.0 was used as the washing solution. In Example 6, nucleic acids were recovered in the same manner as in Example 1, except that water (water-HCl) with pHs of 2.0, 3.0, and 4.0 was used as the washing solution. Subsequently, the recovery rate of RNA recovered in the recovery solution was calculated in the same manner as in Example 1. The results are shown in FIG. 5.

[0099] As is clear from FIG. 5, in Examples 3 to 6, RNA can be recovered with a good recovery rate.

[0100] In addition, the relationship between the number of PCR cycles and the fluorescence intensity was determined for each sample.

[0101] Fig. 7 shows the relationship between the PCR cycle number and fluorescence intensity when a hydrochloric acid-potassium chloride buffer solution of pH 2.0 was used as the washing solution in Example 3. Fig. 8 shows the relationship between the PCR cycle number and fluorescence intensity when water of pH 2.0 was used as the washing solution in Example 6.

[0102] As is clear from a comparison of Figures 7 and 8, when a potassium chloride-hydrochloric acid buffer solution at pH 2.0 was used as the washing solution, the fluorescence intensity and Ct value (the cycle number at which the amplification curve began to rise) were almost equal compared to when water at pH 2.0 was used as the washing solution. This indicates that the nucleic acid amplification reaction was not inhibited even when a hydrochloric acid-potassium chloride buffer solution at pH 2.0 was used as the washing solution. The Ct value for each example was determined in the same manner. The results are shown in Table 1 below. Note that the recovery rate and Ct value in Table 1 below are the average values of three measurements (N=3). Regarding water, the results for water at pH 5.0 as the washing solution are also shown. For comparison, the results for a pH 6.0 buffer solution (Tris-HCl) are also shown as Comparative Example 5.

[0103] [Table 1]

[0104] As shown in Table 1, in Examples 3 to 6, the nucleic acid recovery rate was 5% or more, indicating that nucleic acids could be recovered satisfactorily. Furthermore, in Examples 3 to 6, the Ct values were less than 38.0, indicating that no significant increase in the Ct value was observed, and that the nucleic acid amplification reaction was unlikely to be inhibited. On the other hand, in Comparative Example 5, the nucleic acid recovery rate was less than 5%, indicating that the nucleic acid recovery rate was insufficient. Furthermore, in Comparative Example 5, the Ct value became 38.0 or more as the recovery rate decreased, indicating that the nucleic acid amplification reaction was delayed.

[0105] In addition, 1 μL of a sample containing nucleic acid (RNA) (virus; 30,000 copies) was added to 150 μL of an extraction solution (aqueous solution containing urea 4 mol / L, guanidine hydrochloride 4 mol / L, calcium chloride 2 mol / L, and Tris-HCl buffer (pH 7.0)) in which nasal mucus was suspended, and the RNA recovery rate was determined in the same manner. The results are shown in Figure 6. In Examples 3 to 6, it can be seen that good RNA recovery rates were achieved even when samples containing nasal mucus were used.

[0106] (Examples 7 to 22 and Comparative Example 6) In Examples 7 to 22 and Comparative Example 6, a hydrochloric acid-potassium chloride buffer solution with a concentration of 10 mmol / L and pH 2.0 was used as the washing solution. Furthermore, in Examples 7 to 13, trishydroxymethylaminomethane (Tris) was used as the recovery solution at the pH and concentration shown in Table 2 below. In Examples 14 to 17, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) was used as the recovery solution at the pH and concentration shown in Table 2 below. In Examples 18 to 21, Tricine was used as the recovery solution at the pH and concentration shown in Table 2 below. In Example 22, 4-fold diluted TaqPath 1-Step Multiplex MasterMix (ThermoFisher, 1x Master Mix, pH 8.0) was used as the recovery solution. In Comparative Example 6, trishydroxymethylaminomethane (Tris) with a pH of 5.0 and a concentration of 10 mmol / L (10 mM) was used as the recovery solution. Other than that, the RNA recovery rate and Ct value were determined in the same manner as in Example 3. The results are shown in Table 2 below. The recovery rate and Ct value in Table 2 below are the average values of three measurements (N=3).

[0107] [Table 2]

[0108] As shown in Table 2, it can be seen that in Examples 7 to 22, nucleic acids could be recovered with a good nucleic acid recovery rate. Furthermore, in Examples 7 to 22, the Ct values were less than 38.0, and no significant increase in the Ct values was observed, indicating that the nucleic acid amplification reaction was unlikely to be inhibited. On the other hand, in Comparative Example 6, the nucleic acid recovery rate was less than 5%, indicating that the nucleic acid recovery rate was insufficient. Furthermore, in Comparative Example 6, nucleic acids could not be recovered, and therefore no nucleic acid amplification reaction was observed.

[0109] (Examples 23 to 29) In Examples 23 to 29, a hydrochloric acid-potassium chloride buffer solution with a concentration of 10 mmol / L and a pH of 2.0 was used as the washing solution. In Examples 23 to 29, trishydroxymethylaminomethane (Tris) with a pH of 8.0 and a concentration of 10 mmol / L (10 mM) was used as the recovery solution. In Examples 23 to 29, the metal cations shown in Table 3 below were used. The results are shown in Table 3 below. The recovery rates and Ct values in Table 3 below are the average values of three measurements with N=3.

[0110] [Table 3]

[0111] As shown in Table 3, it can be seen that nucleic acids can be recovered with a better recovery rate when divalent CaCl2, MgCl2, or SrCl2 is used as the metal cation.

[0112] (Reference example) In the reference example, metal cations (metal cations for RNA adsorption) are NaCl (sodium ion), CaCl2 (calcium ion), and MgCl2 (magnesium ion). 、 The RNA recovery rates were compared when KCl (potassium ion), LiCl (lithium ion), and RbCl (rubidium ion) were used.

[0113] The recovery rate was measured using the same chip 1 as in Example 1 as follows.

[0114] First, 1 μL of a sample (virus; 5000 copies) containing nucleic acid (RNA) was added to 60 μL of nucleic acid extraction solution (aqueous solution containing 2 mol / L metal cations and 50 mmol / L Tris-HCl Buffer (pH 8.0)) to extract the nucleic acid.

[0115] Next, the mixed solution after nucleic acid extraction was sent from the upstream flow path 2 a to the recovery unit 3 , whereby the nucleic acid was adsorbed onto the silica fiber of the recovery unit 3 .

[0116] Next, 200 μL of cleaning solution was sent to the collection unit 3 to wash the silica fiber loaded with nucleic acid. The collection unit 3 was then heated at 80°C for 3 minutes using a heater to dry the cleaning solution. The cleaning was performed twice: once with 1.8 mol / L guanidine thiocyanate, 40% ethanol, and 30 mmol / L Tris-HCl (pH 7.0), and once with a cleaning solution containing 90% ethanol. After removing the cleaning solution, 28 μL of recovery solution (1× Master Mix) was sent to the collection unit 3, and the nucleic acid (RNA) loaded in the collection unit 3 was isolated and collected.

[0117] The results are shown in Figure 9. As is clear from Figure 9, the recovery rate was increased when divalent CaCl2 and MgCl2 were used as the metal cation. [Explanation of symbols]

[0118] 1. Tip 2...Flow path 2a...Upstream flow path 2b…Downstream flow path 3...Recovery section 4...Anionic adsorbent 5...Substrate 5a…main surface 5b...recess 6...Cover member 7a…Inlet 7b...Collection port

Claims

1. preparing an extraction solution containing nucleic acids by contacting a sample containing nucleic acids with an extraction solution containing metal cations; a step of contacting an extraction solution containing the nucleic acids with an anionic adsorbent to adsorb the nucleic acids to the anionic adsorbent; a step of washing the anionic adsorbent to which the nucleic acids have been adsorbed by bringing a washing solution of pH 5.0 or less into contact with the anionic adsorbent; a step of contacting a recovery solution having a pH of 6.0 or higher with the anionic adsorbent to isolate the nucleic acid from the anionic adsorbent; Equipped with the anionic adsorbent is a silica fiber, a glass fiber, a silica particle, or a porous silica material; A method for purifying nucleic acid, wherein in the step of adsorbing the nucleic acid to the anionic adsorbent, the nucleic acid is adsorbed to the anionic adsorbent via only the metal cation.

2. The method for purifying nucleic acid according to claim 1 , wherein the recovery solution is a solution that does not inhibit a nucleic acid amplification reaction.

3. The method for purifying nucleic acid according to claim 1 or 2, wherein the recovery liquid is a buffer solution.

4. 4. The method for purifying nucleic acid according to claim 3, wherein the concentration of the buffer solution is 5 mmol / L or more and 100 mmol / L or less.

5. 3. The method for purifying nucleic acid according to claim 1, wherein the recovery solution is a solution containing a reagent for nucleic acid amplification.

6. The method for purifying nucleic acid according to any one of claims 1 to 5, wherein the washing solution having a pH of 5.0 or less is at least one selected from the group consisting of water, hydrochloric acid-potassium chloride buffer, glycine-hydrochloric acid buffer, citric acid-sodium citrate buffer, and citric acid-phosphate buffer.

7. 7. The method for purifying nucleic acid according to claim 1, wherein the concentration of the metal cation in the extraction solution is 0.5 mol / L or more.

8. The method for purifying nucleic acid according to any one of claims 1 to 7, wherein the metal cation is an alkali metal ion, an alkaline earth metal ion, a transition metal ion, or a Group 12 metal ion.

9. The method for purifying nucleic acid according to any one of claims 1 to 8, wherein the valence of the metal cation is divalent or greater.

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