JP2020124129A - Nucleic acid isolation method and nucleic acid isolation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for isolating a nucleic acid, in which the nucleic acid can be simply and efficiently recovered, and the reaction of the nucleic acid is less likely to be inhibited in a test or analysis in a subsequent step.
SOLUTION: A step of adsorbing a nucleic acid on a cationic solid phase carrier 4 having a pKa of 6 or less, and an aqueous solution having a pH higher than the pKa of 8.5 or less and adsorbed on the cationic solid phase carrier 4 are used. A method of isolating a nucleic acid, comprising the step of isolating the nucleic acid.
[Selection diagram] Figure 1

Description

The present invention relates to a method for isolating a nucleic acid and a device for isolating a nucleic acid used in the method for isolating the nucleic acid.

Conventionally, as a method for separating and purifying nucleic acids such as RNA and DNA contained in viruses and cells, there have been used a chloroform extraction method, a Boom method, or a purification method using an anion exchanger or a cationic solid phase carrier. Are known.

For example, in Patent Document 1 below, after a nucleic acid-containing aqueous solution obtained by lysing cells with a lysing solution is contacted with an anion exchanger, nucleic acids are eluted from the anion exchanger using an aqueous mobile phase. A method for separating and/or purifying nucleic acid is disclosed. In Patent Document 1, a composition for adjusting the pH of the aqueous mobile phase to about pH 9 to about pH 13 is added to the nucleic acid eluent constituting the aqueous mobile phase.

Further, in Patent Document 2 below, after a cationic solid phase carrier is brought into contact with a sample containing nucleic acid to capture the nucleic acid on the cationic solid phase carrier, the cationic solid phase carrier on which the nucleic acid is captured is removed from the sample. A method of separating is disclosed. In Patent Document 2, the separated cationic solid phase carrier is further treated with an anionic substance, and the nucleic acid is separated from the cationic solid phase carrier to recover the nucleic acid.

Japanese Patent Publication No. 2010-504738 JP 2001-352979 A

When the present invention isolates and recovers a nucleic acid with a highly basic solution having a large pH as in Patent Document 1 or Patent Document 2, in the subsequent steps of inspection and analysis, a nucleic acid reaction such as an enzymatic reaction is performed. Have been found to be hindered.

The object of the present invention is to be used for a method for isolating a nucleic acid and a method for isolating the nucleic acid, which enables simple and efficient recovery of the nucleic acid and is less likely to inhibit the reaction of the nucleic acid in a test or analysis in a subsequent step. It is to provide a device for nucleic acid isolation.

The method for isolating a nucleic acid according to the present invention comprises a step of adsorbing a nucleic acid on a cationic solid phase carrier having a pKa of 6 or less, and an aqueous solution having a pH higher than the pKa of 8.5 or less and using the cationic Isolating the nucleic acid adsorbed on the solid phase carrier.

In one specific aspect of the method for isolating a nucleic acid according to the present invention, the cationic solid phase carrier is an amine compound or an imine compound.

In another specific aspect of the method for isolating a nucleic acid according to the present invention, the cationic solid phase carrier is selected from the group consisting of an alkylamine compound, a phenylamine compound, a nitrogen-containing heterocyclic compound, and an aminosilane compound. It is at least one selected.

In still another specific aspect of the method for isolating a nucleic acid according to the present invention, an aqueous solution having a pH of more than 6 and 8.5 or less is used to isolate the nucleic acid adsorbed on the cationic solid phase carrier.

The nucleic acid isolation device according to the present invention is a nucleic acid isolation device including a chip provided with a channel for sending a fluid, and a cationic solid phase having a pKa of 6 or less in the middle of the channel. A carrier is arranged.

In one specific aspect of the nucleic acid isolation device according to the present invention, the cationic solid phase carrier is immobilized in the middle of the channel.

In another specific aspect of the device for nucleic acid isolation according to the present invention, the cationic solid phase carrier is retained in the flow path together with a liquid.

INDUSTRIAL APPLICABILITY According to the present invention, a nucleic acid can be simply and efficiently recovered, and is used in a method for isolating a nucleic acid and a method for isolating the nucleic acid, which is difficult to inhibit the reaction of the nucleic acid in a test or analysis in a subsequent step. A device for nucleic acid isolation can be provided.

FIG. 1 is a schematic plan view showing a main part of a nucleic acid isolation device according to an embodiment of the present invention. FIG. 2 is a schematic cross-sectional view of a portion along the line AA in FIG. FIG. 3( a) is a schematic diagram for explaining a state in which the nucleic acid is adsorbed on the cationic solid phase carrier in the method for recovering nucleic acid according to one embodiment of the present invention, and FIG. 3( b) is FIG. 3 is a schematic diagram for explaining a state in which a nucleic acid is isolated from a cationic solid phase carrier. FIG. 4 is a diagram showing the relationship between the number of cycles and fluorescence intensity in Examples 3 to 5 and Comparative Examples 3 to 5.

Hereinafter, the present invention will be clarified by describing specific embodiments of the present invention with reference to the drawings.

[Method for isolating nucleic acid]
The method for isolating a nucleic acid according to the present invention is a method for isolating a nucleic acid adsorbed on a cationic solid-phase carrier having a pKa of 6 or less using an aqueous solution having a pH of 8.5 or more and 8.5 or less. It is related.

Hereinafter, a method for isolating a nucleic acid according to an embodiment of the present invention will be described with reference to FIGS. 1 and 2.

FIG. 1 is a schematic plan view showing a main part of a nucleic acid isolation device according to an embodiment of the present invention. 2 is a schematic cross-sectional view of a portion taken along the line AA in FIG.

The nucleic acid isolation device of this embodiment includes the chip 1 shown in FIGS. 1 and 2. The chip 1 has a flow path 2 through which a fluid is sent. A collecting unit 3 is provided in the middle of the flow path 2. Therefore, the flow channel 2 has an upstream flow channel 2 a provided upstream of the collection unit 3 and a downstream flow channel 2 b provided downstream of the collection unit 3.

Further, a cationic solid phase carrier 4 for adsorbing and recovering nucleic acid is arranged in the recovery unit 3. The cationic solid-phase carrier 4 is a compound having a functional group that does not form a cation by itself but easily forms a cation by binding a proton at neutral or lower (pH is 6 or lower). Examples of such a functional group include an amino group and an imino group.

The form of the cationic solid phase carrier 4 is not particularly limited, but it may be used in the form of, for example, a membrane, a filter, a plate, particles, fibers, a tube, or the like. The cationic solid phase carrier 4 may be fixed to the recovery part 3 or may remain in the recovery part 3 together with the liquid.

The pKa of the cationic solid phase carrier 4 is 6 or less. Therefore, the negatively charged nucleic acid can be adsorbed. Therefore, the cationic solid phase carrier 4 acts as a carrier for nucleic acid. The cationic solid phase carrier 4 is not particularly limited, but for example, a cationic compound such as an amine compound or an imine compound can be used. As the amine compound or the imine compound, for example, an alkylamine compound, a phenylamine compound, a nitrogen-containing heterocyclic compound, an aminosilane compound, or the like can be used. More specifically, histidine, phenylamine or the like can be used as such a cationic compound. These cationic compounds may be used alone or in combination of two or more.

The pKa of the cationic solid phase carrier 4 is preferably 5.5 or less, more preferably 5 or less. In this case, the nucleic acid can be more surely adsorbed by the cationic solid phase carrier 4. The lower limit of pKa of the cationic solid phase carrier 4 can be set to 4, for example.

Further, the cationic solid phase carrier 4 may be one obtained by modifying an anionic solid phase carrier such as a silica-based solid phase carrier with a cationic compound. Further, the cationic solid phase carrier 4 and the anionic solid phase carrier may be provided separately. For example, an anionic solid phase carrier may be further provided on the upstream side of the cationic solid phase carrier 4. In that case, the nucleic acid can be purified more accurately. The anionic solid phase carrier is not particularly limited, and examples thereof include glass fiber and hydroxyapatite. Among them, the anionic solid phase carrier is preferably silica fiber.

In the nucleic acid isolation method of the present embodiment, first, a sample containing nucleic acid is injected into the upstream channel 2a from the injection port 7a of the chip 1 and is sent to the recovery unit 3. At this time, since the nucleic acid is negatively charged, when a sample containing the nucleic acid is sent to the recovery unit 3, an ionic bond is formed between the nucleic acid and the cationic solid phase carrier 4, as shown in FIG. It is formed. Thereby, the nucleic acid can be adsorbed on the cationic solid phase carrier 4.

As the sample containing the nucleic acid, for example, a liquid sample after nucleic acid extraction can be used. Specifically, as the liquid sample after nucleic acid extraction, a liquid sample obtained by adding a nucleic acid extraction solution to a sample such as a virus or a cell can be used. The sample containing the nucleic acid may be a liquid sample after nucleic acid extraction in which alcohol such as ethanol is added to precipitate the nucleic acid.

As the nucleic acid extraction solution, for example, a solution containing a protein denaturant and a polar solvent such as water can be used. As the protein denaturing agent, for example, a guanidine derivative, thiourea, urea, a surfactant or the like can be used. Further, the protein denaturing agent may be a salt such as guanidine hydrochloride. These protein denaturants may be used alone or in combination of two or more.

Next, an aqueous solution having a pH higher than the pKa of the cationic solid phase carrier 4 and 8.5 or less is sent to the recovery unit 3. At this time, since the pH of the aqueous solution is higher than the pKa of the cationic solid phase carrier 4, the cationic solid phase carrier 4 is in a non-ionized state in the aqueous solution. Therefore, when the aqueous solution is sent to the recovery unit 3, the ionic bond between the nucleic acid and the cationic solid phase carrier 4 is disrupted, as shown in FIG. Therefore, when the above aqueous solution is sent to the recovery unit 3, the nucleic acid can be isolated from the cationic solid phase carrier 4 and the nucleic acid can be recovered from the recovery port 7b.

In the method for isolating a nucleic acid according to the present embodiment, a nucleic acid adsorbed on a cationic solid-phase carrier 4 having a pKa of 6 or less is treated with an aqueous solution having a pH higher than the pKa of the cationic solid-phase carrier 4 and a pH of 8.5 or less. Used to isolate from the cationic solid phase carrier 4. Therefore, in the method for isolating a nucleic acid of the present embodiment, the nucleic acid can be simply and efficiently recovered simply by adjusting the pH. In particular, in the nucleic acid isolation method of the present embodiment, the nucleic acid is isolated and recovered using a neutral aqueous solution having a pH higher than the pKa of the cationic solid phase carrier 4 and 8.5 or less. be able to. Therefore, it is possible to suppress the inhibition of the reaction of nucleic acid such as enzyme reaction in the inspection and analysis in the subsequent process.

An aqueous solution having a pH higher than the pKa of the cationic solid phase carrier 4 and 8.5 or less is not particularly limited. However, from the viewpoint of more reliably isolating the nucleic acid from the cationic solid phase carrier 4, the pH of the aqueous solution is preferably 6 or higher, more preferably higher than 6, and further preferably 7 or higher. Further, from the viewpoint of further suppressing the inhibition of the reaction in the inspection and analysis in the subsequent step, the pH of the above aqueous solution is preferably 8 or less, more preferably 7.5 or less.

Hereinafter, the details of the nucleic acid isolation device used in the method for isolating nucleic acid of the present embodiment will be described.

(Device for nucleic acid isolation)
The nucleic acid isolation device of this embodiment includes the chip 1 shown in FIGS. 1 and 2. The chip 1 has a flow path 2 through which a fluid is sent. A collecting unit 3 is provided in the middle of the flow path 2. Therefore, the flow path 2 has an upstream flow path 2 a provided upstream of the collection unit 3 and a downstream flow path 2 b provided downstream of the collection unit 3. A cationic solid phase carrier 4 for adsorbing and recovering nucleic acid is arranged in the recovery unit 3. The cationic solid phase carrier 4 may be fixed to the recovery part 3 or may be retained in the recovery part 3 together with the liquid. In the nucleic acid isolation device of the present embodiment, the cationic solid phase carrier 4 is arranged in the recovery unit 3, so that the nucleic acid can be easily and efficiently recovered, and the nucleic acid can be easily used for inspection and analysis in the subsequent steps. Reaction is difficult to be hindered.

Although not particularly limited, the chip 1 has a plate-shaped substrate 5 and a cover member 6 in the present embodiment, as shown in FIG. The substrate 5 has a main surface 5a. A concave portion 5b is provided on the main surface 5a side of the substrate 5. The recess 5b is provided so as to open on the main surface 5a side of the substrate 5.

The material forming the substrate 5 is not particularly limited, and for example, synthetic resin, rubber, metal or the like can be used. The synthetic resin is not particularly limited, but a thermoplastic resin is preferable. Among them, as the thermoplastic resin, for example, cycloolefin polymer, cycloolefin copolymer, polycarbonate, polymethylmethacrylate, polypropylene or the like can be used. These may be used alone or in combination of two or more.

The substrate 5 is preferably made of a molded body of the above 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-assisted injection molding, extrusion molding, multilayer extrusion molding, rotational molding, hot press molding, blow molding, and foam molding. Of these, injection molding is preferable.

The substrate 5 may be formed by stacking a plurality of synthetic resin sheets. The substrate 5 may be composed of a base sheet and a substrate body having a through hole provided on the base sheet.

A cover member 6 is provided on the main surface 5 a of the substrate 5. The cover member 6 is provided so as to close the recess 5b of the substrate 5. The cover member 6 closes the recess 5 b of the substrate 5 to form the recovery unit 3. In the present embodiment, the upstream flow passage 2a and the downstream flow passage 2b are also configured by the cover member 6 closing the recess 5b of the substrate 5 in the same manner.

The cover member 6 can be made of, for example, a flexible material such as a resin film. As the resin film, for example, a thermoplastic resin such as cycloolefin polymer, cycloolefin copolymer, polycarbonate, polymethylmethacrylate, or polypropylene can be used.

Further, the cover member 6 may be made of an elastic member. The elastic member is not particularly limited, but is preferably an elastomer. In addition, in the present invention, the substrate 5 and the cover member 6 may be integrally configured.

The method of attaching the cover member 6 to the main surface 5a of the substrate 5 is not particularly limited, and a known laminating method can be used. Examples of the laminating method include methods such as press-bonding and roll laminating. The laminating conditions are not particularly limited, but it is preferable to laminate at a temperature of 50° C. or less, for example. It is also preferable to laminate at a pressure of 10 MPa or less. By laminating under such conditions, it is possible to make it more difficult to embed the cover member 6 in the flow path 2.

In the substrate 5, the above-mentioned flow path 2 through which the fluid is sent is provided. Here, the flow path 2 is a micro flow path. The flow channel 2 may be a flow channel having a larger cross-sectional area than the micro flow channel instead of the micro flow channel. However, it is preferably a micro flow channel. Thereby, various analyzes can be performed with a very small amount of sample.

By the way, the micro flow path means a fine flow path that causes a micro effect when a fluid is conveyed. In such a micro flow channel, the liquid is strongly affected by the surface tension, and exhibits a behavior different from that of the liquid flowing in a normal large-sized flow channel.

The cross-sectional shape and size of the micro channel are not particularly limited as long as the channel produces the above micro effect. For example, when a pump or gravity is used when flowing a fluid through the micro flow channel, from the viewpoint of reducing the flow channel resistance, when the cross sectional shape of the micro flow channel is generally rectangular (including square), it is small. The dimension of one side 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 using the chip 1, the dimension of the smaller side is preferably 5 mm or less, more preferably 1 mm or less, and further preferably 500 μm or less.

Further, when the cross-sectional shape of the microchannel is roughly circular, the diameter (in the case of an ellipse, the minor axis) 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, the diameter (in the case of an ellipse, the short diameter) is preferably 5 mm or less, more preferably 1 mm or less, and further preferably 500 μm or less.

On the other hand, for example, when effectively utilizing the capillary phenomenon when flowing a fluid through a microchannel, when the cross-sectional shape of the microchannel is roughly rectangular (including square), the dimension of the smaller side And is preferably 5 μm or more, more preferably 10 μm or more, still more preferably 20 μm or more. In addition, the dimension of the smaller side is preferably 200 μm or less, and more preferably 100 μm or less.

In the present embodiment, the recovery unit 3 described above is provided in the middle of the flow path 2. In addition, the flow path 2 has an upstream flow path 2a and a downstream flow path 2b.

One end of the upstream flow path 2a is connected to the recovery unit 3. On the other hand, a fluid or gas can be made to flow in from the inlet side where the other end of the upstream channel 2a is provided. When a plurality of fluids or gases are made to flow into the recovery unit 3, they may all be made to flow in from the same inlet, or a separate inlet or upstream channel 2a may be provided for each inflowing fluid or gas. ..

The fluid sent from the upstream channel 2a to the recovery unit 3 can be sent by a solution sending means provided inside or outside the chip 1. The liquid feeding means is not particularly limited, and examples thereof include a micro pump. Specifically, there is a means for sending the liquid to the recovery unit 3 side by sending a liquid, air, or a predetermined gas into the upstream channel 2a using a micropump. In this case, the micropump may be provided inside the chip 1 or may be provided outside the chip 1. The liquid is sent to the recovery unit 3 by such means.

Further, as another liquid feeding means, a gas generating member arranged in a space connected to the upstream side of the upstream 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. The gas can be generated by applying an external force to the gas generating member at a predetermined timing, and the gas can be sent to the upstream flow path 2a. Thereby, the liquid can be sent from the upstream channel 2a to the recovery section 3 side. Examples of the gas generating member include a gas generating tape.

One end of the downstream flow path 2b is connected to the recovery unit 3. From the other end provided on the downstream side of the downstream flow path 2b, the fluid or gas can be sent to another part. In addition to the upstream channel 2a and the downstream channel 2b, other channels may be connected as necessary. In the downstream side flow passage 2b, a flow passage, a mixing/reaction part, etc. can be arranged according to the intended inspection and reaction.

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 examples below.

(Example 1)
In Example 1, the chip 1 shown in FIGS. 1 and 2 was manufactured as follows.

Cycloolefin polymer was used as the material for forming the substrate 2, and this was injection-molded to produce the substrate 5 having the recess 5b. A sealing tape was used for the cover member 6, and the recess 5b of the substrate 5 was closed with the sealing tape to fabricate the chip 1. The histidine-modified particles (average particle size 1 μm) were arranged as the cationic solid phase carrier 4 in the recovery unit 3.

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

First, a reagent (pH: 5, adjusted with hydrochloric acid) containing a virus (50000 copies) was sent to the collection unit 3 to carry a nucleic acid on histidine-modified particles as the cationic solid phase carrier 4.

Next, an aqueous solution having a pH of 7 as a recovery liquid was sent to the recovery unit 3 to isolate and recover the nucleic acid.

Next, the recovered nucleic acid (RNA) is converted into cDNA by reverse transcription reaction (45° C., 5 minutes), and heated and cooled between a plurality of temperature ranges (60° C., 90° C.) for PCR. I went. Thereby, the cDNA obtained from the nucleic acid (RNA) was amplified by the PCR reaction. After amplifying the nucleic acid (cDNA), the amplified nucleic acid was detected by a method using a Q probe, and the amount of recovered RNA was calculated. Then, when the RNA recovery rate was determined from the relationship with the amount of virus (50,000 copies) passed through the recovery unit 3, it was 80% in Example 1.

In addition, instead of the virus (50,000 copies), RNA recovery was similarly obtained using a reagent containing a nasal wipe component collected using a nasal swab swab (manufactured by Sekisui Medical Co., Ltd., kit accessory). However, in Example 1, it was 40%.

(Example 2)
Including a virus (nucleic acid solution) and a nasal wipe component in the same manner as in Example 1 except that phenylamine-modified particles (average particle size 1 μm) were used instead of the histidine-modified particles as the cationic solid-phase carrier. The RNA recovery rate of each reagent (including the actual sample) was determined.

(Comparative Example 1)
Including the virus (nucleic acid solution) and the nasal wipe component in the same manner as in Example 1 except that the alkylamine-modified particles (average particle size 1 μm) were used as the cationic solid-phase carrier instead of the histidine-modified particles. The RNA recovery rate of each reagent (including the actual sample) was determined.

(Comparative example 2)
A reagent containing a virus (nucleic acid solution) and a nasal wipe component in the same manner as in Example 1 except that chitosan-modified particles (average particle size 5 μm) were used as the cationic solid phase carrier instead of the histidine-modified particles. The RNA recovery rate of each (including the actual sample) was determined.

The results are shown in Table 1 below.

(Example 3)
PCR was performed in the same manner as in Example 1 except that the reagent containing the virus (1×10 ⁵ copies) was used.

(Example 4)
PCR was performed in the same manner as in Example 1 except that the reagent containing the virus (1×10 ⁴ copies) was used.

(Example 5)
PCR was performed in the same manner as in Example 1 except that a reagent containing virus (1×10 ³ copies) was used.

(Comparative example 3)
PCR was performed in the same manner as in Example 3 except that an aqueous solution having a pH of 9 was used as the recovery liquid.

(Comparative example 4)
PCR was performed in the same manner as in Example 4 except that an aqueous solution having a pH of 9 was used as the recovery liquid.

(Comparative example 5)
PCR was performed in the same manner as in Example 5 except that an aqueous solution having a pH of 9 was used as the recovery liquid.

The relationship between the number of cycles and the fluorescence intensity in Examples 3 to 5 and Comparative Examples 3 to 5 is shown in FIG.

As shown in FIG. 4, in Examples 3 to 5 using the aqueous solution of pH 7 as the recovery liquid, the fluorescence intensity was higher and the reaction efficiency was higher than in Comparative Examples 3 to 5 using the aqueous solution of pH 9 (inhibition of reaction inhibition was suppressed. Have been confirmed). Further, in Examples 3 to 5 using the aqueous solution of pH 7 as the recovery liquid, the Ct value (the number of cycles at which the amplification curve started to rise) was smaller than that of Comparative Examples 3 to 5 using the aqueous solution of pH 9, and the reaction efficiency was enhanced. It was confirmed that the reaction was suppressed (the inhibition of the reaction was suppressed).

DESCRIPTION OF SYMBOLS 1... Chip 2... Flow path 2a... Upstream flow path 2b... Downstream flow path 3... Collection part 4... Cationic solid phase carrier 5... Substrate 5a... Main surface 5b... Recess 6... Cover member 7a... Injection port 7b... Collection port

Claims (7)

a step of adsorbing a nucleic acid onto a cationic solid phase carrier having a pKa of 6 or less,
a step of isolating the nucleic acid adsorbed on the cationic solid-phase carrier using an aqueous solution having a pH higher than pKa and 8.5 or less;
A method for isolating a nucleic acid, comprising: The method for isolating nucleic acid according to claim 1, wherein the cationic solid phase carrier is an amine compound or an imine compound. The cationic solid phase carrier is at least one selected from the group consisting of an alkylamine-based compound, a phenylamine-based compound, a nitrogen-containing heterocyclic compound, and an aminosilane-based compound, according to claim 1 or 2. Method for isolating nucleic acid. The method for isolating a nucleic acid according to any one of claims 1 to 3, wherein the nucleic acid adsorbed on the cationic solid-phase carrier is isolated using an aqueous solution having a pH of more than 6 and 8.5 or less. A device for nucleic acid isolation, comprising a chip provided with a channel for sending a fluid, comprising:
A device for nucleic acid isolation, wherein a cationic solid phase carrier having a pKa of 6 or less is arranged in the middle of the flow path. The nucleic acid isolation device according to claim 5, wherein the cationic solid phase carrier is immobilized in the middle of the flow channel. The nucleic acid isolation device according to claim 5, wherein the cationic solid-phase carrier is retained in the flow path along with a liquid.

Images