JP2020191785A - Method of recovering nucleic acid, support for nucleic acid bonding and nucleic acid recovery kit - Google Patents

Abstract

To provide a method of recovering a nucleic acid that can recover a nucleic acid by convenient operation and with high efficiency even when the nucleic acid is long in length, and a support for nucleic acid bonding and a nucleic acid recovery kit suitably used for the recovery method.SOLUTION: A method of recovering a nucleic acid includes causing a nucleic acid to be adsorbed or bonded to a porous silica particle with D50 of volume-based particle size distribution of 6-50 μm, a pore volume by nitrogen adsorption of 0.3-5 cm3/g, and an average pore diameter by nitrogen adsorption of 60-500Å, to form a complex; and causing the nucleic acid to be eluted from the complex for recovery.SELECTED DRAWING: None

Description

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, a kit for proteins (antigens, antibodies, etc.) has been used. However, there are problems such as low accuracy except for some infectious diseases such as influenza, and false positives due to past infections when targeting antibodies. Therefore, in recent years, infectious disease diagnostic kits targeting nucleic acids have been studied. By targeting nucleic acids, improvement in accuracy is expected.
Specimens, which are biological samples, contain various components, and it is necessary to isolate nucleic acids from the samples in order to make a diagnosis.

As a method for isolating nucleic acid from a biological sample, a method is known in which nucleic acid is adsorbed or bound to a carrier containing silica, other components are removed by washing, and then the nucleic acid is eluted from the carrier. Nucleic acid purification kits using this method have already been put into practical use, and silica membranes are widely used as carriers (for example, Non-Patent Documents 1 to 3). Further, in order to simplify and automate the isolation operation, it has been proposed to use magnetic silica particles in which the surface of the magnetic material is coated with porous silica as a carrier (for example, Patent Document 1).

Japanese Unexamined Patent Publication No. 2000-300262

"Nucleic acid purification kit" (catalog), [online], Nippon Genetics Co., Ltd., [Search on June 26, 2017], Internet <URL: http://www.sns-kk.co.jp/data/ pdf / 2012_6 / 2011769944.pdf> "DNA Cleanup" (catalog), [online], Takara Bio Inc., [Search on June 26, 2017], Internet <URL: http://www.takara-bio.co.jp/goods/catalog /pdf/mach2012_15-28.pdf> "Genomic DNA from blood" (catalog), [online], Macherey-Nagel, [Search on June 26, 2017], Internet <URL: http: http: www.mn-net.com /.../8/ ... / Genomic% 20DNA / UM_gDNABlood.pdf>

However, the isolation method of the conventional nucleic acid, not necessarily high nucleic acid extraction rate, especially in the case of 10 5 ^bp or longer nucleic acid, or can not be extracted, the extraction can be extracted ratio was less problems. If the extraction rate is low, it is necessary to perform the amplification process for a long time using an expensive device, the facilities that can be performed are limited, and it takes time.

An object of the present invention is to provide a nucleic acid recovery method capable of recovering nucleic acid with high efficiency by a simple operation even when the nucleic acid length is long, a nucleic acid binding carrier and a nucleic acid recovery kit preferably used in the recovery method. There is.

The present invention provides a nucleic acid recovery method, a nucleic acid binding carrier, and a nucleic acid recovery kit having the following configurations [1] to [12].
[1] Porous medium having a volume-based particle size distribution D ₅₀ of 6 to ₅₀ μm, a pore volume of 0.3 to ⁵ cm ³ / g due to nitrogen adsorption, and an average pore diameter of 60 to 500 Å due to nitrogen adsorption. Adsorb or bind the nucleic acid to the quality silica particles to form a complex,
A method for recovering a nucleic acid, which comprises eluting and recovering the nucleic acid from the complex.
[2] The method for recovering nucleic acid according to [1], wherein D ₉₀ / D ₁₀ of the particle size distribution based on the volume of the porous silica particles is 10 or less.
[3] The method for recovering nucleic acid according to [1] or [2], wherein the specific surface area of the porous silica particles is 150 to 1000 m ² / g.
[4] The method for recovering nucleic acid according to any one of [1] to [3], wherein the silica purity of the porous silica particles is 90% by mass or more.
[5] The method for recovering a nucleic acid according to any one of [1] to [4], wherein the nucleic acid is adsorbed or bound to the porous silica particles in the presence of a chaotropic substance solution.
[6] The method for recovering a nucleic acid according to any one of [1] to [5], wherein the complex is washed before eluting the nucleic acid from the complex.
[7] the length of the nucleic acid is ¹⁰ 5 ^~10 11 bp, one method of recovering nucleic acids of [1] to [6].
[8] Porous medium having a volume-based particle size distribution D ₅₀ of 6 to ₅₀ μm, a pore volume of 0.3 to ⁵ cm ³ / g due to nitrogen adsorption, and an average pore diameter of 60 to 500 Å due to nitrogen adsorption. A carrier for binding nucleic acids composed of quality silica particles.
[9] Porous medium having a volume-based particle size distribution D ₅₀ of 6 to ₅₀ μm, a pore volume of 0.3 to ⁵ cm ³ / g due to nitrogen adsorption, and an average pore diameter of 60 to 500 Å due to nitrogen adsorption. The first container containing the quality silica particles and
A second container containing a binding solution that adsorbs or binds nucleic acid to the porous silica particles, and
Nucleic acid recovery kit comprising.
[10] The nucleic acid recovery kit of [9], wherein the binding solution is a chaotropic substance solution.
[11] The nucleic acid recovery kit according to [9] or [10], further comprising a third container containing an eluate that elutes the nucleic acid from a complex in which the porous silica particles and the nucleic acid are adsorbed or bound.
[12] The nucleic acid recovery kit according to any one of [9] to [11], further comprising a fourth container containing a washing liquid for washing the complex in which the porous silica particles and the nucleic acid are adsorbed or bound.

According to the method for recovering nucleic acid of the present invention, nucleic acid can be recovered with high efficiency by a simple operation even when the length of nucleic acid is long.
Each of the nucleic acid binding carrier and the nucleic acid recovery kit of the present invention can be suitably used for the above recovery method.

Example is a graph showing the relationship between _{D 50} and DNA recovery of porous silica particles in 1 to 10. Is a graph showing the relationship between _D 90 _{/ D 10} and the DNA recovery of porous silica particles in Examples 1 to 10. 3 is a graph showing the relationship between the pore volume of the porous silica particles and the DNA recovery rate in Examples 1 to 10. 3 is a graph showing the relationship between the average pore size of the porous silica particles and the DNA recovery rate in Examples 1 to 10. 3 is a graph showing the relationship between the specific surface area (SSA) of the porous silica particles and the DNA recovery rate in Examples 1 to 10.

The meanings of the following terms in the present specification are as follows.
“D ₅₀ ” is the particle diameter at the point where the cumulative volume distribution curve with the total volume of the particle size distribution on a volume basis as 100% is 50%, that is, the volume-based cumulative 50% diameter.
“D ₁₀ ” is the particle diameter at the point where the total volume of the particle size distribution on a volume basis is 100% in the cumulative volume distribution curve, that is, the cumulative volume on a volume basis of 10%.
“D ₉₀ ” is the particle diameter at the point where the cumulative volume distribution curve with the total volume of the particle size distribution on a volume basis as 100% is 90%, that is, the volume-based cumulative 90% diameter.
The "volume-based particle size distribution" is obtained from the frequency distribution and the cumulative volume distribution curve measured by a laser scattering particle size distribution measuring device (for example, a laser diffraction / scattering particle size distribution measuring device or the like). The measurement is performed by sufficiently dispersing the powder in an aqueous medium by ultrasonic treatment or the like.
The "pore volume due to nitrogen adsorption" (hereinafter, also simply referred to as "pore volume") is the pore volume obtained from the adsorption isotherm by the BJH method. Nitrogen gas is used as the adsorption gas in the measurement of the adsorption isotherm.
The "average pore diameter due to nitrogen adsorption" (hereinafter, also simply referred to as "average pore diameter") is the average pore diameter obtained from the adsorption isotherm by the BJH method. Nitrogen gas is used as the adsorption gas in the measurement of the adsorption isotherm.
The "BJH method" is a method for determining the mesopore size distribution by Barrett, Joiner and Hallenda, which is one of the methods for obtaining the pore size distribution from the adsorption isotherm.
The "specific surface area" is the specific surface area obtained from the adsorption isotherm by the BET (Brunauer, Emmet, Teller) method. Nitrogen gas is used as the adsorption gas in the measurement of the adsorption isotherm.
"Silica purity" is the ratio of the mass of silica (SiO ₂ ) to the total mass of the porous silica particles. Silica purity is measured by the Sotohara No. 2006 "Silica anhydride" quantification method.
"~" Indicating a numerical range means that the numerical values described before and after the numerical range are included as the lower limit value and the upper limit value.

[Nucleic acid recovery method]
The nucleic acid recovery method of the present invention includes the following binding step and recovery step. After the binding step and before the recovery step, the following cleaning steps can be further included.
Binding step: Nucleic acid is adsorbed or bound to porous silica particles having D ₅₀ of 6 to ₅₀ μm, pore volume of 0.3 to ⁵ cm ³ / g, and average pore diameter of 60 to 500 Å to form a complex. The process of forming.
Cleaning step: A step of cleaning the composite.
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, the porous silica particles are used as a carrier for nucleic acid binding. Porous silica particles contain silica. In addition, silica is present on the surface of the porous silica particles. The porous silica particles may further contain components other than silica.

The silica purity of the porous silica particles is preferably 90% by mass or more, more preferably 95% by mass or more, and particularly preferably 98% by mass or more. The upper limit of silica purity is not particularly limited and may be 100% by mass.
When other components are contained in the porous silica particles as in the invention described in Patent Document 1, other components may be eluted from the porous silica particles at the time of nucleic acid recovery, which may adversely affect the measurement. Further, magnetic silica particles in which the surface of a magnetic material is coated with porous silica are costly as raw materials and production, and are expensive.
When the silica purity is at least the above lower limit value, even if other components are eluted from the porous silica particles during the recovery of nucleic acid, the measurement is less likely to be adversely affected and the cost is low.
The porous silica particles are preferably not magnetic silica particles from the viewpoint of cost and chemical resistance. For example, the entire particle may be a particle made of porous silica.

In the particle size distribution of the porous silica particles on a volume basis, D ₅₀ is 6 to ₅₀ μm, preferably 6 to 45 μm, and particularly preferably 7 to 30 μm. When D ₅₀ is within the above range, the nucleic acid recovery efficiency is excellent.
D ₉₀ / D ₁₀ is preferably 10 or less, more preferably 8 or less, and particularly preferably 6 or less. When D ₉₀ / D ₁₀ is not more than the above upper limit value, the nucleic acid recovery efficiency is more excellent. The lower limit of D ₉₀ / D ₁₀ is not particularly limited and may be 1, for example.
D ₉₀ may be, for example, 10 to 100 μm.
D ₁₀ may be, for example, 1 to 50 μm.

The pore volume of the porous silica particles is 0.3 to ⁵ cm ³ / g, preferably 0.4 to ³ cm ³ / g, and particularly preferably 0.5 to 2.5 cm ³ / g. When the pore volume is at least the above lower limit value, the nucleic acid recovery efficiency is excellent. When the pore volume is not more than the upper limit value, the detergency is excellent.

The average pore size of the porous silica particles is 60 to 500 Å, preferably 60 to 450 Å, and particularly preferably 90 to 450 Å. When the average pore diameter is at least the lower limit, the nucleic acid recovery efficiency is excellent. When the average pore diameter is equal to or less than the upper limit, the recovery rate is excellent even for short nucleic acids.

The specific surface area of porous silica particles is preferably 150~1000m ² / g, more preferably ^{150~800m 2 / g, 170~650m 2 /} g is particularly preferred. When the specific surface area is at least the above lower limit value, the nucleic acid recovery efficiency is more excellent. When the specific surface area is not more than the upper limit value, the detergency is excellent.

As the porous silica particles, those satisfying desired characteristics (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 general term for DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).
The nucleic acid to be adsorbed on the porous silica particles is not particularly limited, and may be DNA, RNA, or a mixture thereof. The types and structures of DNA and RNA are not particularly limited. For example, the DNA may be a single-stranded DNA or a double-stranded DNA.
The nucleic acid may be a nucleic acid derived from a eukaryote (animal, plant, insect, nematode, yeast, mold, etc.) or a nucleic acid derived from a prokaryote (bacteria such as Escherichia coli, bacillus, orchid algae, mycoplasma). It may be a nucleic acid derived from an inanimate object (virus, chloroplast, mitochondria, etc.).

The length of the nucleic acid, typically a ^{10 to 10} 11 ^bp, preferably 10 ^{5 to 10} 11 ^bp, particularly preferably ¹⁰ 5 ~10 9 bp.
The length of the nucleic acid to be targeted by nucleic acid purification kit using a conventional silica membrane is usually 10 6 ^bp or less. As the length of the nucleic acid is longer, especially equal to or greater than 10 5 ^bp, recovery efficiency is low. It is considered that this is because as the length of the nucleic acid increases, the nucleic acid is strongly adsorbed on the carrier and becomes difficult to elute.
The recovery method of the present invention, can be recovered by the recovery efficiency of a length superior even 10 5 ^bp or more nucleic acids. Therefore, highly useful, particularly the present invention when the length of the nucleic acid is 10 5 ^bp or more. When the length of the nucleic acid is 10 ¹¹ bp or less, the nucleic acid recovery efficiency is more excellent.
The naturally occurring nucleic acid having a length of 10 ⁵ to 10 ¹¹ bp is DNA.

(Joining process)
In the binding step, nucleic acids are adsorbed or bound to the porous silica particles to form a complex.
The bonding step can be carried out by using a known method except that the porous silica particles are used as the carrier.
As an example of a method of adsorbing or binding nucleic acid to porous silica particles, a sample containing nucleic acid and a binding solution are mixed to obtain a first mixed solution, and the first mixed solution and the porous silica particles are brought into contact with each other. There is a way to make it.

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

The binding liquid is a liquid having an action of adsorbing or binding nucleic acid to the porous silica particles. By the action of the binding solution, the nucleic acid in the sample is adsorbed or bound to the porous silica particles.
As the binding solution, a chaotropic substance solution is typically used. Therefore, in the binding step, the nucleic acid is typically adsorbed or bound to the porous silica particles in the presence of a chaotropic substance solution.

Chaotropic substances generate chaotropic ions in a solvent such as water. The action of chaotropic ions facilitates adsorption or binding between nucleic acids and silica. In addition, chaotropic ions can change the secondary, tertiary, and quaternary structures without changing the primary structure of proteins and nucleic acids. Therefore, by mixing the biological sample and the chaotropic substance solution, the 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 that elutes nucleic acids from cells and the like in a biological sample.

Examples of the chaotropic substance include guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, sodium perchlorate, urea and the like. One type of chaotropic substance may be used alone, or two or more types may be used in combination. Of these, guanidine thiocyanate and guanidine hydrochloride are preferable because 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 liquid may be appropriately set according to the chaotropic substance as long as a sufficient chaotropic effect can be obtained. It is usually 1.0 to 8.0 M, preferably 4.0 to 7.5 M when the chaotropic substance is guanidine hydrochloride, and 3.0 to 5 when the chaotropic substance is guanidine thiocyanate. .5M is preferable.

The binder preferably contains a buffer. The buffering agent may be generally used and is not particularly limited, but a buffering agent having a buffering ability near neutrality, that is, at pH 5.0 to 9.0 is preferable, and for example, Tris-hydrochloride buffer, TE. Examples thereof include a buffer solution (containing Tris and ethylenediaminetetraacetic acid (EDTA)), a sodium tetraborate-hydrochloride buffer solution, and a potassium dihydrogen phosphate-sodium tetraborate buffer solution.
The buffer concentration of the binder is preferably 1 to 500 mM in terms of solid content.
The pH of the binder is preferably 6.0 to 9.0. pH is a value at 25 ° C.

The binding solution may further contain a surfactant for the purpose of destroying the cell membrane and denaturing the protein contained in the cell. The surfactant may be generally used for extracting nucleic acid from cells or the like, and is not particularly limited, but for example, a polyoxyethylene alkylphenyl ether-based surfactant (for example, a triton-based surfactant), poly. Examples thereof include nonionic surfactants such as oxyethylene sorbitan fatty acid ester-based surfactants (for example, Tween surfactants), and anionic surfactants such as N-lauroyl sarcosine sodium. One type of surfactant may be used alone, or two or more types may be used in combination.
The surfactant concentration of the binder is preferably 1 to 500 mM in the case of a nonionic surfactant, for example.

The binding solution may further contain a reducing agent for the purpose of denaturing and inactivating proteins contained in the sample, particularly ribonuclease. The reducing agent may be generally used for extracting nucleic acid from cells or the like, and is not particularly limited, and examples thereof include 2-mercaptoethanol and dithiothreitol.

Alcohol may be further mixed when mixing the sample and the binder. Alcohol can promote insolubilization of nucleic acids. Examples of the alcohol include ethanol, propanol, isopropanol, butanol and the like.
Alcohol may be contained in the binding liquid in advance.

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 mixed solution is 10 to 90% by mass with respect to the sample. It may be there.
The temperature condition at the time of mixing may be, for example, 4 to 80 ° C.
The mixing time varies depending on the mixing method, but may be, 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 in order to reduce components other than nucleic acids (solid contaminants, etc.). May be good. The conditions for centrifugation may be, for example, 100 to 20000 rpm for 0.1 to 60 minutes.

As a method of contacting the first mixed solution with the porous silica particles, for example, (1) the first mixed solution and the porous silica particles are mixed to obtain a second mixed solution, and the second mixed solution is porous. A method of recovering silica particles (that is, a composite), (2) a method of passing the first mixed solution through a liquid-permeable container (for example, a column) held so that the porous silica particles do not flow out of the container. And so on.
The temperature condition for bringing the first mixed solution into contact with the porous silica particles may be, for example, 4 to 80 ° C. The contact time between the first mixed solution and the porous silica particles may be, for example, 0.1 to 60 minutes.

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

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

(Washing process)
In the washing step, the complex obtained by adsorbing or binding the porous silica particles and the nucleic acid obtained in the binding step is washed.
Components other than nucleic acids (proteins, sugars, lipids, etc.) may be attached to the complex. By washing the complex, components other than nucleic acids can be reduced.
The number of washings may be once or twice or more.

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

As the cleaning liquid, a chaotropic substance solution and alcohol or an aqueous solution thereof may be used in combination. For example, after cleaning with a chaotropic substance solution, cleaning with alcohol or an aqueous solution thereof may be performed.
As the cleaning liquid, two or more kinds of alcohols having different alcohol concentrations or an aqueous solution thereof may be used in combination.

The method for cleaning the complex is not particularly limited. For example, (1') a method of mixing the complex recovered by the method of (1) above in the bonding step to obtain a third mixed solution and recovering the complex from the third mixed solution, (2'). ) In the bonding step, a method of passing the cleaning liquid through the container after discharging the first mixed liquid by the method (2) above can be mentioned.
The temperature condition at the time of washing may be, for example, 4 to 80 ° C.
One washing time may be, for example, 0.1 to 60 minutes.

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

The method (2') may be, for example, a method in which a cleaning liquid is put into the container, held for an arbitrary time (cleaning time), and then discharged from the container.
The amount of the cleaning liquid to be put in the container may be, for example, 10 to 10000 parts by mass with respect to 100 parts by mass of the composite.
Stirring or the like may be performed while the cleaning liquid is held in the container.
Examples of the method of discharging the cleaning liquid from the container include the same method as the method of discharging the first mixed liquid from the container.

After washing, the complex may be dried if necessary.
Examples of the drying method include a constant temperature bath and the like.
The drying conditions may be, for example, 30 to 80 ° C. for 0.1 to 3000 minutes.

(Elution process)
In the elution step, the nucleic acid is eluted from the complex after the binding step or the washing step, and the nucleic acid is recovered.
The number of elutions may be once or twice or more.

The elution step can be carried out using a known method. For example, a method of contacting the complex with the eluate and then recovering the eluate can be mentioned.
The eluate is a solution that has the function of eluting nucleic acids adsorbed or bound to porous silica particles. By the action of the eluate, nucleic acid is eluted from the complex, and an eluate containing the nucleic acid is obtained.
Examples of the eluate include water, TE buffer (10 mM Tris-hydrochloride, 1.0 mM EDTA, pH 8.0) and the like.

The method of contacting the complex with the eluate and then recovering the eluate is not particularly limited. For example, the complex recovered by the method (1) in the (1 ") bonding step or the complex washed by the method (1") in the washing step is mixed with the eluate to obtain a fourth mixed solution. After obtaining and removing the complex (that is, porous silica particles) in which the nucleic acid is eluted from the fourth mixed solution, the first mixed solution is discharged by the method (2) in the (2 ") binding step. A method of passing the eluate through the container or the container after the cleaning solution is discharged by the method (2 ") in the cleaning step can be mentioned.
The temperature condition at the time of contact between the complex and the eluate may be, for example, 4 to 80 ° C. The contact time between the complex and the eluate may be, for example, 0.1 to 60 minutes.

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

The method (2 ") may be, for example, a method in which the eluate is put into the container, held for an arbitrary time (contact time), and then discharged from the container.
The amount of eluate to be placed in the container may be, for example, 10 to 10000 with respect to 100 parts by mass of the complex.
Stirring or the like may be performed while the eluate is held in the container.
Examples of the method for discharging the eluate from the container include the same method as the method for discharging the first mixed solution from the container.

In the method for recovering nucleic acid of the present invention, as a carrier for nucleic acid binding, D ₅₀ is 6 to ₅₀ μm, the pore volume is 0.3 to ⁵ cm ³ / g, and the average pore diameter is 60 to 500 Å. Since certain porous silica particles are used, nucleic acid can be recovered with high efficiency even when the length of nucleic acid is long. In addition, the nucleic acid can be recovered simply by adsorbing or binding the nucleic acid to the porous silica particles and eluting the nucleic acid, which is convenient.
In the case of the porous silica particles, D ₅₀ , the pore volume and the average pore diameter are within the above ranges, so that the nucleic acid adsorption or binding efficiency to the porous silica particles in the binding step and the dissolution step Since the elution efficiency of the nucleic acid from the complex is excellent, the nucleic acid can be recovered with high efficiency.
The reason why the adsorption or binding efficiency is high is considered to be that a part of the nucleic acid easily fits into the pores opened on the surface of the porous silica particles. The reason for the high elution efficiency is that the contact area with nucleic acid is relatively small due to the pores opened on the surface of the porous silica particles, and the individual pores are opened at multiple locations. It is conceivable that when the nucleic acid fitted in the pores is desorbed, the eluate wraps around the pores and the nucleic acid is easily desorbed.

[Nucleic acid recovery kit]
The nucleic acid recovery kit of the present invention includes a first container containing the above-mentioned porous silica particles and a second container containing a binding liquid for adsorbing or binding nucleic acid to the porous silica particles.
The nucleic acid recovery kit of the present invention may further include a third container containing an eluate that elutes the nucleic acid from the complex in which the porous silica particles and the nucleic acid are adsorbed or bound, if necessary. Good.
If necessary, the nucleic acid recovery kit of the present invention may further include a fourth container containing a washing liquid for washing the complex in which the porous silica particles and the nucleic acid are adsorbed or bound.

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

Examples of the binding liquid contained in the second container include the binding liquid mentioned in the binding step, and a chaotropic substance solution is preferable.
When the binding liquid 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 the eluate contained in the third container is used in the elution step, it may be diluted with a solvent such as water.

Examples of the cleaning liquid contained in the fourth container include the cleaning liquids mentioned in the cleaning step.
When the cleaning liquid contained in the fourth container is used in the cleaning step, it may be diluted with a solvent such as water.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to the following description. “%” Indicates “mass%” unless otherwise specified.
Of Examples 1 to 11 described later, Examples 1 to 6 are examples, and other examples are comparative examples.
The evaluation methods and materials used in each example are shown below.

〔Evaluation methods〕
(Particle size distribution)
The porous silica particles are sufficiently dispersed in water by ultrasonic treatment, and measurement is performed with a laser diffraction / scattering type particle size distribution measuring device (MT-3300EX, manufactured by Nikki So Co., Ltd.) to obtain a frequency distribution and a cumulative volume distribution curve. Obtained a volume-based particle size distribution. From the obtained cumulative volume distribution curve, D ₁₀ , D ₅₀ , D ₉₀ , and D ₉₀ / D ₁₀ were obtained.

(Nitrogen adsorption)
The adsorption isotherm was measured using a nitrogen gas as the adsorption gas with a pore distribution measuring device (3FLEX manufactured by micrometrics).
Using the analysis software attached to the pore distribution measuring device, the pore volume (BJH adsorption integrated pore volume) and the pore volume (BJH adsorption integrated pore volume) from 34 points where the relative pressure P / P _{0 on} the adsorption isotherm is 0.01 to 0.995 by the BJH method. The average pore diameter (BJH average pore diameter) was determined. Further, the specific surface area was determined by the BET method from 10 points where the relative pressure P / P ₀ was 0.05 to 0.30.

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

Bound solution: Adsorption buffer (composition: 50 mM tris-hydrochloride, 5.0 M guanidine thiocyanate, 20 mM EDTA, 1.2% polyethylene glycol mono) attached to a nucleic acid extraction kit (manufactured by Qiagen, product name: DNeasy Blood tissue kit). -P-isooctylphenylene 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.
Eluate: An elution buffer included in the nucleic acid extraction kit.
Human Genome Solution: manufactured by Promega, product name human genomic DNA G3041, contains approximately 3 × 10 ⁹ base pairs of DNA in length, DNA concentration 200 μg / mL.
Escherichia coli genomic solution: Contains DNA of approximately 4.65 × 10 ⁶ base pairs in length, prepared by treating the culture solution of Escherichia coli DH5α using the product name GenElute Plasmid Miniprep Kit manufactured by Sigma Aldrich, and has a DNA concentration of 50 μg / mL.

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

[Examples 1 to 10]
Using a human genome solution or an Escherichia coli genome solution as a sample, 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% ethanol were added to an Eppendorf tube having a volume of 1.5 mL and mixed to obtain a first mixed solution.
Next, 20 mg of the 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 entire amount of the second mixture is transferred to a chip column, and the porous silica particles are collected on the filter of the chip column by centrifuging at 3500 rpm for 2 minutes at 20 ° C. using a centrifuge. It was.
Next, the filtrate was discarded, 500 μL of the cleaning solution 1 was added to the chip column, and the mixture was centrifuged under the same conditions as described above. Next, the filtrate was discarded, 500 μL of the washing solution 2 was added to the chip column, and the mixture was centrifuged under the same conditions as described above. Then, 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, left for 1 minute, and then centrifuged at 3500 rpm for 2 minutes at 20 ° C. The obtained filtrate was used as a nucleic acid extract.

The DNA concentration in the nucleic acid extract was measured using Invitrogen's Qubit assay according to the protocol. Specifically, 199 μL of Working solution was added to 1 μL of the 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 Qubit Fluorometer.
From the measured DNA concentration, the mass W ₁ (g) of the DNA in the nucleic acid extract was calculated. From this value and the mass W ₂ (g) of DNA in the sample (220 μL) used, the DNA recovery rate was calculated by the following formula.
DNA recovery rate (%) = W ₁ / W ₂ x 100

[Example 11]
Using a human genome solution or an E. coli genome solution as a sample, a commercially available nucleic acid extraction kit (manufactured by Qiagen, product name: DNeasy Blood tissue kit) equipped with a silica membrane is used to extract nucleic acids according to the protocol, and a nucleic acid extract is used. Got
For the obtained nucleic acid extract, the DNA recovery rate was determined in the same manner as in Examples 1 to 10, and the nucleic acid recovery rate was determined by recovering the nucleic acid from the sample according to the following procedure. The results are shown in Table 1.

Table 1 shows the porous silica particles D ₁₀ , D ₅₀ , D ₉₀ , D ₉₀ / D ₁₀ , silica (SiO ₂ ) purity, specific surface area, pore volume, and average pore diameter used in Examples 1 to 10. Regarding the silica purity, only the porous silica particles 3 and 8 were measured, but the silica purity of the other porous silica particles seems to be 98% by mass or more.
Further, in Examples 1 to 10, the vertical axis is the DNA recovery rate when the human genome solution is used as a sample, and the porous silica particles D ₅₀ , D ₉₀ / D ₁₀ , pore volume, average pore diameter or specific surface area (SSA). ) Is shown on the horizontal axis in FIGS. 1 to 5. 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, nucleic acids could be recovered with high efficiency.
On the other hand, in Example 7, since the D50 of the porous silica particles was less than 6 μm and the pore volume was less than 0.3 cm ³ / g, the nucleic acid recovery efficiency was inferior. In Example 8, 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, so that the nucleic acid recovery efficiency was inferior. In Example 9, the D50 of the porous silica particles was less than 6 μm, the pore volume was less than 0.3 cm ³ / g, and the average pore diameter was less than 60 Å, so that the nucleic acid recovery efficiency was inferior. In Example 10, since the D50 of the porous silica particles was less than 6 μm, the nucleic acid recovery efficiency was inferior.

Claims (12)

Porous silica particles having a volume-based particle size distribution D ₅₀ of 6 to ₅₀ μm, a pore volume of 0.3 to ⁵ cm ³ / g due to nitrogen adsorption, and an average pore diameter of 60 to 500 Å due to nitrogen adsorption. To form a complex by adsorbing or binding nitrogen to
A method for recovering a nucleic acid, which comprises eluting and recovering the nucleic acid from the complex. The method for recovering nucleic acid according to claim 1, wherein D ₉₀ / D ₁₀ of the particle size distribution based on the volume of the porous silica particles is 10 or less. The method for recovering nucleic acid according to claim 1 or 2, 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 any one of claims 1 to 3, 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 4, wherein the nucleic acid is adsorbed or bound to the porous silica particles in the presence of a chaotropic substance solution. The method for recovering nucleic acid according to any one of claims 1 to 5, wherein the complex is washed before eluting the nucleic acid from the complex. A is ¹⁰ 5 ^{to 10} 11 bp length of the nucleic acid, the method of recovering nucleic acid according to any one of claims 1 to 6. Porous silica particles having a volume-based particle size distribution D ₅₀ of 6 to ₅₀ μm, a pore volume of 0.3 to ⁵ cm ³ / g due to nitrogen adsorption, and an average pore diameter of 60 to 500 Å due to nitrogen adsorption. Nucleic acid binding carrier consisting of. Porous silica particles having a volume-based particle size distribution D50 of 6 to ₅₀ μm, a pore volume of 0.3 to ⁵ cm ³ / g due to nitrogen adsorption, and an average pore diameter of 60 to 500 Å due to nitrogen adsorption. The first container that houses the
A second container containing a binding solution that adsorbs or binds nucleic acid to the porous silica particles, and
Nucleic acid recovery kit comprising. The nucleic acid recovery kit according to claim 9, wherein the binding solution is a chaotropic substance solution. The nucleic acid recovery kit according to claim 9 or 10, further comprising a third container containing an eluate that elutes the nucleic acid from a complex in which the porous silica particles and the nucleic acid are adsorbed or bound. The nucleic acid recovery kit according to any one of claims 9 to 11, further comprising a fourth container containing a washing liquid for washing the complex in which the porous silica particles and the nucleic acid are adsorbed or bound.

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