WO2019035335A1

WO2019035335A1 - Nucleic acid collection method, nucleic-acid-binding carrier, and nucleic acid collection kit

Info

Publication number: WO2019035335A1
Application number: PCT/JP2018/028130
Authority: WO
Inventors: 酒井　智弘; 太志原; 哲也小谷; 信介山▲崎▼
Original assignee: Ａｇｃ株式会社
Priority date: 2017-08-18
Filing date: 2018-07-26
Publication date: 2019-02-21

Abstract

Provided are: a method for collecting nucleic acid at a high rate of collection by means of a simple operation, even when the nucleic acid is long; a nucleic-acid-binding carrier that is suitable for use in the collection method; and a nucleic acid collection kit. A nucleic acid collection method that involves: forming composites by making nucleic acid bind to porous silica particles that have a D50 of 6–50 μm in a volume-based particle size distribution, a pore volume of 0.3–5 cm3/g as found by the BJH method, and an average pore diameter of 60–500 Å as found by the BJH method or porous silica particles that have a D50 of 6–50 μm in a volume-based particle size distribution, a pore volume of 0.6–5 cm3/g as found by mercury porosimetry, and an average pore diameter of 200–5,000 Å as found by mercury porosimetry; liquating the nucleic acid from the composites; and collecting the nucleic acid.

Description

Nucleic acid recovery method, nucleic acid binding carrier and nucleic acid recovery kit

The present invention relates to a nucleic acid recovery method, a nucleic acid binding carrier and a nucleic acid recovery kit.

Conventionally, as an infectious disease diagnostic kit, one intended for a protein (antigen, antibody, etc.) has been used. However, there is a problem that the accuracy is low except for a part of infectious diseases such as influenza, and in the case of targeting an antibody, false positive due to past infection. Therefore, in recent years, infectious disease diagnostic kits for nucleic acids have been studied. By targeting nucleic acid, an improvement in accuracy is expected.

The sample which is a biological sample contains various components, and it is necessary to isolate the nucleic acid from the sample for diagnosis.
As a method of isolating nucleic acid from a biological sample, there is known a method of adsorbing or binding nucleic acid to a carrier containing silica, removing components other than nucleic acid by washing, and eluting nucleic acid from the carrier. Nucleic acid purification kits using this method have already been put to practical use, and silica membranes are widely used as carriers (eg, non-patent documents 1 to 3). In addition, in order to simplify and automate the isolation operation, it has been proposed to use, as a carrier, magnetic silica particles in which the surface of a magnetic material is coated with porous silica (for example, Patent Document 1).

Japanese Patent Laid-Open No. 2000-300262

However, conventional nucleic acid isolation methods do not always have high extraction rates of nucleic acids, and in the case of nucleic acids having a length of 10 ⁵ bp or more in particular, they can not be extracted or extraction is low even if extraction is possible. there were. In addition, when the extraction rate is low, it is necessary to perform an amplification process for a long time using an expensive apparatus, which limits the facilities that can be implemented and also takes a long time.

An object of the present invention is to provide a method for recovering nucleic acid with high recovery rate by a simple operation even when nucleic acid is long, a carrier for binding to nucleic acid and a nucleic acid recovery kit suitably used for the recovery method.

The present invention provides a nucleic acid recovery method, a nucleic acid binding carrier and a nucleic acid recovery kit, having the following aspects [1] to [16].
[1] is a _{D 50} of 6 ~ 50 [mu] m particle size distribution based on volume, the pore volume determined by the BJH method is the 0.3 ~ ^{5 cm} 3 / g, an average pore diameter determined by BJH method from 60 to A nucleic acid is bound to porous silica particles (hereinafter also referred to as porous silica particles I) having a thickness of 500 Å to form a complex,
A method for recovering nucleic acid, which comprises eluting and recovering the nucleic acid from the complex.
[2] a _{D 50} of 6 ~ 50 [mu] m particle size distribution based on volume, the pore volume determined by mercury porosimetry is 0.6 ~ ^5cm 3 / g, 200 average pore diameter determined by the mercury porosimeter is ~ A nucleic acid is bound to a porous silica particle (hereinafter, also referred to as porous silica particle II) which is 5000 Å to form a complex,
A method for recovering nucleic acid, which comprises eluting and recovering the nucleic acid from the complex.
[3] The method for recovering a nucleic acid according to [1] or [2], wherein D ₉₀ / D ₁₀ of the particle size distribution based on the volume of the porous silica particles is 10 or less.
[4] The method for recovering nucleic acid according to [1] or [3], wherein the specific surface area of the porous silica particles is 150 to 1000 m ² / g.
[5] The method for recovering nucleic acid according to [2] or [3], wherein the specific surface area of the porous silica particles is 8 to 400 m ² / g.
[6] The method for recovering nucleic acid according to any one of [1] to [5], wherein the purity of the porous silica particles is 90% by mass or more.
[7] The method for recovering nucleic acid according to any one of [1] to [6], wherein the nucleic acid is bound to the porous silica particle in the presence of a chaotropic substance solution.
[8] The method for recovering nucleic acid according to any one of [1] to [7], wherein the complex is washed before eluting the nucleic acid from the complex.
[9] The method for recovering a nucleic acid according to any one of [1] to [8], wherein the length of the nucleic acid is 10 ⁵ to 10 ¹¹ bp.
[10] a _{D 50} of 6 ~ 50 [mu] m particle size distribution based on volume, the pore volume determined by the BJH method is 0.3 ~ ^{5 cm} 3 / g, an average pore diameter determined by BJH method from 60 to A carrier for nucleic acid binding comprising porous silica particles of 500 Å.
[11] a _{D 50} of 6 ~ 50 [mu] m particle size distribution based on volume, the pore volume determined by mercury porosimetry is 0.6 ~ ^{5 cm} 3 / g, an average pore diameter determined by the mercury porosimeter is 200 ~ A carrier for nucleic acid binding comprising porous silica particles of 5000 Å.
[12] a _{D 50} of 6 ~ 50 [mu] m particle size distribution based on volume, the pore volume determined by the BJH method is the 0.3 ~ ^{5 cm} 3 / g, an average pore diameter determined by BJH method from 60 to A first container containing porous silica particles that is 500 Å;
A second container containing a binding solution for binding nucleic acid to the porous silica particles.
[13] a particle size _{D 50} of 6 ~ 50 [mu] m in the distribution of a volume basis, the pore volume determined by mercury porosimetry is the 0.6 ~ ^5cm 3 / g, 200 average pore diameter determined by the mercury porosimeter is ~ A first container containing porous silica particles of 5000 Å;
A second container containing a binding solution for binding nucleic acid to the porous silica particles.
[14] The nucleic acid recovery kit according to [12] or [13], wherein the binding solution is a chaotropic substance solution.
[15] The container according to any one of [12] to [14], further comprising a third container containing an eluate for eluting the nucleic acid from the complex in which the porous silica particle and the nucleic acid are bound to each other. Nucleic acid recovery kit.
[16] The nucleic acid recovery kit according to any one of [12] to [15], further comprising a fourth container containing a washing solution for washing the complex in which the porous silica particles and the nucleic acid are bound. .

According to the nucleic acid recovery method of the present invention, even when the nucleic acid is long, the nucleic acid can be recovered at a high recovery rate by a simple operation.
The nucleic acid binding carrier and the nucleic acid recovery kit of the present invention can be suitably used for the above recovery method, respectively.

FIG. 16 is a graph showing the relationship between D ₅₀ of the porous silica particles I and DNA recovery in Examples 1 to 10 of Example A. FIG. FIG. 16 is a graph showing the relationship between D ₉₀ / D ₁₀ of the porous silica particles I and the DNA recovery in Examples 1 to 10 of Example A. FIG. FIG. 16 is a graph showing the relationship between the pore volume of porous silica particles I and the DNA recovery in Examples 1 to 10 of Example A. FIG. 7 is a graph showing the relationship between the average pore size of porous silica particles I and the DNA recovery in Examples 1 to 10 of Example A. 7 is a graph showing the relationship between the specific surface area (SSA) of porous silica particles I and the DNA recovery in Examples 1 to 10 of Example A. FIG. 16 is a graph showing the relationship between D ₅₀ of the porous silica particles II and the DNA recovery in Examples 12 to 26 of Example B. FIG. FIG. 16 is a graph showing the relationship between D ₉₀ / D ₁₀ of the porous silica particles II and the DNA recovery in Examples 12 to 26 of Example B. FIG. FIG. 16 is a graph showing the relationship between the pore volume of porous silica particles II and the DNA recovery in Examples 12 to 26 of Example B. FIG. FIG. 16 is a graph showing the relationship between the average pore size of porous silica particles II and the DNA recovery in Examples 12 to 26 of Example B. FIG. FIG. 16 is a graph showing the relationship between the specific surface area (SSA) of porous silica particles II and the DNA recovery in Examples 12 to 26 of Example B. FIG.

The meanings of the following terms in the present specification are as follows.
“D ₅₀ ” is a particle diameter at a point of 50% in a cumulative volume distribution curve where the total volume of particle size distribution on a volume basis is 100%, that is, a volume based cumulative 50% diameter.
“D ₁₀ ” is the particle diameter at a point of 10% in the cumulative volume distribution curve where the total volume of particle size distribution on a volume basis is 100%, that is, the volume based cumulative 10% diameter.
“D ₉₀ ” is a particle diameter at a point of 90% in the cumulative volume distribution curve where the total volume of particle size distribution on a volume basis is 100%, that is, a volume-based cumulative 90% diameter.

The “particle size distribution on a volume basis” is obtained from the frequency distribution and the cumulative volume distribution curve measured by a laser scattering particle size distribution measuring apparatus (for example, a laser diffraction / scattering type particle size distribution measuring apparatus etc.). The measurement is carried out by sufficiently dispersing the powder in an aqueous medium by ultrasonic treatment or the like.
The “pore volume determined by the BJH method” is a pore volume determined from the adsorption isotherm by the BJH method. In the measurement of the adsorption isotherm, nitrogen gas is used as the adsorption gas.
The "average pore size determined by the BJH method" is an average pore size determined from the adsorption isotherm by the BJH method. In the measurement of the adsorption isotherm, nitrogen gas is used as the adsorption gas.
The “BJH method” is a method of determining mesopore diameter distribution by Barrett, Joyner and Halenda, which is one of methods for determining pore diameter distribution from adsorption isotherm.

The "pore volume determined by mercury porosimetry" is a pore volume measured using a mercury porosimeter.
The "average pore size determined by mercury porosimetry" is an average pore size measured using a mercury porosimeter.
In the measurement of the pore volume or average pore diameter by a mercury porosimeter, a physical property value of mercury is a contact angle of 140 °, a surface tension of 480 dynes / cm, and a density of 13.5335 g / mL. Measurement conditions are a pressure of 50 mm Hg, an evacuation time of 5 minutes, an injection pressure of 1.49 psi, and an equilibration time of 10 seconds.
The "specific surface area" is a specific surface area determined from an adsorption isotherm by a BET (Brunauer, Emmet, Teller) method. In the measurement of the adsorption isotherm, nitrogen gas is used as the adsorption gas.
"Silica purity" is the ratio of the mass of the silica (SiO ₂₎ with respect to the total weight of the porous silica particles. Silica purity is measured by the quasi-drug raw material standard 2006 "anhydrous silicic acid" determination method.
“-” Indicating a numerical range is used in the meaning including the numerical values described before and after that as the lower limit value and the upper limit value.

[Method of recovering nucleic acid]
The nucleic acid recovery method of the present invention comprises the following binding and elution steps. After the binding step, the following washing step may be further included before the elution step.
Bonding step: a step of binding a nucleic acid to porous silica particles I or porous silica particles II to form a complex.
Washing step: a step of washing the complex.
Elution step: a step of eluting and recovering nucleic acid from the complex.

(Porous silica particles)
In the method for recovering nucleic acid of the present invention, porous silica particles are used as a carrier for binding nucleic acid. Porous silica particles have silica present on their surface. The porous silica particles may further contain components other than silica (hereinafter, also referred to as other components).

90 mass% or more is preferable, as for the silica purity of porous silica particle, 95 mass% or more is more preferable, and 98 mass% or more is especially preferable. The upper limit of the silica purity is not particularly limited, and may be 100% by mass.
When other components are contained in the porous silica particles as described in Patent Document 1, the other components may be eluted from the porous silica particles at the time of recovery of the nucleic acid to adversely affect the measurement. Moreover, the magnetic silica particle which coat | covered the surface of the magnetic body with porous silica is expensive for a raw material and manufacture, and is expensive.
If the silica purity is at least the above lower limit, even if other components are eluted from the porous silica particles at the time of recovery of nucleic acid, the measurement is unlikely to adversely affect the measurement and is inexpensive.
The porous silica particles are preferably not magnetic silica particles in terms of cost and chemical resistance. For example, the whole particle may be a particle which consists of porous silica.

In the particle size distribution on a volume basis of the porous silica _{particles, D 50} is 6 ~ 50 [mu] m, preferably 6 ~ 45μm, 7 ~ 30μm are particularly preferred. If D ₅₀ is within the above range, the nucleic acid recovery rate is excellent.
D ₉₀ / _{D 10} is preferably 10 or less, more preferably 8 or less, particularly preferably 6 or less. If D ₉₀ / _{D 10} is less than the upper limit, the nucleic acid recovery more excellent. The lower limit of D ₉₀ / D ₁₀ is not particularly limited, and may be 1, for example.
D ₉₀ is, for example, 10 to 100 μm, and D ₁₀ is, for example, 1 to 50 μm.

Pore volume of porous silica particles I is a pore volume determined by the BJH method, the range is 0.3 ~ ^5cm 3 / g, preferably 0.4 ~ ^{3cm 3} / g, 0.5 Particular preference is given to ̃2.5 cm ³ / g.
Further, the pore volume of the porous silica particles II is a pore volume determined by mercury porosimeter, the range is 0.6 ~ ^5cm 3 / g, preferably 0.6 ~ ^4cm 3 / g, 0 6 to 2.5 cm ³ / g are particularly preferred. If the pore volume is equal to or more than the lower limit value, the nucleic acid recovery rate is excellent. If the pore volume is equal to or less than the upper limit value, the washability is excellent.

The average pore size of the porous silica particles I is an average pore size determined by the BJH method, and the range is 60 to 500 Å, preferably 60 to 450 Å, and particularly preferably 90 to 450 Å. If the average pore diameter of the porous silica particles I is not less than the above lower limit value, the nucleic acid recovery rate is excellent. If the average pore diameter is equal to or less than the upper limit value, the recovery rate is excellent even for short nucleic acids.
The average pore size of the porous silica particles II is an average pore size determined by a mercury porosimeter, and the range is 200 to 5000 Å, preferably 200 to 4000 Å, and particularly preferably 200 to 2500 Å. If the average pore diameter of the porous silica particles II is not less than the lower limit value and not more than the upper limit value, the nucleic acid recovery efficiency is excellent.

The specific surface area of porous silica particles I is a specific surface area as determined by BET method is preferably 150 ~ 1000m ² / g, more preferably 150 ~ 800m ² / g, particularly preferably 170 ~ 650m ² / g.
The specific surface area of porous silica particles II is a specific surface area as determined by the BET method, preferably 8 ~ 400m ² / g, more preferably 8 ~ 350m ² / g, particularly preferably 8 ~ 300m ² / g . When the specific surface area is equal to or more than the lower limit value, the nucleic acid recovery rate is more excellent. When the specific surface area is equal to or less than the upper limit value, the washability is excellent.

As the porous silica particles, those having desired properties (D ₅₀ , pore volume, average pore diameter, etc.) can be appropriately selected and used from commercially available porous silica particles.

(Nucleic acid)
Nucleic acid is a generic term for DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).
The type of nucleic acid to be bound to the porous silica particle is not particularly limited, and may be DNA, RNA, or a mixture thereof. The type and structure of DNA and RNA are not particularly limited. For example, the DNA may be single stranded DNA or double stranded DNA.
The nucleic acid may be nucleic acid derived from a eukaryote (animal, plant, insect, nematode, yeast, mold etc.) or nucleic acid derived from a prokaryote (bacteria such as E. coli, Bacillus subtilis, cyanobacteria, mycoplasma etc.) It may be a nucleic acid derived from an inanimate substance (virus, chloroplast, mitochondria, etc.).

The length of the nucleic acids is preferably 10 ^{~ 10} 11 ^bp, more preferably from ^{10 5 ~} 10 11 ^bp, particularly preferably ¹⁰ 5 ~ 10 9 bp.
The length of the nucleic acid targeted by the nucleic acid purification kit using a conventional silica membrane is usually 10 ⁶ bp or less. The longer the nucleic acid, the lower the recovery, especially at 10 ⁵ bp or more, the lower the recovery. It is considered that this is because as the nucleic acid becomes longer, the nucleic acid becomes strongly bound to the carrier and becomes difficult to elute.
According to the recovery method of the present invention, even a nucleic acid having a length of 10 ⁵ bp or more can be recovered with an excellent recovery rate. Therefore, the utility of the present invention is particularly high when the length of the nucleic acid is 10 ⁵ bp or more. If the length of the nucleic acid is 10 ¹¹ bp or less, the nucleic acid recovery rate is more excellent.
The naturally occurring nucleic acid of 10 ⁵ to 10 ¹¹ bp in length is DNA.

(Bonding process)
In the binding step, nucleic acids are bound to the porous silica particles to form a complex.
The bonding step can be carried out using known methods, except that the porous silica particles are used as a carrier.
As an example of a method for binding nucleic acid to porous silica particles, a sample containing nucleic acid and a binding solution are mixed to obtain a mixed solution (hereinafter also referred to as a first mixed solution), and the first mixed solution and the pores are obtained. And a method of contacting with the porous silica particles.

Usually, a biological sample is used as a sample containing nucleic acid. Examples of biological samples include blood, serum, puncture fluid, airway wiping fluid, urine, feces, pus, hair, nails and the like.

The binding solution is a solution having an action of binding a nucleic acid to the porous silica particles. The action of the binding solution causes the nucleic acids in the sample to bind to the porous silica particles.
Preferably, a chaotropic substance solution is used as the binding solution. Thus, in the binding step, preferably the nucleic acids are bound to the porous silica particles in the presence of a chaotropic substance solution.

The chaotropic substance generates chaotropic ions in a solvent such as water. The action of the chaotropic ion facilitates binding of the nucleic acid to the porous silica particle. In addition, chaotropic ions can change the secondary, tertiary or quaternary structure without changing the primary structure of proteins or nucleic acids. Therefore, by mixing the biological sample and the chaotropic substance solution, cells or the like containing the nucleic acid in the biological sample can be destroyed and the nucleic acid can be eluted. Therefore, the chaotropic substance solution also functions as an eluent for eluting nucleic acids from cells or the like in a biological sample.

Examples of chaotropic substances include guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, sodium perchlorate, urea and the like. The chaotropic substances may be used alone or in combination of two or more. Among these, guanidine thiocyanate or guanidine hydrochloride is preferable in view of the strength of the chaotropic effect and the magnitude of the inhibitory effect on nucleolytic enzymes such as ribonuclease.

The concentration of the chaotropic substance in the binding solution may be set appropriately depending on the chaotropic substance, as long as sufficient chaotropic effect can be obtained, preferably 1.0 to 8.0 M, and the chaotropic substance is guanidine hydrochloride. 4.0 to 7.5 M is preferable, and 3.0 to 5.5 M is preferable when the chaotropic substance is guanidine thiocyanate.

The binding solution preferably contains a buffer. The buffer is not particularly limited as long as it is generally used, and is not particularly limited, but one having a buffer ability near neutral, ie, pH 5.0 to 9.0 is preferable, for example, Tris-HCl buffer, TE Buffers (containing Tris and ethylenediaminetetraacetic acid (EDTA)), sodium tetraborate-hydrochloric acid buffer, potassium dihydrogenphosphate-sodium tetraborate buffer and the like can be mentioned.
The buffer concentration of the binding solution is preferably 1 to 500 mM in terms of solid content. The pH of the binding solution is preferably 6.0 to 9.0. The pH is the value at 25 ° C.

The binding solution may further contain a surfactant for the purpose of breaking down cell membranes and denatured proteins contained in the cells. The surfactant is not particularly limited as long as it is generally used for nucleic acid extraction from cells and the like, and examples thereof include polyoxyethylene alkyl phenyl ether surfactant (for example, triton surfactant), poly Nonionic surfactants such as oxyethylene sorbitan fatty acid ester surfactants (for example, Tween surfactants), anionic surfactants such as N-lauroyl sarcosine sodium, and the like can be mentioned. One surfactant may be used alone, or two or more surfactants may be used in combination.
The surfactant concentration of the binding solution is preferably 1 to 500 mM, for example, in the case of a nonionic surfactant.

The binding solution may further contain a reducing agent in order to denature and inactivate proteins contained in the sample, particularly ribonuclease. The reducing agent is not particularly limited as long as it is generally used for nucleic acid extraction from cells and the like, and examples thereof include 2-mercaptoethanol, dithiothreitol and the like.

The alcohol may be further mixed when mixing the sample and the binding solution. Alcohol can promote the insolubilization of nucleic acids. Examples of the alcohol include ethanol, propanol, isopropanol, butanol and the like.
The binding solution may contain an alcohol beforehand.

When alcohol is mixed with the sample and the binding solution, the amount of alcohol mixed with the sample and the binding solution is, for example, an amount such that the content of alcohol in the first mixture is 10 to 90% by mass with respect to the sample It is.
The temperature condition at the time of mixing is, for example, 4 to 80.degree. The mixing time varies depending on the mixing method, but is, for example, 0.1 to 60 minutes.

Before contacting the first mixed solution with the porous silica particles, the first mixed solution is subjected to pretreatment such as centrifugation for reduction of components other than nucleic acids (solid impurities and the like). It is also good. The conditions for centrifugation are, for example, 100 to 20000 rpm and 0.1 to 60 minutes.

As a method of contacting the first mixed solution with the porous silica particles, for example, the first mixed solution and the porous silica particles are mixed to obtain a mixed solution (hereinafter also referred to as a second mixed solution). A method of recovering porous silica particles (that is, composite) from the liquid mixture (hereinafter, also referred to as method (1)), or a flowable container (for example, a porous member in which porous silica particles are retained so as not to flow out of the container) A method (hereinafter, also referred to as method (2)) or the like in which the first mixed solution is passed through a column or the like can be mentioned.
The contact temperature at the time of contacting the first mixed solution with the porous silica particles is, for example, 4 to 80 ° C., and the contact time is, for example, 0.1 to 60 minutes.

In the method (1), the amount of porous silica particles mixed with the first liquid mixture is preferably 2 to 200 parts by mass with respect to 100 parts by mass of the first liquid mixture.
The time from when the first liquid mixture and the porous silica particles are mixed to when the porous silica particles are recovered from the second liquid mixture corresponds to the contact time. During this time, the second mixed liquid may be stirred or the like.
As a method of recovering porous silica particles from the second mixed solution, various solid-liquid separation methods such as centrifugation and filtration can be used. As an example, the second mixed solution is put in the upper part of the container divided up and down by a filter, and it is made to flow by centrifugation, suction or the like, and the porous silica particles remaining on the filter are recovered.

The method (2) may be, for example, a method in which the first mixed solution is put in the container, held for an arbitrary time (contact time), and then discharged from the container.
The amount of the first liquid mixture contained in the container is, for example, preferably 50 to 5000 parts by mass with respect to 100 parts by mass of the porous silica particles.
While holding the first mixed solution in the container, stirring or the like may be performed.
The method for discharging the first mixed solution from the container is not particularly limited, and examples thereof include centrifugation and suction.

(Washing process)
In the washing step, the complex in which the porous silica particles and the nucleic acid are bound obtained in the binding step is washed.
Components other than nucleic acids (proteins, saccharides, lipids, etc.) may be attached to the complex. By washing the complex, components other than nucleic acids can be reduced. The washing may be performed once or twice or more.

The washing solution used for washing is not particularly limited as long as it does not elute nucleic acid from the complex and can promote the detachment of components other than nucleic acid. As a washing | cleaning liquid, a chaotropic substance solution, alcohol, its aqueous solution etc. are mentioned, for example.
The chaotropic substance solution may be the same as that described above for the binding solution, and examples include 4.0 to 7.5 M guanidine hydrochloride solution. The chaotropic substance solution may contain a buffer, a surfactant, a reducing agent and the like.
As the above-mentioned alcohol, ethanol, propanol, isopropanol, butanol and the like are preferable, and ethanol is particularly preferable. The alcohol concentration of the alcohol or its aqueous solution is preferably 40 to 100% by mass, and more preferably 60 to 100% by mass. If the alcohol concentration is above the lower limit value, it is difficult for the nucleic acid to elute.

As the washing solution, a chaotropic substance solution and an alcohol or an aqueous solution thereof may be used in combination. For example, washing with a chaotropic substance solution may be followed by washing with an alcohol or an aqueous solution thereof.
As the washing solution, two or more kinds of alcohols having different alcohol concentrations or their aqueous solutions may be used in combination.

The method of washing the complex is not particularly limited. For example, a method of recovering a complex from a third liquid mixture by obtaining a mixed liquid (hereinafter also referred to as a third mixed liquid) by mixing the complex and the washing liquid recovered in the method (1) in the bonding step Hereinafter, a method (hereinafter, also referred to as method (2 ′)), etc., in which a cleaning solution is passed through a container after the first mixed solution is discharged in the method (2) in the method (1 ′) Can be mentioned.
The temperature condition at the time of washing is preferably 4 to 80.degree.
The washing time per wash is preferably 0.1 to 60 minutes.

In the method (1 ′), the amount of the washing solution mixed with the complex in one washing is preferably 10 to 10000 parts by mass with respect to 100 parts by mass of the complex.
The time from when the complex and the washing liquid are mixed to when the complex is recovered from the third mixed solution corresponds to the washing time. During this time, the second mixed liquid may be stirred or the like.
As a method of recovering the complex from the third mixed solution, the same method as the method of recovering porous silica particles from the second mixed solution can be mentioned.

The method (2 ′) may be, for example, a method of putting a cleaning solution into the container, holding it for an arbitrary time (cleaning time), and then discharging it from the container.
The amount of the washing solution contained in the container is preferably 10 to 10000 parts by mass with respect to 100 parts by mass of the complex.
While holding the cleaning solution in the container, stirring or the like may be performed.
As a method of discharging the cleaning liquid from the container, the same method as the method of discharging the first mixed liquid from the container may be mentioned.

After washing, if necessary, the complex may be dried. As a drying method, a thermostat etc. are mentioned, for example. As the drying conditions, preferably, the drying temperature is 30 to 80 ° C., and the drying time is 0.1 to 3000 minutes.

(Elution step)
In the elution step, the nucleic acid is eluted from the complex after the binding step or the washing step to recover the nucleic acid. The elution may be performed once or twice or more.
The elution step can be carried out using a known method. For example, there is a method in which the complex is brought into contact with the eluate, and then the eluate is recovered.
The eluate is a solution having an action of eluting nucleic acids bound to the porous silica particles. The action of the eluate elutes the nucleic acid from the complex to obtain an eluate containing the nucleic acid.
As an elution solution, for example, water, TE buffer (10 mM Tris-hydrochloride, 1.0 mM EDTA, pH 8.0) and the like can be mentioned.

There is no particular limitation on the method of bringing the complex into contact with the elution solution and then recovering the elution solution. For example, the complex recovered in the method (1) in the binding step or the complex washed in the method (1 ′) in the washing step and the eluate are mixed and a mixed solution (hereinafter also referred to as a fourth mixed solution) ) And removing the complex (that is, porous silica particles) from which the nucleic acid is eluted from the fourth mixture (hereinafter, also referred to as method (1 ′ ′)), in the binding step in the method (2) A method (hereinafter, also referred to as method (2 ′ ′)) in which the eluate is passed through a container after discharging the first liquid mixture or a container after discharging the washing liquid by the method (2 ′) in the washing step Can be mentioned.
The contact temperature at the time of contact between the complex and the eluate is preferably 4 to 80 ° C., and the contact time is preferably 0.1 to 60 minutes.

In the method (1 ′ ′), the amount of eluate mixed with the complex is, for example, 10 to 10000 parts by mass with respect to 100 parts by mass of the complex.
The time from when the complex and the eluate are mixed to when the porous silica particles are recovered from the fourth mixture corresponds to the contact time. During this time, the fourth liquid mixture may be stirred or the like.
As a method of removing porous silica particles from the fourth mixed solution, various solid-liquid separation methods such as centrifugation and filtration can be used.

Method (2 ′ ′) may be, for example, a method of putting the eluate into the container, holding it for an arbitrary time (contact time), and then discharging it from the container.
The amount of eluate contained in the container is, for example, 10 to 10000 parts by mass with respect to 100 parts by mass of the complex.
While holding the eluate in the container, stirring or the like may be performed.
As a method of discharging the eluate from the container, the same method as the method of discharging the first mixed liquid from the container may be mentioned.

In the present invention, as the nucleic acid binding _{carrier, D 50} is 6 ~ 50 [mu] m, a pore volume determined by the BJH method is 0.3 ~ ^5cm 3 / g, average pore diameter as determined by the BJH method Porous silica particles having a diameter of 60 to 500 Å, or having a D ₅₀ of 6 to 50 μm, a pore volume of 0.6 to 5 cm ³ / g as determined by mercury porosimetry, and an average pore diameter as determined by mercury porosimetry Since porous silica particles of 200 to 5000 Å are used, nucleic acids can be recovered with high recovery rate even when the nucleic acids are long. Further, the nucleic acid can be recovered only by binding the nucleic acid to the porous silica particle and eluting it, and the operation is simple.
In the porous silica particle, the binding efficiency of the nucleic acid to the porous silica particle in the binding step and the compounding in the elution step, because the D ₅₀ , the pore volume and the average pore diameter are in the above ranges Because the elution efficiency of nucleic acid from the body is both excellent, nucleic acid can be recovered with high recovery rate.
As a reason for the high binding efficiency, it is considered that a part of the nucleic acid easily fits into the pores opened on the surface of the porous silica particle. The reason why the elution efficiency is high is that the contact area with the nucleic acid is relatively reduced by the pores opened on the surface of the porous silica particles, and the individual pores are opened at a plurality of locations, When the nucleic acid fitted in the pore is detached, it is conceivable that the elution solution wraps around from inside the pore and the nucleic acid is easily detached.

[Nucleic acid recovery kit]
The nucleic acid recovery kit of the present invention comprises a first container containing the porous silica particles described above, and a second container containing a binding solution for binding nucleic acids to the porous silica particles.
The nucleic acid recovery kit of the present invention may further include a third container containing an eluate for eluting the nucleic acid from the complex in which the porous silica particles and the nucleic acid are bound, as necessary.
The nucleic acid recovery kit of the present invention may further include a fourth container containing a washing solution for washing the complex in which the porous silica particles and the nucleic acid are bound, as necessary.

The nucleic acid recovery kit of the present invention can be used in the above-described method of recovering a nucleic acid of the present invention. The bonding step can be carried out, for example, using porous silica particles contained in a first container and a binding liquid contained in a second container.
If the third container is further provided, the elution step can be further performed using the eluate contained in the third container.
When the fourth container is further provided, the washing step can be further performed using the washing liquid stored in the fourth container.

When the porous silica particles contained in the first container are used in the bonding step, the porous silica particles may be removed from the first container, and the bonding step may be performed in another container. The bonding step may be performed in the first container while the particles are contained in the first container.

The binding solution contained in the second container includes the binding solution mentioned in the binding step, and a chaotropic substance solution is preferable.
When the binding solution contained in the second container is used in the binding step, it may be diluted with a solvent such as water.

Examples of the eluate contained in the third container include the eluate mentioned in the elution step.
When using the eluate stored in the third container in the elution step, it may be diluted with a solvent such as water.

The washing liquid contained in the fourth container includes the washing liquid mentioned in the washing step.
When using the washing | cleaning liquid accommodated in the 4th container at the washing | cleaning process, you may dilute with solvent, such as water.

Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited to these examples.

Example A
With respect to the following porous silica particles 1 to 10, the particle size distribution, pore volume, average pore diameter and specific surface area were determined according to the following evaluation method, and nucleic acids (DNA) were further obtained based on Examples 1 to 11 below. The recovery rate of Of Examples 1 to 11 of Example A, Examples 1 to 6 are Examples, and Examples 7 to 11 are Comparative Examples.
The evaluation methods and materials used in each example are shown below.

〔Evaluation method〕
(Particle size distribution)
The porous silica particles are sufficiently dispersed in water by ultrasonication, and measurement is performed using a laser diffraction / scattering particle size distribution measuring device (MT-3300EX, manufactured by Nikkiso Co., Ltd.) to obtain frequency distribution and cumulative volume distribution curve. Volume-based particle size distribution was obtained. From the cumulative volume distribution curve obtained was determined _{_{_{_{D 10, D 50, D 90}}}} , D 90 / D 10.

(Measurement of pore volume and average pore diameter by BJH method)
An adsorption isotherm was measured with a pore distribution measuring device (manufactured by mirometrics, 3FLEX) using nitrogen gas as an adsorption gas.
Pore volume (BJH adsorption integrated pore volume) and BJH method from the 34 points of relative pressure P / P ₀ in the adsorption isotherm from 0.01 to 0.995 using analysis software attached to the pore distribution measuring device The average pore size (BJH average pore diameter) was determined.
(Specific surface area)
Further, the specific surface area was determined by the BET method from 10 points at a relative pressure P / P ₀ of 0.05 to 0.30.

〔material〕
Porous silica particle 1: manufactured by AGC S.I. Tech., Model number: L-52.
Porous silica particle 2: manufactured by AGC S.I. Tech., Model number: L-203.
Porous silica particle 3: manufactured by AGC S.I. Tech., Model number: EP-DM-20-120A.
Porous silica particles 4: manufactured by Osaka Soda, model number: SP-120-20P.
Porous silica particles 5: manufactured by Osaka Soda, model number: IR-60-25 / 40.
Porous silica particles 6: manufactured by Fuji Silysia, model number: SMB100-20.
Porous silica particles 7: manufactured by AGC S.I. Tech., Model number: Sun Lovely.
Porous silica particles 8: manufactured by AGC S.I. Tech., Model number: NP-200.
Porous silica particles 9: manufactured by Fuji Silysia, model number: BW-820MH.
Porous silica particles 10: manufactured by Fuji Silysia, model number: MB100-75 / 200.

Binding solution: Adsorption buffer (Composition: 50 mM Tris-hydrochloride, 5.0 M guanidine thiocyanate, 20 mM EDTA, 1.2% by mass polyethylene) attached to the nucleic acid extraction kit (Qiagen, trade name: DNeasy Blood tisue kit) Glycol mono-p-isooctyl phenylene ether).
Washing solution 1: Washing buffer 1 attached to the nucleic acid extraction kit.
Washing solution 2: Washing buffer 2 attached to the nucleic acid extraction kit.
Eluent: Elution buffer supplied with the above nucleic acid extraction kit.
Human genome solution: manufactured by Promega, product name human genomic DNA G3041, containing DNA approximately 3 × 10 ⁹ base pairs in length, DNA concentration 200 μg / mL.
E. coli genome solution: prepared by treating a culture solution of E. coli DH5α using Sigma-Aldrich, product name GenElute Plasmid Miniprep Kit, containing DNA of about 4.65 × 10 ⁶ base pairs in length, 50 μg / DNA concentration mL.

Tip column: Two pipette tips (manufactured by BioPointe Scientific, trade name: Pipette tip 1250 μl) are each cut into two along the circumferential direction, and the portion including the tip of the pipette tip (hereinafter also referred to as “part A”) And a portion including the rear end of the pipette tip (hereinafter, also referred to as “portion B”). At this time, the cutting position (the distance from the tip to the cutting position) of each of the two pipette tips was changed. Because the pipette tip has a smaller diameter toward the tip, the inner diameters at the cutting positions of the two pipette tips are different.
The opening at the rear end of the longer part of the two parts A obtained from each of the two pipette tips (the one with the larger internal diameter at the cutting position) was covered with a 1 μm pore size polytetrafluoroethylene (PTFE) filter . In that state, the part A is inserted from the tip end side through the opening at the rear end of the longer part (smaller inside diameter of the cutting position) of the two parts B, the rear end part of the part A and the filter The outer edge portion of the hook was locked to the tip end of the portion B. This was used as a tip column.

[Examples 1 to 10]
Using a human genome solution or an E. coli genome solution as a sample, the nucleic acid was recovered from the sample according to the following procedure, and the recovery rate of nucleic acid (DNA) was determined. The results are shown in Table 1.
First, 220 μL of a sample, 200 μL of a binding solution and 200 μL of 100% by mass ethanol were added to a 1.5 mL Eppendorf tube, and mixed to obtain a first mixed solution.
Next, 20 mg of porous silica particles shown in Table 1 were added to 620 μL of the first mixed solution, and mixed to obtain a second mixed solution.
Next, the whole amount of the second mixed solution is transferred to the chip column, and the porous silica particles are collected on the filter of the chip column by centrifuging under the conditions of 20 ° C. for 2 minutes at 3500 rpm using a centrifuge. The
Next, the filtrate was discarded, 500 μL of washing solution 1 was added to the tip column, and centrifuged under the same conditions as described above. Next, the filtrate was discarded, 500 μL of washing solution 2 was added to the tip column, and centrifuged under the same conditions as described above. Thereafter, the filtrate was discarded and centrifuged at 3500 rpm for 7 minutes at 20 ° C.
Next, 200 μL of the eluate was added to the chip column, allowed to stand for 1 minute, and centrifuged under conditions of 3500 rpm, 2 minutes, and 20 ° C. The obtained filtrate was used as a nucleic acid extract.

The DNA concentration in the nucleic acid extract was measured according to the protocol using an assay kit (manufactured by invitrogen, trade name Qubit assay). Specifically, 199 μL of Working solution was added to 1 μL of nucleic acid extract, vortexed for 2 to 3 seconds, reacted at room temperature for 2 minutes, and then the DNA concentration was measured with a fluorometer (invitrogen, trade name Qubit Fluorometer) .
The mass W ₁ (g) of the DNA in the nucleic acid extract was calculated from the measured DNA concentration. From this value and the mass W ₂ (g) of DNA in the used sample (220 μL), the DNA recovery rate was determined by the following equation.

DNA recovery

_{_{(%) = W 1 / W}} 2 × 100

[Example 11]
Using a commercially available nucleic acid extraction kit (manufactured by Qiagen, trade name: DNeasy Blood tisue kit) using a human genome solution or E. coli genome solution as a sample, the nucleic acid is extracted according to the protocol and a nucleic acid extract I got
With respect to the obtained nucleic acid extract, in the same manner as in Examples 1 to 10, the nucleic acid was recovered from the sample by the following procedure, and the recovery rate of the nucleic acid (DNA) was determined. The results are shown in Table 1.

The D ₁₀ , D ₅₀ , D ₉₀ , D ₉₀ / D ₁₀ , silica (SiO ₂ ) purity, specific surface area, pore volume, and average pore diameter of the porous silica particles used in Examples 1 to 10 are shown in Table 1. . In addition, although only the porous silica particles 3 and 8 were measured about the silica purity, the silica purity of the other porous silica particle is all considered to be 98 mass% or more.
Also, in Examples 1-10, taken ordinate DNA recovery rate when the human genome solution as a sample, _D 50 of the porous silica _particles, D 90 _{/ D 10} pore volume, average pore diameter or the specific surface area (SSA Figures 1 to 5 show graphs in which the horizontal axis is taken). In each graph, the results of Examples 1 to 6 are shown as Examples, and the results of Examples 7 to 10 are shown as Comparative Examples.

In Examples 1 to 6, the recovery of nucleic acid was high.
On the other hand, in Example 7, since the porous silica particles had a D ₅₀ of less than 6 μm and a pore volume of less than 0.3 cm ³ / g, the nucleic acid recovery rate was poor. In Example 8, since the pore volume of the porous silica particles was less than 0.3 cm ³ / g and the average pore diameter was less than 60 Å, the recovery rate of nucleic acid was inferior. In Example 9, since the D50 of the porous silica particles is greater than ₅₀ μm, the pore volume is less than 0.3 cm ³ / g, and the average pore diameter is less than 60 Å, the nucleic acid recovery rate is poor. In Example 10, the recovery rate of the nucleic acid was poor because the D ₅₀ of the porous silica particles was larger than ₅₀ μm.

Example B
The particle size distribution, pore volume, average pore diameter and specific surface area of the following porous silica particles 12 to 26 were determined according to the following evaluation method, and nucleic acids (DNA) were further obtained based on Examples 12 to 27 of Example B below. The recovery rate of Of the examples 12 to 27 of the example B, the examples 12 to 21 are examples and the examples 22 to 27 are comparative examples.
The evaluation methods and materials used in each example are shown below.

〔Evaluation method〕
(Particle size distribution)
With respect to the porous silica particles 12 to 26, in the same manner as in Example A, a volume-based particle size distribution was obtained. From the cumulative volume distribution curve obtained was determined _{_{_{_{D 10, D 50, D 90}}}} , D 90 / D 10.

(Specific surface area)
An adsorption isotherm was measured with a pore distribution measuring device (manufactured by mirometrics, 3FLEX) using nitrogen gas as an adsorption gas.
The specific surface area was determined by the BET method from ten points with a relative pressure P / P ₀ of 0.05 to 0.30 in the adsorption isotherm using analysis software attached to the pore distribution measuring apparatus.

(Measurement of pore volume and average pore size by mercury porosimetry)
Pore volume and average pore size were measured using a mercury porosimeter (manufactured by Mirometrics, AutoPore IV 9500).
The physical properties of mercury used were a contact angle of 140 °, a surface tension of 480 dynes / cm, and a density of 13.5335 g / mL. The measurement conditions were a pressure of 50 mmHg, an evacuation time of 5 minutes, an injection pressure of 1.49 psi, and an equilibration time of 10 seconds.

〔material〕
Porous silica particles 12: manufactured by AGC S.I. Tech., Model number: L-52.
Porous silica particles 13: manufactured by AGC S.I. Tech., Model number: EP-DM-10-1000AW.
Porous silica particles 14 AG manufactured by SSI Tech, model number: EP-DM-10-700AW.
Porous silica particles 15 manufactured by AGC SITEC Co., Ltd., model number: EP-DM-20-1000AW.
Porous silica particle 16 AG manufactured by SSI Tech, model number: L-203.
Porous silica particles 17 manufactured by Osaka Soda, model number: SP-1000-10.
Porous silica particles 18: manufactured by Osaka Soda, model number: SP-1000-20.
Porous silica particles 19 manufactured by Osaka Soda, model number: SP-2000-20.
Porous silica particles 20 manufactured by Fuji Silysia, model number: SMB1000-10.
Porous silica particle 21 manufactured by Fuji Silysia, model number: SMB1000-20.
Porous silica particles 22: manufactured by AGC S.I. Tech., Model number: EP-DF-5-1000AW2.
Porous silica particle 23 AGC SCITECH Co., Ltd. make, model number: NP-200.
Porous silica particle 24 AGC SCITECH Co., Ltd. make, model number: D-75-1000AW.
Porous silica particle 25 AGC SCITECH Co., Ltd. make, model number: D-150-1000AW.
Porous silica particles 26: manufactured by AGC S.I. Tech., Model number: D-250-1000 AW.

The same type as in Example A was used for the binding solution, washing solution 1, washing solution 2, eluate, human genome solution, E. coli genome solution and chip column.

[Examples 12 to 26]
Using a human genome solution or E. coli genome solution as a sample, the nucleic acid was recovered from the sample in the same manner as in Examples 1 to 10 of Example A, and the recovery rate of nucleic acid (DNA) was determined. The results are shown in Table 2.

[Example 27]
Example 27 shows the results of Example 11 in Table 1 of Example A.
With respect to the obtained nucleic acid extract, in the same manner as in Examples 12 to 26, the nucleic acid was recovered from the sample by the following procedure, and the recovery rate of the nucleic acid (DNA) was determined. The results are shown in Table 2.

The D ₁₀ , D ₅₀ , D ₉₀ , D ₉₀ / D ₁₀ , silica (SiO ₂ ) purity, specific surface area, pore volume, and average pore diameter of the porous silica particles used in Examples 12 to 26 are shown in Table 2. . Although only the porous silica particles 13 to 15 and 22 to 23 were measured as to the silica purity, it is considered that the silica purity of the other porous silica particles is also 98 mass% or more.
In Examples 12 to 26, the DNA recovery rate when using a human genome solution as a sample is indicated on the vertical axis as D ₅₀ , D ₉₀ / D ₁₀ , pore volume, average pore diameter or specific surface area (SSA) of porous silica particles. 6 to 10 show graphs in which the abscissa axis is taken). In each graph, the results of Examples 12 to 21 are shown as Examples, and the results of Examples 22 to 27 are shown as Comparative examples.

In Examples 12-21, the recovery of nucleic acid was high.
On the other hand, in Example 22, since the porous silica particles had a D ₅₀ of less than 6 μm, the nucleic acid recovery rate was poor. In Example 23, since the pore volume of the porous silica particles is less than 0.6 cm ³ / g and the average pore diameter is less than 200 Å, the recovery rate of the nucleic acid is poor. In Examples 24 to 26, since the porous silica particles had a D ₅₀ of more than ₅₀ μm, the recovery rate of nucleic acid was poor.
The entire contents of the specification, claims, abstract and drawing of Japanese Patent Application 2017-157978 and Japanese Patent Application 2017-157968 filed on Aug. 18, 2017 are incorporated herein by reference. It is incorporated as a disclosure of the specification of the invention.

Claims

A D 50 of 6 ~ 50 [mu] m particle size distribution based on volume, the pore volume determined by the BJH method is 0.3 ~ 5cm 3 / g, an average pore diameter determined by the BJH method is a 60 ~ 500 Å Binding nucleic acid to porous silica particles to form a complex;
A method for recovering nucleic acid, which comprises eluting and recovering the nucleic acid from the complex.
A D 50 of 6 ~ 50 [mu] m particle size distribution based on volume, the pore volume determined by mercury porosimetry is the 0.6 ~ 5cm 3 / g, average pore diameter is 200 ~ 5000 Å determined by mercury porosimeter Binding nucleic acid to porous silica particles to form a complex;
A method for recovering nucleic acid, which comprises eluting and recovering the nucleic acid from the complex.
The porous D 90 / D 10 of the particle size distribution based on the volume of the silica particles is 10 or less, a method of recovering nucleic acids according to claim 1 or 2.
The method for recovering nucleic acid according to claim 1 or 3, wherein the specific surface area of the porous silica particles is 150 to 1000 m 2 / g.
The method for recovering nucleic acid according to claim 2 or 3, wherein the specific surface area of the porous silica particles is 8 to 400 m 2 / g.
The method for recovering nucleic acid according to any one of claims 1 to 5, wherein the silica purity of the porous silica particles is 90% by mass or more.
The method for recovering nucleic acid according to any one of claims 1 to 6, wherein the nucleic acid is bound to the porous silica particle in the presence of a chaotropic substance solution.
The method for recovering nucleic acid according to any one of claims 1 to 7, wherein the complex is washed before eluting the nucleic acid from the complex.
The method for recovering nucleic acid according to any one of claims 1 to 8, wherein the length of the nucleic acid is 10 5 to 10 11 bp.
A D 50 of 6 ~ 50 [mu] m particle size distribution based on volume, the pore volume determined by the BJH method is 0.3 ~ 5cm 3 / g, an average pore diameter determined by the BJH method is a 60 ~ 500 Å A carrier for nucleic acid binding comprising porous silica particles.
A D 50 of 6 ~ 50 [mu] m particle size distribution based on volume, the pore volume determined by mercury porosimetry is the 0.6 ~ 5cm 3 / g, average pore diameter is 200 ~ 5000 Å determined by mercury porosimeter A carrier for nucleic acid binding comprising porous silica particles.
A D 50 of 6 ~ 50 [mu] m particle size distribution based on volume, the pore volume determined by the BJH method is 0.3 ~ 5cm 3 / g, an average pore diameter determined by the BJH method is a 60 ~ 500 Å A first container containing porous silica particles;
A second container containing a binding solution for binding nucleic acid to the porous silica particles.
A D 50 of 6 ~ 50 [mu] m particle size distribution based on volume, the pore volume determined by mercury porosimetry is the 0.6 ~ 5cm 3 / g, average pore diameter is 200 ~ 5000 Å determined by mercury porosimeter A first container containing porous silica particles;
A second container containing a binding solution for binding nucleic acid to the porous silica particles.
The nucleic acid recovery kit according to claim 12 or 13, wherein the binding solution is a chaotropic substance solution.
The nucleic acid recovery kit according to any one of claims 12 to 14, further comprising a third container containing an eluate for eluting the nucleic acid from a complex in which the porous silica particle and the nucleic acid are bound.
The nucleic acid recovery kit according to any one of claims 12 to 15, further comprising a fourth container containing a washing solution for washing a complex in which the porous silica particle and the nucleic acid are bound.