JP2020162485A - Method of recovering nucleic acid, nucleic acid amplification method, and microfluidic device - Google Patents

Abstract

To provide a method of recovering nucleic acid that can improve a recovery rate of nucleic acid effectively.SOLUTION: A method of recovering nucleic acid includes the steps of causing nucleic acid to be adsorbed to an adsorbent 4, and of using a recovery liquid containing an aprotic polar compound to recover the nucleic acid adsorbed to the adsorbent 4.SELECTED DRAWING: Figure 1

Description

The present invention relates to nucleic acid recovery methods, nucleic acid amplification methods, and microfluidic devices.

Conventionally, as a method for recovering nucleic acids such as RNA and DNA contained in a sample such as a virus, a bacterium, a fungus, or a cell, the nucleic acid is precipitated by extracting the nucleic acid from the sample and then adding a precipitation reagent such as alcohol. The method of making and filtering is widely used.

Patent Document 1 below discloses a method for recovering nucleic acid from a biological sample. In Patent Document 1, a water-soluble neutral polymer adsorbs nucleic acid on a carrier adsorbed on the surface of cerium oxide, and an eluate is added to the carrier on which the nucleic acid is adsorbed to recover the nucleic acid. Further, Patent Document 1 describes that a buffer solution is preferable as an eluate for recovering nucleic acid. Further, Non-Patent Document 1 describes that chitosan is used as a carrier for adsorbing nucleic acids.

International Publication No. 2017/159763

Weidong Cao et al. Analytical Chemistry, Vol. 78, No. 20, 7222-7228, October 15, 2006

By the way, when recovering the nucleic acid adsorbed on the adsorbent, a buffer solution as in Patent Document 1 or water is often used.

When the present inventors use a buffer solution or water as the recovery liquid when recovering nucleic acid, it is difficult to recover the nucleic acid firmly adsorbed on the adsorbent, and the nucleic acid recovery rate cannot be sufficiently increased. I found that there is. Further, when a polyamine compound is used as an adsorbent as in Non-Patent Document 1, it is necessary to make the recovery solution basic in order to put the amine in a non-ionized state and reduce the binding force with the nucleic acid. In this case, it has been found that nucleic acid amplification reactions such as the polymerase chain reaction (PCR) may be inhibited in the examination and analysis in the subsequent step.

An object of the present invention is to provide a nucleic acid recovery method, a nucleic acid amplification method, and a microfluidic device capable of efficiently increasing the nucleic acid recovery rate.

The method for recovering nucleic acid according to the present invention includes a step of adsorbing the nucleic acid on the adsorbent and a step of recovering the nucleic acid adsorbed on the adsorbent using a recovery solution containing an aprotic polar compound. ..

In certain aspects of the nucleic acid recovery method according to the invention, the recovery solution containing the aprotic polar compound further comprises water.

In another specific aspect of the nucleic acid recovery method according to the present invention, the concentration of the aprotic polar compound in the recovery liquid is 0.01 mol / L or more.

In yet another specific aspect of the nucleic acid recovery method according to the present invention, the concentration of the aprotic polar compound in the recovery solution is 10 mol / L or less.

In yet another specific aspect of the method for recovering nucleic acids according to the present invention, the aprotic polar compound is a pyrrolidone, formamide, sulfoxide, acetamide, acetonitrile and derivatives thereof, ketones such as acetone, cyclohexanone, methyl ethyl ketone, etc. It is at least one selected from the group consisting of ethers such as THF and dioxane, glycols such as ethylene glycol and propylene glycol, and esters such as ethyl acetate, butyl acetate, and triphosphate ester.

In yet another particular aspect of the method for recovering nucleic acids according to the present invention, the aprotic polar compound is an aprotic polar solvent.

In yet another specific aspect of the method for recovering nucleic acids according to the present invention, the adsorbent is composed of at least one selected from the group consisting of silica, glass, hydroxyapatite, talc, montmorillonite, and zeolite. ..

In yet another particular aspect of the method for recovering nucleic acids according to the present invention, the adsorbent is composed of at least one of chitosan and histidine polymers.

The nucleic acid amplification method according to the present invention includes a recovery step of recovering nucleic acid by a nucleic acid recovery method configured according to the present invention, and a nucleic acid amplification step of performing an amplification reaction of the nucleic acid contained in the recovery liquid recovered in the recovery step. , Equipped with.

In a specific aspect of the nucleic acid amplification method according to the present invention, a dilution step of diluting the recovery liquid recovered in the recovery step is further provided before the nucleic acid amplification step.

In another specific aspect of the nucleic acid amplification method according to the present invention, the concentration of the aprotic polar compound in carrying out the amplification reaction of the nucleic acid is set to 2 mol / L or less by the dilution step.

In a wide range of aspects of the microfluidic device according to the present invention, the microfluidic device used in the nucleic acid recovery method or nucleic acid amplification method configured according to the present invention is a flow path for feeding a fluid and the flow path in the flow path. It comprises a chip that is arranged and has an adsorbent for adsorbing and isolating nucleic acids.

In another broad aspect of the microfluidic device according to the present invention, a chip comprising a flow path for delivering a fluid and an adsorbent located in the flow path for adsorbing and isolating nucleic acids. A recovery liquid containing an aprotic polar compound is arranged in the flow path.

In certain aspects of the microfluidic device according to the invention, the chip further has a reaction field that amplifies the recovered nucleic acid.

In another particular aspect of the microfluidic device according to the invention, the chip further comprises a dilution channel for diluting the recovered solution.

According to the present invention, it is possible to provide a nucleic acid recovery method, a nucleic acid amplification method, and a microfluidic device that can efficiently increase the nucleic acid recovery rate.

FIG. 1 is a schematic plan view showing a chip used in the nucleic acid recovery method according to the embodiment of the present invention. FIG. 2 is a schematic cross-sectional view of a portion along the line AA in FIG.

The details of the present invention will be described below.

In the method for recovering nucleic acid according to the present invention, first, the nucleic acid is adsorbed on an adsorbent. Next, the nucleic acid adsorbed on the adsorbent is recovered using a recovery solution containing an aprotic polar compound.

Since the nucleic acid recovery method according to the present invention has the above-mentioned structure, the nucleic acid recovery rate can be effectively increased.

Conventionally, when a buffer solution or water is used as the recovery liquid when recovering nucleic acid, it is difficult to recover the nucleic acid firmly adsorbed on the adsorbent, and there is a problem that the recovery rate of nucleic acid cannot be sufficiently increased. In addition, when a polyamine compound is used as the adsorbent, it is necessary to make the recovery solution basic in order to put the amine in a non-ionized state and reduce the binding force with the nucleic acid. In this case, inspection in a subsequent step or In the analysis, there is a problem that the reaction such as PCR is inhibited.

The present inventors have focused on a recovery solution containing an aprotic polar compound, and have found that the recovery rate of nucleic acid can be effectively increased by using a solution containing an aprotic polar compound as the recovery solution. It was. Specifically, this point can be explained as follows.

Nucleic acid has a phosphate base in the main chain, and while this part is compatible with polar solvents, when protons are supplied, it becomes non-ionizing and difficult to dissolve, so the recovery rate of nucleic acid is sufficient. It may not be possible to increase it. On the other hand, in the present invention, since a recovery solution containing an aprotic polar compound is used, the solubility of nucleic acid can be increased, and as a result, the recovery rate of nucleic acid can be effectively increased. ..

Further, by using a recovery solution containing an aprotic polar compound, nucleic acids can be efficiently recovered even when a polyamine-based compound is used as an adsorbent without making the recovery solution basic. Therefore, it is difficult for a reaction such as PCR to be inhibited in a test or analysis in a subsequent process.

Further, by using a recovery solution containing an aprotic polar compound, it is possible to improve the specificity at the time of PCR and lower the heat denaturation temperature.

In the present invention, it is desirable to bring a sample containing nucleic acid into contact with a nucleic acid extraction solution to extract the nucleic acid prior to adsorbing the nucleic acid on the adsorbent. Further, a liquid sample in which an alcohol such as ethanol is further added to precipitate nucleic acid may be used.

Examples of the sample containing nucleic acid include viruses, bacteria, fungi, cells and the like containing nucleic acids such as DNA and RNA.

As the nucleic acid extraction solution, for example, a solution containing a protein denaturing agent, a polar solvent, and a nucleic acid flocculant can be used.

A protein denaturant has a role of disrupting the higher-order structure of a protein by interacting with the protein. As the protein denaturing agent, for example, a surfactant, a reducing agent, a guanidine derivative, thiourea, urea, or a salt thereof or the like can be used. As the surfactant, for example, sodium dodecyl sulfate (SDS), polyoxyethylene sorbitan monolaurate (Tween 20) and the like can be used. As the reducing agent, for example, 2-mercaptoethanol, dithiothreitol (DTT) and the like can be used. Further, as the salt, for example, a salt such as guanidine hydrochloride can be used. These protein denaturants may be used alone or in combination of two or more.

The concentration of the protein denaturant in the nucleic acid extract solution is not particularly limited, but is preferably 2 mol / L or more, more preferably 4 mol / L or more, still 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 acid can be extracted more reliably. When two or more kinds of protein denaturing agents are contained, the total concentration is preferably within the above range.

The polar solvent is not particularly limited, but for example, water, dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), dimethylacetamide (DMA), methoxypropanol, polyethylene glycol, pentanediol, propanediol, aminoethanol. , Diethanolamine and the like can be used. These polar solvents may be used alone or in combination of two or more. The nucleic acid extraction solution may not contain a polar solvent. Further, the nucleic acid extraction solution may contain a solvent other than the polar solvent. However, from the viewpoint of more reliably extracting nucleic acid, it is preferable that the nucleic acid extraction solution contains a polar solvent such as water. The polar solvent is preferably contained in the nucleic acid extraction solution in an amount of 50% or more.

Examples of the nucleic acid flocculant include carrier DNA, carrier RNA, polyadenylation and the like. These may be used alone or in combination of two or more. By adding a nucleic acid flocculant, a plurality of nucleic acids can be aggregated, and the nucleic acids can be more efficiently adsorbed by an adsorbent described later.

The concentration of the nucleic acid flocculant in the nucleic acid extraction solution is not particularly limited, but is preferably 0.1 mg / L or more, more preferably 1 mg / L or more, still more preferably 10 mg / L or more, preferably 1 g / L or less, and more. It is preferably 0.1 g / L or less. By setting the concentration of the nucleic acid flocculant within the above range, the nucleic acid can be more efficiently adsorbed by the adsorbent described later.

As a method for recovering the nucleic acid extracted in this manner, for example, the first method (nucleic acid recovery step-1) and the second method (nucleic acid recovery step-2) shown below can be used. ..

(Nucleic acid recovery step-1)
In the first method, a step of recovering the nucleic acid extracted as described above will be described with reference to FIGS. 1 and 2.

FIG. 1 is a schematic plan view showing a chip used in the nucleic acid recovery method according to the embodiment of the present invention. Further, FIG. 2 is a schematic cross-sectional view taken along the line AA of FIG.

As shown in FIGS. 1 and 2, the chip 1 has a flow path 2 through which a fluid is fed. A recovery unit 3 is provided in the middle of the flow path 2. Therefore, the flow path 2 has an upstream side flow path 2a provided on the upstream side of the recovery unit 3 and a downstream side flow path 2b provided on the downstream side of the recovery unit 3.

In addition, an adsorbent 4 for adsorbing and recovering nucleic acid is arranged in the recovery unit 3. The adsorbent 4 is a carrier for supporting nucleic acid. In the present embodiment, the adsorbent 4 is composed of a cellulose filter unit 5 and a plurality of particles 6 filled inside the cellulose filter unit 5. In this embodiment, the particle 6 is a silica particle.

However, the form of the adsorbent 4 is not particularly limited, and for example, it can be used in the form of a membrane, a filter, a plate, a tube, a fibrous form, a particulate form, a porous form, or the like. The adsorbent 4 is preferably fibrous, particulate, or porous. As in this embodiment, a plurality of forms may be used in combination.

The adsorbent 4 is not particularly limited, but can be composed of, for example, silicon compounds, phosphate minerals, silicate minerals, aluminosilicate minerals, and the like. Examples of silicon compounds include silica and glass. Examples of phosphate minerals include hydroxyapatite and the like. Examples of silicate minerals include talc and montmorillonite. Examples of the aluminosilicate minerals include zeolites and the like. Further, the adsorbent 4 may be a polyamine-based compound such as chitosan or a histidine polymer. One of these may be used alone, or a plurality of types may be used in combination.

In the recovery method of the present embodiment, the liquid sample containing the nucleic acid extracted as described above is injected into the upstream flow path 2a from the injection port of the chip 1 and sent to the recovery unit 3. As a result, the nucleic acid is adsorbed on the adsorbent 4 in the recovery unit 3.

Next, the cleaning liquid is sent to the recovery unit 3. Thereby, the nucleic acid adsorbed on the adsorbent 4 is washed. The cleaning liquid is not particularly limited, and examples thereof include alcohols such as ethanol. Next, the recovered liquid is sent to the recovery unit 3, the nucleic acid is isolated from the adsorbent 4, the liquid is sent to the downstream flow path 2b side, and the nucleic acid is recovered from the recovery port.

In the recovery method of the present embodiment, the nucleic acid is recovered using a recovery solution containing an aprotic polar compound. Therefore, as described above, the nucleic acid recovery rate can be efficiently increased. Further, even when a polyamine-based compound is used for the adsorbent 4, nucleic acids can be efficiently recovered without making the recovery liquid basic. Therefore, it is difficult for a reaction such as PCR to be inhibited in a test or analysis in a subsequent process.

The aprotic polar compound is not particularly limited, and for example, pyrrolidone, formamide, sulfoxide, acetamide, acetonitrile or a derivative thereof, ketones such as acetone, cyclohexanone and methyl ethyl ketone, ethers such as THF and dioxane, and ethylene glycol. , Glycos such as propylene glycol, and esters such as ethyl acetate, butyl acetate, or triphosphate ester. Among them, the aprotonic polar compound is preferably N-methylpyrrolidone, dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), N, N-dimethylformamide (DMF), or THF, and N-methylpyrrolidone or dimethyl sulfoxide is preferable. (DMSO) is more preferred. When N-methylpyrrolidone or dimethyl sulfoxide (DMSO) is used, it is possible to more effectively increase the recovery rate of nucleic acid and make it more difficult to inhibit reactions such as PCR. As these aprotic polar compounds, one type may be used alone, or a plurality of types may be used in combination.

The aprotic polar compound may be a solid, but is preferably a liquid at room temperature, i.e. an aprotic polar solvent. The boiling point of the aprotic polar solvent is preferably 90 ° C. or higher. When the boiling point of the aprotic polar solvent is at least the above lower limit, it can be made more difficult to volatilize during PCR. The upper limit of the boiling point of the aprotic polar solvent is not particularly limited, but can be, for example, 300 ° C.

The recovery solution containing the aprotic polar compound may be a mixed solution of the aprotic polar compound and water. The concentration of the aprotic polar compound contained in the recovered solution is preferably 0.01 mol / L or more, more preferably 0.1 mol / L or more, still more preferably 0.5 mol / L or more. When the concentration of the aprotic polar compound is equal to or higher than the above lower limit, the nucleic acid recovery rate can be further effectively increased. The water may be a buffer solution.

The upper limit of the concentration of the aprotic polar compound contained in the recovered solution is not particularly limited, but is preferably 10 mol / L or less, more preferably 7 mol / L or less, and further preferably 5 mol / L or less. When the concentration of the aprotic polar compound is not more than the above upper limit value, excessive dilution for lowering the final concentration of the aprotic polar compound brought into the nucleic acid amplification step described later becomes unnecessary, so that nucleic acid amplification The efficiency can be further increased.

Hereinafter, the details of the chips constituting the microfluidic device used in the recovery method of the present embodiment will be described.

The chip 1 is not particularly limited, but in the present embodiment, as shown in FIG. 2, it has a plate-shaped substrate 7.

The material constituting the substrate 7 is not particularly limited, and for example, synthetic resin, rubber, metal, or the like can be used. The synthetic resin is not particularly limited, and examples thereof include silicone resins such as dimethylpolysiloxane (PDMS), curable resins such as phenol resins and polyurethane resins, and thermoplastic resins. From the viewpoint of productivity and the like, a thermoplastic resin is particularly preferable. Among them, as the thermoplastic resin, for example, cycloolefin polymer, cycloolefin copolymer, polycarbonate, polymethylmethacrylate, polystyrene, polypropylene and the like can be used. One of these may be used alone, or a plurality of types may be used in combination.

The substrate 7 is preferably made of the above-mentioned molded body of the thermoplastic resin. The molding method is not particularly limited, and a known molding method can be used. Examples of the molding method include injection molding, injection compression molding, gas assist method injection molding, extrusion molding, multi-layer extrusion molding, rotary molding, hot press molding, blow molding, and foam molding. Of these, injection molding is preferable.

The substrate 7 may be formed by laminating a plurality of synthetic resin sheets. The substrate 7 may be composed of a base sheet, a substrate main body having a through hole provided on the base sheet, and a cover sheet provided on the substrate main body.

Each of the base sheet and the cover sheet can be made of a flexible material such as a resin film. As the resin film, for example, a thermoplastic resin such as a cycloolefin polymer, a cycloolefin copolymer, polycarbonate, polymethylmethacrylate, polystyrene, or polypropylene can be used.

Further, the base sheet and the cover sheet may each be composed of elastic members. The elastic member is not particularly limited, but is preferably an elastomer.

The above-mentioned flow path 2 through which the fluid is sent is provided in the substrate 7. Here, the flow path 2 is a micro flow path. The flow path 2 may not be a micro flow path but a flow path having a larger cross-sectional area than the micro flow path. However, it is preferably a microchannel. Thereby, various analyzes can be performed with a small amount of sample.

By the way, the micro flow path means a fine flow path that causes a micro effect when the fluid is conveyed. In such a microchannel, the liquid is strongly affected by surface tension and behaves differently from the liquid flowing through a normal large-sized channel.

The cross-sectional shape and size of the microchannel are not particularly limited as long as the channel produces the above-mentioned micro effect. For example, when a pump or gravity is used to flow a fluid through a microchannel, it is small when the cross-sectional shape of the microchannel is approximately rectangular (including a square) from the viewpoint of reducing channel resistance. The size of the side is preferably 20 μm or more, more preferably 50 μm or more, and further preferably 100 μm or more. From the viewpoint of further miniaturization of the microfluidic device using the chip 1, the size of the smaller side is preferably 5 mm or less, more preferably 1 mm or less, still more preferably 500 μm or less.

Further, when the cross-sectional shape of the microchannel is generally circular, the diameter (minor diameter in the case of an ellipse) is preferably 20 μm or more, more preferably 50 μm or more, still more preferably 100 μm or more. From the viewpoint of further miniaturization of the microfluidic device, the diameter (minor diameter in the case of an ellipse) is preferably 5 mm or less, more preferably 1 mm or less, and further preferably 500 μm or less.

In the recovery method of the present embodiment, nucleic acid can be recovered by using a microfluidic device including such a chip 1. The recovery liquid containing the aprotic polar compound may be arranged in the flow path 2 of the microfluidic device or may be arranged outside the microfluidic device.

In addition, such a microfluidic device may further include a liquid feeding means. The liquid feeding means is not particularly limited, and examples thereof include a micropump. Specifically, it is a means for sending a fluid to the recovery unit 3 side by sending a liquid, air, or a predetermined gas to the upstream side flow path 2a using a micropump. In this case, the micropump may be provided inside the chip 1 or may be provided outside the chip 1.

Further, as another liquid feeding means, a gas generating member arranged in a space connected to the upstream side from the upstream side flow path 2a can be mentioned. The gas generating member is a member that generates gas by an external force such as light or heat. By applying an external force to the gas generating member at a predetermined timing, gas can be generated and the gas can be sent to the upstream flow path 2a. As a result, the fluid can be sent from the upstream flow path 2a to the recovery unit 3 side. Examples of the gas generating member include a gas generating tape.

(Nucleic acid recovery step-2)
In the second method, the step of recovering the nucleic acid extracted as described above is performed as follows.

An adsorbent for adsorbing and recovering nucleic acid is placed in a plastic container such as polypropylene. The adsorbent is a carrier for supporting nucleic acid. The form of the adsorbent is not particularly limited, but can be used, for example, in the form of particles, plates, fibers, or the like.

The adsorbent is not particularly limited, but may be composed of, for example, silicon compounds, phosphate minerals, silicate minerals, aluminosilicate minerals and the like. Examples of silicon compounds include silica and glass. Examples of phosphate minerals include hydroxyapatite and the like. Examples of silicate minerals include talc and montmorillonite. Examples of the aluminosilicate minerals include zeolites and the like. Further, the adsorbent may be a polyamine-based compound such as chitosan or a histidine polymer. One of these may be used alone, or a plurality of types may be used in combination.

In the recovery method of the present embodiment, the mixed solution after nucleic acid extraction in the nucleic acid extraction step is injected into the container prepared as described above, stirred with the adsorbent, and the nucleic acid is adsorbed on the adsorbent. The stirring method is not particularly limited, but for example, a vortex mixer, ultrasonic waves, or the like can be used.

Next, after taking out the solution in the container, the cleaning liquid is injected and stirred. Thereby, the nucleic acid adsorbed on the adsorbent is washed. The cleaning liquid is not particularly limited, and examples thereof include alcohols such as ethanol. Next, after taking out the cleaning liquid, the recovered liquid is injected and stirred. This causes the nucleic acid to be isolated from the adsorbent. As the recovery liquid, for example, an aqueous solution containing an additive such as water or a buffer can be used.

In the recovery method of the present embodiment, the nucleic acid is recovered using a recovery solution containing an aprotic polar compound. Therefore, as described above, the nucleic acid recovery rate can be efficiently increased. Further, even when a polyamine-based compound is used as the adsorbent, the nucleic acid can be efficiently recovered without making the recovery liquid basic. Therefore, it is difficult for a reaction such as PCR to be inhibited in a test or analysis in a subsequent process.

The aprotic polar compound is not particularly limited, and is, for example, pyrrolidone, formamide, sulfoxide, acetamide, acetonitrile or a derivative thereof, ketones such as acetone and cyclohexanone, ethers such as THF and ethylene glycol, and ethyl acetate. Esters such as. Among them, as the aprotonic polar compound, N-methylpyrrolidone, dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), N, N-dimethylformamide (DMF), or THF is preferable, and N-methylpyrrolidone or dimethyl sulfoxide is preferable. (DMSO) is more preferred. When N-methylpyrrolidone or dimethyl sulfoxide (DMSO) is used, it is possible to more effectively increase the recovery rate of nucleic acid and make it more difficult to inhibit reactions such as PCR. As these aprotic polar compounds, one type may be used alone, or a plurality of types may be used in combination.

The aprotic polar compound may be a solid, but is preferably a liquid at room temperature, i.e. an aprotic polar solvent. The boiling point of the aprotic polar solvent is preferably 90 ° C. or higher. When the boiling point of the aprotic polar solvent is at least the above lower limit, it can be made more difficult to volatilize during PCR. The upper limit of the boiling point of the aprotic polar solvent is not particularly limited, but can be, for example, 300 ° C.

The recovery solution containing the aprotic polar compound may be a mixed solution of the aprotic polar compound and water. The concentration of the aprotic polar compound contained in the recovered solution is preferably 0.01 mol / L or more, more preferably 0.1 mol / L or more, still more preferably 0.5 mol / L or more. When the concentration of the aprotic polar compound is equal to or higher than the above lower limit, the nucleic acid recovery rate can be further effectively increased. The water may be a buffer solution.

The upper limit of the concentration of the aprotic polar compound contained in the recovered solution is not particularly limited, but is preferably 10 mol / L or less, more preferably 7 mol / L or less, and further preferably 5 mol / L or less. When the concentration of the aprotic polar compound is not more than the above upper limit value, excessive dilution for lowering the final concentration of the aprotic polar compound brought into the nucleic acid amplification step becomes unnecessary, so that the nucleic acid amplification efficiency is improved. It can be further enhanced.

The nucleic acid amplification method according to the present invention includes a recovery step of recovering nucleic acid by the above-mentioned nucleic acid recovery method, and a nucleic acid amplification step of performing an amplification reaction of nucleic acid contained in the recovery liquid recovered in the recovery step. Therefore, for example, in the above-mentioned microfluidic device, a reaction field for further amplifying the recovered nucleic acid may be provided on the downstream side of the recovery unit. In the reaction field, for example, a reaction such as PCR can be allowed to proceed by performing a thermal cycle treatment in which heating and cooling at 60 ° C. to 90 ° C. are repeated.

Further, a dilution step of diluting the recovery liquid recovered in the recovery step may be further provided before the nucleic acid amplification step. In this case, for example, in the above-mentioned microfluidic device, a dilution flow path for diluting the recovered liquid may be provided between the recovery unit and the reaction field.

Examples of the method for diluting the recovered liquid include a method of adding water. The concentration (final concentration) of the aprotic polar compound in carrying out the amplification reaction of nucleic acid by the dilution step is preferably 2 mol / L or less, more preferably 1.5 mol / L or less. It is more preferably 0 mol / L or less. When the concentration (final concentration) of the aprotic polar compound is not more than the above upper limit value, nucleic acid amplification inhibition is more unlikely to occur. The lower limit of the concentration (final concentration) of the aprotic polar compound when carrying out the amplification reaction of nucleic acid is not particularly limited, but can be, for example, 0.001 mol / L.

Hereinafter, the present invention will be clarified by giving specific examples and comparative examples of the present invention. The present invention is not limited to the following examples.

(Example 1)
Using the Qiagen QIAamp Virus RNA Mini Kit, 1 μL of Escherichia coli (NBRC 12713 strain, 10,000 copies) and 2 μg of carrier RNA were added to 50 μL of Buffer AVL, vortexed for 15 seconds and incubated at room temperature for 10 minutes. Further, 50 μL of 98% ethanol was added and vortexed for 15 seconds to prepare a sample solution, and a syringe pump was placed on chip 1 (FIGS. 1 and 2) of a microfluidic device filled with silica particles having an average particle size of 5 μm. The sample DNA was adsorbed by sending the sample solution using the above sample solution. Similarly, 100 μL of cleaning liquid 1 (Buffer AW1) and cleaning liquid 2 (Buffer AW2) are sent using a syringe pump, and then the liquid is heated for 3 minutes while sending air using a syringe pump on a plate at 100 ° C. Evaporated. Similarly, using a syringe pump, DNA can be recovered by sending 30 μL of a buffer solution (Buffer AVE) to which N-methylpyrrolidone (recovery solution additive) added as a recovery solution has a concentration of 4 mol / L. went.

To prepare the PCR reaction solution, take out 2.5 μL of the above recovered solution and mix it with 4.5 μL of water to obtain 7 μL of diluted recovered solution, 10 μL of PCR reagent, 1 μL of Forward Primer (10 μM), and 1 μL of Revere Primer (10 μM). , TAKARA, Syber Green (250-fold diluted) 1 μL was mixed. As the PCR reagent, "Taq HS Fast Detector" was used. In addition, as a standard, 7 μL of an Escherichia coli NBRC 12713 strain-extracted nucleic acid solution whose nucleic acid concentration was known in advance was prepared at four levels of 100,000, 50,000, 5,000, and 500 copies instead of the diluted recovery solution.

The amount of recovered nucleic acid was measured by using the above-mentioned PCR reaction solution and standard PCR reaction solution using a thermal cycler "Light Cycler (Roche)". The measurement was carried out by heating at 95 ° C. for 20 seconds, followed by 45 temperature cycles of 95 ° C. for 3 seconds and 60 ° C. for 5 seconds.

As a result of the measurement, the nucleic acid recovery rate was calculated using the following formula based on the amount of nucleic acid derived from the standard.

Nucleic acid recovery rate (%) = {(amount of nucleic acid in Light Cycler) x 30 / (amount of recovery liquid taken out)} / 10,000 x 100

(Examples 2 to 11)
The aprotic polar compounds shown in Table 1 are added to the recovery liquid to adjust the concentration to a predetermined value, and a predetermined amount is taken out from the recovery liquid obtained by feeding the liquid, and water is added so that the total volume of the diluted recovery liquid becomes 7 μL. The nucleic acid recovery rate was calculated in the same manner as in Example 1 except that it was prepared.

(Comparative Example 1)
The nucleic acid recovery rate was calculated in the same manner as in Example 1 except that the aprotic polar compound was not added.

(Example 12)
Escherichia coli (NBRC 12713 strain, 10,000 copies) in 50 μL of nucleic acid extraction solution (aqueous solution containing urea 4 mol / L, guanidine hydrochloride 4 mol / L, glycine 1 mol / L, carrier RNA 3 μg, Tris-HCl Buffer (PH 7.0)) ) 1 μL and 2 μg of carrier RNA were added and vortexed for 15 seconds. Then, a sample solution was prepared by incubating at room temperature for 10 minutes. Sample DNA was adsorbed by feeding the above sample solution to chip 1 (FIGS. 1 and 2) of a microfluidic device filled with chitosan-modified particles having an average particle size of 5 μm using a syringe pump. Similarly, 200 μL of water prepared with hydrochloric acid at pH = 4 was sent using a syringe pump, and then the liquid was evaporated by heating for 3 minutes while sending air using a syringe pump on a plate at 100 ° C. Similarly, using a syringe pump, N-methylpyrrolidone (recovery solution additive) as a recovery solution is added to a concentration of 4 mol / L, and 30 μL of water prepared at pH = 7 is sent to the DNA. It was recovered. The PCR reaction solution was prepared, the recovered nucleic acid was measured, and the nucleic acid recovery rate was calculated in the same manner as in Example 1.

(Examples 13 to 15)
The aprotic polar compounds shown in Table 1 are added to the recovery liquid to adjust the concentration to a predetermined value, and a predetermined amount is taken out from the recovery liquid obtained by feeding the liquid, and water is added so that the total volume of the diluted recovery liquid becomes 7 μL. The nucleic acid recovery rate was calculated in the same manner as in Example 12 except that it was prepared.

(Comparative Example 2)
The nucleic acid recovery rate was calculated in the same manner as in Example 12 except that the aprotic polar compound was not added.

1 ... Chip 2 ... Flow path 2a ... Upstream side flow path 2b ... Downstream side flow path 3 ... Recovery unit 4 ... Adsorbent 5 ... Cellulose filter unit 6 ... Particle 7 ... Substrate

Claims (15)

The process of adsorbing nucleic acid to the adsorbent and
A step of recovering the nucleic acid adsorbed on the adsorbent using a recovery solution containing an aprotic polar compound, and
A method for recovering nucleic acid. The method for recovering nucleic acid according to claim 1, wherein the recovery liquid containing the aprotic polar compound further contains water. The method for recovering nucleic acid according to claim 1 or 2, wherein the concentration of the aprotic polar compound in the recovery liquid is 0.01 mol / L or more. The method for recovering nucleic acid according to any one of claims 1 to 3, wherein the concentration of the aprotic polar compound in the recovery liquid is 10 mol / L or less. The aprotic polar compounds are from pyrrolidone, formamide, sulfoxide, acetamide, acetonitrile and derivatives thereof, acetone, cyclohexanone, methyl ethyl ketone, THF, dioxane, ethylene glycol, propylene glycol, ethyl acetate, butyl acetate, and triphosphate ester. The method for recovering a nucleic acid according to any one of claims 1 to 4, which is at least one selected from the group. The method for recovering nucleic acid according to any one of claims 1 to 5, wherein the aprotic polar compound is an aprotic polar solvent. The recovery of the nucleic acid according to any one of claims 1 to 6, wherein the adsorbent is composed of at least one selected from the group consisting of silica, glass, hydroxyapatite, talc, montmorillonite, and zeolite. Method. The method for recovering nucleic acid according to any one of claims 1 to 6, wherein the adsorbent is composed of at least one of chitosan and histidine polymer. A recovery step of recovering nucleic acid by the nucleic acid recovery method according to any one of claims 1 to 8.
A nucleic acid amplification step of performing an amplification reaction of the nucleic acid contained in the recovery liquid recovered in the recovery step, and a nucleic acid amplification step.
A nucleic acid amplification method comprising. The nucleic acid amplification method according to claim 9, further comprising a dilution step of diluting the recovery liquid recovered in the recovery step before the nucleic acid amplification step. The nucleic acid amplification method according to claim 10, wherein the concentration of the aprotic polar compound when carrying out the amplification reaction of the nucleic acid by the dilution step is 2 mol / L or less. A microfluidic device used in the nucleic acid recovery method according to any one of claims 1 to 8 or the nucleic acid amplification method according to any one of claims 9 to 11.
A microfluidic device comprising a chip having a flow path for delivering a fluid and an adsorbent located in the flow path for adsorbing and isolating nucleic acids. A chip comprising a flow path for delivering a fluid and an adsorbent located in the flow path for adsorbing and isolating nucleic acid.
A microfluidic device in which a recovery liquid containing an aprotic polar compound is arranged in the flow path. The microfluidic device of claim 12 or 13, wherein the chip further comprises a reaction field that amplifies the recovered nucleic acid. The microfluidic device of claim 14, wherein the chip further comprises a dilution channel for diluting the recovered liquid.

Images