JP2022097368A - Recovery kit and method for preparing nucleic acid-containing solution - Google Patents

Abstract

To provide a recovery kit that can efficiently prepare a nucleic acid-containing solution without using magnetic beads, and without performing centrifugal operation for B/F separation, and a method for preparing a nucleic acid-containing solution.SOLUTION: A recovery kit is used to recover a nucleic acid from a nucleic acid-containing substance in which a nucleic acid is enclosed in a nucleic acid-covering substance. The recovery kit has a particulate carrier that is packed in a solution-permeable bag-like material, has non-adsorptivity to a nucleic acid, and has adsorptivity to the nucleic acid-covering substance, and a tubular container for housing the bag-like material. The bag-like material has a volume of 100 μl to 5 ml.SELECTED DRAWING: Figure 1

Description

The present invention relates to a recovery kit for recovering nucleic acid and a method for preparing a nucleic acid-containing solution from a nucleic acid-containing substance using the recovery kit.

Exosomes are a type of extracellular vesicle that contains nucleic acids inside biological membranes that contain lipids and proteins. Nucleic acids in exosomes, which are particularly capable of amplification, are used as cell identification biomarkers because they are formed from molecules derived from the cell of origin and contain some unique molecules. As a method for extracting nucleic acid from exosomes, for example, microvesicles are isolated from biological samples by an ultracentrifugation method, and the isolated microvesicles are treated with DNase or the like, centrifuged and pelletized, and then pelletized. There is a method of extracting nucleic acid from microvesicles (Patent Document 1). In the examples of Patent Document 1, after centrifuging the serum to obtain pellets of nucleic acid-containing particles, the cells are suspended in a cell lysate and treated with a Qiagen RNeasy Plus kit featuring a DNA removal column to obtain RNA derived from the nucleic acid-containing particles. It has gained.

Further, as a method for separating nucleic acid from a sample containing a protein or the like, there is a method using highly magnetized magnetic beads (Patent Document 2). In Patent Document 2, a lysing binding solution is added to a blood sample to dissolve a biological sample, a protein solubilizer is added to destroy the nuclear membrane and nuclear protein, and highly magnetized magnetic beads are added to adsorb nucleic acids, and then the nucleic acid is adsorbed. Each time the cleaning solution, ethanol, and eluent are added, the adsorbed substance and the non-adsorbed substance are separated (B / F separation), and the nucleic acid adsorbed on the magnetic beads is extracted.

There is also a test tube containing magnetic beads for efficiently stirring and recovering the substance adsorbed on the magnetic beads (Patent Document 3). The test tube includes a hollow shaft body that can reciprocate in the longitudinal direction of the container and a magnetic force generating means arranged in the hollow of the shaft body, and the magnetic beads are moved into a fluid body by reciprocating the shaft body. This is a magnetic bead recovery agitator capable of recovering magnetic beads from a fluid body by means of generating a magnetic force.

If the bag-shaped biological membrane that encloses the nucleic acid of an exosome is referred to as a "nucleic acid-encapsulating substance," the exosome can be said to be a nucleic acid-containing substance in which the nucleic acid is encapsulated in the nucleic acid-encapsulating substance. In addition to extracellular vesicles such as exosomes, viruses have nucleic acids inside such nucleic acid-encapsulating substances. The virus has a viral genome within an envelope derived from a capsid protein or biological membrane. Viruses having an envelope include, for example, DNA viruses such as simple herpes virus and hepatitis B virus; hepatitis C virus, SARS corona virus, SARS-CoV-2, MERS corona virus, influenza virus, ebora virus, human immunodeficiency virus. There are RNA viruses such as. By collecting blood, saliva, or pharyngeal swab, or other sample from a person suffering from a viral disease, extracting the viral genome from this sample, and determining the sequence or the like, the causative virus of the disease can be identified.

For example, in order to detect Legionella bacteria, there is a method of immersing a bag in a liquid to be treated contained in a container (Patent Document 4). The bag contains a carrier composed of a calcium phosphate-based compound having a high affinity for Legionella, and has an opening smaller than the average particle size of the carrier. The liquid to be treated passes through the opening of the bag and infiltrates into the bag, comes into contact with the carrier, and Legionella bacteria adhere to the surface of the carrier. Subsequently, by bringing the buffer solution into contact with the carrier to which Legionella bacteria are attached, the Legionella bacteria attached to the carrier can be released into the buffer solution and recovered.

Japanese Patent Publication No. 2013-545463 Japanese Unexamined Patent Publication No. 2009-33995 Japanese Unexamined Patent Publication No. 2001-170513 Japanese Unexamined Patent Publication No. 2006-296417

In the event of a viral disease epidemic, it is necessary to establish a rapid testing system and an efficient sample processing system. However, depending on the pathogenicity of the virus, it is necessary to inactivate the virus in a facility with a high biosafety level and extract the gene fraction containing the viral nucleic acid. Chromatography is effective for adsorbing virus particles and fractionating free nucleic acid, but in the flow path chromatographic separation method in which a sample is charged from one end of a chromatographic carrier and the separation liquid is collected from the other end, the pathogenic sample is an injector. The contamination range is wide because it moves in the carrier flow path and the detection device. On the other hand, in the batch chromatographic separation method in which the carrier and the liquid sample are mixed and separated in the container, a centrifugation operation is indispensable in order to separate the carrier and the liquid after mixing the carrier with the liquid sample by pipetting or the like. Aerosols containing sex viruses are likely to occur. In this respect, the method using magnetic beads does not require centrifugal operation, but the unit price of beads used per acquired virus particle unit is high, and it is not realistic to use them for adsorbing all viruses in a liquid sample.

The batch chromatographic separation method requires that the liquid sample flow sufficiently to expose the nucleic acid inclusion material to the carrier. In the method disclosed in Patent Document 4, the efficiency of adhesion of the legionella bacteria to the carrier is improved by applying vibration to the bag or the liquid to be treated via a string connected to the bag, but the liquid to be treated The volume unit of this is relatively large, 500 mL, and it takes time to sufficiently flow the liquid to be treated. Therefore, there is room for improvement in the efficiency of attachment of Legionella bacteria to the carrier.

The present invention has been made in view of the above circumstances, and is a recovery kit and nucleic acid capable of efficiently preparing a nucleic acid-containing solution without using magnetic beads and without performing a centrifugation operation for B / F separation. It is an object of the present invention to provide a method for preparing a containing solution.

The present inventor has found that when a particulate carrier is packed with a solution-permeable bag-like substance, the supernatant can be recovered without centrifugation after mixing the liquid sample containing a virus or the like with the particulate carrier. Was completed.

That is, the recovery kit according to the first aspect of the present invention is
A recovery kit for recovering the nucleic acid from a nucleic acid-containing substance contained in a nucleic acid-encapsulating substance.
A particulate carrier packed in a solution-permeable bag-like substance that has non-adsorbability to the nucleic acid and has adsorptivity to the nucleic acid-encapsulating substance.
A tubular container for storing the bag-shaped object and
Equipped with
The volume of the bag-shaped object is
It is 100 μl to 5 ml.

The particulate carrier is
Has adsorptivity for one or more selected from the group consisting of proteins, sugar chains and lipids.
It may be that.

The particulate carrier
Clay carrier, agarose carrier, dextran carrier, hydroxyapatite carrier, silica gel carrier, polystyrol resin carrier, polyphenol resin carrier, polyacrylic resin carrier, polyalkyl resin carrier, polyvinyl resin carrier, derivatives of these carriers, and anti-nucleic acid coating on the surface. One or more selected from the group consisting of a carrier in which at least one of a packaging substance antibody and a surface sugar chain recognition molecule is immobilized.
It may be that.

The average particle size of the particulate carrier is
0.5 μm to 2 mm,
It may be that.

In the tubular container, the solvent is stored together with the particulate carrier.
It may be that.

The recovery kit according to the first aspect of the present invention is
Further equipped with a solution for nucleic acid extraction,
It may be that.

The method for preparing a nucleic acid-containing solution according to the second aspect of the present invention is as follows.
A liquid sample containing a nucleic acid-containing substance is added to the tubular container of the recovery kit according to the first aspect of the present invention.
The liquid sample is pipeted to adsorb the nucleic acid-encapsulating substance constituting the nucleic acid-containing substance on the particulate carrier.
Discard the supernatant and
A nucleic acid extraction solution is added to the tubular container to destroy the nucleic acid-encapsulating substance and release the encapsulated nucleic acid.
The supernatant containing the nucleic acid is collected.

The liquid sample is
A solution containing a virus as the nucleic acid-containing substance.
It may be that.

The liquid sample is
A solution containing extracellular vesicles as the nucleic acid-containing substance.
It may be that.

According to the present invention, a nucleic acid-containing solution can be efficiently prepared without using magnetic beads and without performing a centrifugation operation for B / F separation.

It is a figure which shows the appearance after adding the sample liquid to the recovery kit in Example 2. FIG.

The recovery kit according to the present embodiment is a recovery kit for recovering nucleic acid from a nucleic acid-containing substance in which nucleic acid is contained in a nucleic acid-encapsulating substance. The recovery kit includes a particulate carrier packed in a solution-permeable bag-like material and a tubular container for storing the bag-like material.

Nucleic acid means DNA, RNA, microRNA, mRNA, non-coding RNA and the like. The "nucleic acid-encapsulating substance" is a bag-shaped substance that does not contain nucleic acid in itself. Further, the "nucleic acid-containing substance" means a substance in which nucleic acid is stored inside the nucleic acid-encapsulating substance. "Nucleic acid-containing substances" include naturally occurring exosomes, microvesicles, extracellular vesicles such as Apoptotic Bodies, and viruses. Further, as the "nucleic acid-containing substance", there is a DDS preparation containing a nucleic acid drug as a non-naturally-derived substance. As the nucleic acid-containing substance, for example, an antisense nucleic acid and a DDS preparation in which a nucleic acid drug such as siRNA is encapsulated with an artificial lipid membrane such as a liposome can be exemplified.

The particulate carrier has non-adsorptive properties to nucleic acids and has adsorptive properties to nucleic acid-encapsulating substances. The particulate carrier has non-adsorptive properties to nucleic acids under certain solution conditions and has adsorptive properties to nucleic acid-encapsulating substances under the solution conditions. Nucleic acid inclusions are composed of proteins, lipids, glycolipids, glycoproteins and the like. Therefore, even if the starting solution conditions have adsorptivity to nucleic acids in addition to proteins, lipids, glycolipids, and glycoproteins, the carrier is subsequently replaced with a solution condition in which the nucleic acids show non-adsorbability, and only the nucleic acids are released. A carrier that can be used may be used. For example, hydroxyapatite is adsorptive to both proteins and nucleic acids, but in the presence of 100 mM EDTA, it is adsorptive only to proteins and non-adsorbent to nucleic acids, so that only nucleic acids can be released. When a protein having an isoelectric point 5 is dissolved in a buffer solution having a pH of 7, it has a negative charge, and this protein binds to a positively charged carrier such as diethylaminoethanol. On the other hand, since the protein at the isoelectric point 7 is positively charged in a buffer solution having a pH of 5, it binds to a negatively charged carrier such as a cation exchange column. Therefore, "adsorbability" means a property that can be adsorbed to a target substance under specific adsorption conditions.

Similarly, "non-adsorptive" means non-adsorbent under certain conditions for nucleic acids. Conventionally, as a particulate carrier having non-adsorptive properties to nucleic acids under such specific conditions and having adsorptive properties to one or more selected from the group consisting of proteins, sugar chains and lipids constituting the nucleic acid-encapsulating substance. Various carriers used in the batch chromatographic method for nucleic acid extraction can be preferably used. As the particulate carrier, for example, clay carrier, agarose carrier, dextran carrier, hydroxyapatite carrier, silica gel carrier, polystyrene resin carrier, polyphenol resin carrier, polyacrylic resin carrier, polyalkyl resin carrier, polyvinyl resin carrier, and these carriers. There are derivatives. For example, as a derivative of the agarose carrier, there is Sepharose, which is a gel obtained by chemically cross-linking agarose. In addition, as a derivative of dextran, there is Sephadex in which dextran is chemically crosslinked. Further, as a derivative of hydroxyapatite, there is hydroxyapatite carbonate and the like. Further, the polystyrene resin, the polyphenol resin, the polyacrylic resin, the polyalkyl resin, and the polyvinyl resin have non-adsorption property to nucleic acid under specific conditions and have adsorptivity to nucleic acid encapsulating substance. Shall contain a derivative of. Further, the particulate carrier is used on the surface of organic particles composed of resin bead particles, diatomaceous soil, alumina and other inorganic particles, activated carbon, etc., against the anti-nucleic acid encapsulant antibody, and the target protein on the surface of the nucleic acid encapsulant. A surface sugar chain recognition molecule such as an antibody or a nucleic acid may be immobilized via an epoxy or other chemically modifying group. The particulate carrier used in the present invention can be appropriately selected depending on the nucleic acid-encapsulating substance and the characteristics of the nucleic acid.

The average particle size of the particulate carrier is 0.5 μm to 2 mm, preferably 1 μm to 1.5 mm, and more preferably 1 μm to 1 mm. Within this range, the particulate carrier and the liquid can be easily separated. The average particle size is measured by the light scattering method.

The particulate carrier is characterized in that it is packed in a solution-permeable bag. The solution means various solutions including a liquid sample to be added to a tubular container, a washing solution, a buffer solution, a nucleic acid extraction solution, and a protein denaturing or decomposition solution. If the solution is permeable, it is possible to ensure contact between the components contained in the liquid sample such as the sample liquid and the particulate carrier. The size of extracellular vesicles such as viruses and exosomes, which are nucleic acid-containing substances, is about 100 nm. Therefore, it is important that the bag-like material has pores through which nucleic acid-containing substances such as viruses and extracellular vesicles can pass, and that the pores do not allow the particulate carrier to leak. For this reason, the bag-like material is solution permeable and has pores that allow the nucleic acid-containing substance to pass through and not the particulate carrier. By packing the particulate carrier with a bag-like substance, the nucleic acid-containing substance contained in the liquid sample can be brought into contact with the particulate carrier, while the dispersion of the particulate carrier in the liquid is made into the bag-like substance. It can be suppressed and the separation of the liquid and the particulate carrier can be facilitated. Since the average particle size of the particulate carrier is 0.5 μm to 2 mm, the pore diameter of the bag-shaped product should be appropriately selected in the range of 100 nm to 2 mm according to the average particle size of the particulate carrier to be used. Can be done.

The material of the bag-like material does not adsorb nucleic acid, nucleic acid encapsulating substance, and nucleic acid-containing substance, for example, glass fiber, aramid fiber, cellulose fiber, nylon fiber, vinylon fiber, polyester fiber, polyethylene fiber, polyolefin fiber. , Rayon fibers, and halogen-containing fibers such as polytetrafluoroethylene, mixtures or laminates thereof. In the case of non-woven fabrics composed of these, well-known techniques such as dry method, wet method, spun bond method, thermal bond method, chemical bond method, stitch bond method, needle punch method, melt blow method, spun lace method, and steam jet are used. It can be prepared by a method or the like. Further, it can be molded into a bag-shaped product by a method suitable for the material. For example, when a heat-meltable substance such as polypropylene is used, each side may be bonded by heat-melting to form a bag-like product, or may be molded by a needle punching method or the like. In addition, by adjusting the mesh of the above-mentioned material, the density of the non-woven fabric, etc., the bag-shaped material can ensure the property of passing through nucleic acid-containing substances such as viruses or extracellular vesicles and preventing the particulate carrier from passing through. can.

The size and shape of the bag-shaped object are not particularly limited. For example, the shape of the bag-like object is a plane-bonded two non-woven fabrics, a three-dimensional molding, for example, a hollow triangular pyramid, a hexahedron, an octahedron, a dodecahedron, or an icosahedron. Other examples include polyhedrons, cones, columns, and spheres. It is preferable to appropriately select such a shape according to the shape of the bottom of the tubular container for storing the particulate carrier stored in the bag-shaped material. Further, it is preferable that the size is such that the particulate carrier and the nucleic acid-encapsulating substance can be efficiently adsorbed in the solution. Therefore, the volume of the particulate carrier is 0.01 to 0.9 times, preferably 0.05 to 0.7 times, more preferably 0.1 times the volume of the bag-shaped material with respect to the volume of the bag-shaped material. Store in a bag so that it is up to 0.5 times larger.

The liquid sample containing the nucleic acid-containing material added to the tubular container is preferably stirred by pipetting. In this case, the volume of the liquid sample added to the tubular container is about 1 to 100 ml. The volume of the bag-shaped object is appropriately adjusted according to the volume of the liquid sample. The volume of the bag is, for example, 10 μl to 10 ml, 40 μl to 8 ml, 60 μl to 6 ml or 80 μl to 5 ml, preferably 100 μl to 5 ml, 100 μl to 3 ml, 100 μl to 2 ml, 100 μl to 1 ml, 200 μl to 3 ml, 200 μl to 2 ml. , 200 μl to 1 ml or 500 μl to 3 ml.

As the tubular container, the container used in the conventional batch chromatographic separation method can be widely used. Therefore, the shape of the bottom of the tubular container may be a flat bottom, a U-shape, or a V-shape. Further, the shape may be such that the diameter in the vicinity of the bottom is narrow and the diameter in the vicinity of the opening is widened. There is no limit to the length or capacity of the tubular container. The length and capacity suitable for pipetting can be selected. The tubular container may have a lid. Therefore, regardless of the name, test tubes, centrifuge tubes, Spitz tubes, microtubes, PCR tubes, microplates, ELISA plates, culture plates and the like can be preferably used. The material of the tubular container may be any of plastic such as polypropylene, hard glass, Pyrex (registered trademark) glass, quartz glass, and metal.

The tubular container may contain the solvent together with the particulate carrier. Examples of the solvent include physiological saline and a buffer solution. It is advantageous to store the solvent together with the particulate carrier in advance because the liquid sample can be pipetted with the solvent. If the tubular container contains a solvent, for example, when the nucleic acid-encapsulating substance and the free nucleic acid contained in the liquid sample are adsorbed on the particulate carrier, the free nucleic acid can be easily removed by a pipette or a decanter. Can be done.

The recovery kit according to this embodiment may further include one or more nucleic acid extraction solutions. Nucleic acid extraction solutions include, for example, protein denaturants such as guanidine thiocyanate, guanidine hydrochloride and phenol, surfactants such as SDS, nucleic acid degrading enzyme inactivating agents such as diethylpyrocarbonate (DEPC) and ribonuclease inhibitors. 2-Reducing agents such as mercaptoethanol and DTT can be exemplified. These can be combined and used as a solution for nucleic acid extraction.

In another embodiment, there is provided a method of preparing a nucleic acid-containing solution from a sample containing the nucleic acid-containing substance using the recovery kit. For example, a carrier packed with 50 μl of hydroxyapatite having an average particle diameter of 40 μm in a bag-like material prepared with a polypropylene non-woven fabric having a capacity of 100 μl is prepared, and this is stored in a tubular container having a capacity of 2 ml to prepare a recovery kit. The case where an RNA-containing solution is prepared from a solution (sample solution) containing a virus as a nucleic acid-containing substance in a recovery kit will be described.

According to a conventional method, 1 ml of a sample solution containing a nucleic acid-containing substance such as SARS-CoV-2 collected from a subject is added to a tubular container. Since hydroxyapatite has protein adsorptivity, when the sample solution is pipetted in a tubular container, the SARS-CoV-2 virus particles contained in the sample solution are adsorbed on hydroxyapatite. Then, after allowing to stand at a temperature of 2 to 10 ° C. for 1 hour, the supernatant is discarded. The supernatant contains components that were not adsorbed by hydroxyapatite and is removed. Then, 1 ml of a washing solution containing, for example, 100 mM EDTA and adjusted to pH 8 with sodium hydroxide is added to a tubular container and pipetted to wash the hydroxyapatite, and then the supernatant is pipetted or decanted to remove the supernatant. Collect and dispose of. By this washing step, components that have not been adsorbed on hydroxyapatite and substances such as free nucleic acids that are once adsorbed but whose binding to hydroxyapatite is inhibited by EDTA can be removed. Since hydroxyapatite is packed in a bag, B / F separation can be performed by suction or decanting with a pipette without performing centrifugal operation.

After removing the supernatant, add appropriate amounts of RNA-degrading enzyme inactivating agents such as DEPC, protein denaturants such as phenol solution, and nucleic acid extraction solutions such as surfactants such as SDS to a tubular container, and use a vortex mixer or the like. It destroys the envelope (nucleic acid inclusion substance). As a result, RNA is released from the nucleic acid-encapsulating substance and transferred into the solution. The supernatant in the tubular container contains the free RNA, and the RNA-containing solution can be recovered by suction with a pipette or decanting. This RNA-containing solution can be used, for example, for PCR tests and the like.

The above has described the method of preparing an RNA-containing solution from a sample solution containing a virus, but other nucleic acid-containing solutions can be prepared by appropriately selecting the type of washing solution or other solvent or nucleic acid extraction solution. ..

Samples containing virus-derived nucleic acid-containing substances include, for example, blood, serum, plasma, nasopharyngeal swab, saliva, nasal lavage fluid, bronchial alveolar lavage fluid, sputum / nasal juice / fecal suspension, and qi collected from subjects. Examples thereof include a solution in which the virus in the phase or on the surface of the substance is collected and then re-eluting. In addition, as a sample containing a nucleic acid-containing substance derived from a virus, there is a sample containing a virus collected from indoor / outdoor air, rivers, or other environments and held in a solution.

The sample containing the nucleic acid-containing substance derived from the virus may be a virus-containing cell culture medium or the like used in a bio-based laboratory. In viruses, the genes DNA or RNA are covered by capsid protein shells or envelopes, depending on the type. Therefore, when the particulate carrier and the solution containing the virus are mixed, the enveloped protein or the capsid protein binds to the particulate carrier. Since some viruses are pathogenic, it is necessary to treat them in a facility with a high biosafety level, and it is desired that the number of steps can be reduced. It is also important to avoid the generation of aerosols containing pathogenic viruses in the treatment process. The tubular container constituting the recovery kit according to the present embodiment can be used as a sample storage container for a virus-containing test solution or the like, and a container for batch chromatography.

When using a recovery kit filled with a conventional particulate carrier, centrifugation was required for B / F separation, and aerosols were generated in or around the centrifuge. However, according to the recovery kit according to the present embodiment, since the B / F separation can be performed without performing the centrifugation operation, it is possible to suppress the generation of aerosol in the solution containing the virus. Further, since the particulate carrier is packed in a bag-like material, the cleaning liquid can be discarded by suction or decanting with a pipette, so that the operation is simple. The protein is denatured by adding a nucleic acid extraction solution, and the virus released from the nucleic acid-encapsulating substance such as the envelope and the capsid loses its infectivity. According to the recovery kit according to the present embodiment, all the steps up to the preparation of the nucleic acid-containing solution and the virus inactivation can be performed in a tubular container without generating a virus-containing aerosol. Moreover, since the particulate carrier is packed in a bag, the recovery rate of the supernatant containing free nucleic acid is high. Using this nucleic acid-containing solution, the sequences and contents of DNA, RNA, etc. can be analyzed as in the conventional case.

The liquid sample containing the nucleic acid-containing substance may be blood, urine, spinal fluid, bile, pancreatic fluid, body fluids such as sheep's water and ascites, and extracellular vesicle-containing solutions such as cell culture fluid, which contain exosomes. good. Exosomes are membrane-bound extracellular vesicles contained in eukaryotic cells and contain various molecular components derived from the cell of origin, such as proteins, DNA and RNA. According to the recovery kit according to the present embodiment, the tubular container can be used as a storage container for extracellular vesicle solution and a container for batch chromatography. Similar to the solution containing the virus, the solution of extracellular vesicles is added to a tubular container containing the particulate carrier packed in a bag, and the contained protein is bound to the particulate carrier. Since the nucleic acid of the follicle is bound to the protein, the nucleic acid is also present on the particulate carrier side. Next, the components that do not bind to the particulate carrier are discarded by suction or decantation with a pipette without centrifuge operation, a washing solution is added and washed, and then nucleic acid extraction is performed in the same manner as in the case of extracting nucleic acid derived from a virus. The solution is added to break the bond between the protein and the nucleic acid, and then the supernatant containing the nucleic acid eluted in the nucleic acid extraction solution is recovered as a nucleic acid-containing solution. This solution can be used to analyze sequences and contents of DNA, RNA, mRNA and miRNA.

In the recovery kit according to the present embodiment, by setting the volume of the bag-shaped material to 100 μl to 5 ml, the carrier can be sufficiently exposed to the liquid sample in a short time by a simple operation such as pipetting. As a result, the nucleic acid-encapsulating substance in the liquid sample can be efficiently adsorbed on the carrier.

In the recovery kit according to the present embodiment, the particulate carrier may be provided in a state of being packed in a bag-shaped material, or may be provided in a state of being independent of the bag-shaped material. When the particulate carrier is provided in a state independent of the bag-like substance, the bag-like substance containing the particulate carrier prepared by suspending it in phosphate buffered saline or the like is stored in a tubular container at the time of use. You may do so.

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

Example 1
A polypropylene non-woven fabric was formed into a cylindrical shape having an inner diameter of 0.5 cm by bonding by heat melting. One end side of this cylinder was closed by adhesion by thermal melting, and a bag-shaped object having an open other end side was molded. A slurry in which a hydroxyapatite carrier (manufactured by Biorad) having an average particle diameter of 40 μm measured by a light scattering method is suspended in a phosphate buffered saline, and the volume occupied by the particulate carrier in a wet state becomes 50% of the total volume. Was prepared. After storing 100 μl of this slurry in the bag-shaped material, the upper end 1 cm from the one end side that was previously heat-melted is adhered by heat melting so as to be orthogonal to the lower end, and the opening on the other end side is closed to form a tetrahedron. did. This was housed in a polypropylene lidped microtube (Protein LoBind Tubes manufactured by Eppendorf) having a capacity of 2 ml to prepare a recovery kit. The volume of the prepared bag-shaped product was 103 μl.

Example 2
Influenza A virus (A / Puerto Rico / 8/1934 (H1N1) strain) inoculated into MDCK cells, Eagle's minimum essential medium containing 5 μg / ml acetylated trypsin (derived from bovine pancreas, manufactured by Merck). Incubated in (manufactured by Nissui) for 3 days. Next, the culture supernatant of the virus-infected cells was collected and centrifuged at 3000 × g for 5 minutes to remove insoluble components, and the sample solution was used.

After adding 1 ml of the sample solution to the recovery kit prepared in Example 1 and pipetting using a micropipette (Nichiryo Premium 100-1000 manufactured by Nichiryo Co., Ltd.) set to a volume of 1 ml, the mixture is allowed to stand at a temperature of 4 ° C. for 1 hour. did. This state is shown in FIG. The supernatant was removed by pipetting, and then 1 ml of a washing solution having a pH adjusted to 8.0 with sodium hydroxide containing 100 mM EDTA was added and pipetting was performed to remove the supernatant.

Then, 100 μl of 6 mol / L guanidine thiocyanate was added as an RNA extraction reagent and pipetting was performed to release the nucleic acid from the virus. Then, the supernatant was collected with a pipette and used as a nucleic acid-containing solution. This nucleic acid-containing solution was diluted 2-fold with sterile water for molecular biology (manufactured by Nakaraitesk Co., Ltd.) and then subjected to an automatic nucleic acid extractor (magLEAD 12 gC manufactured by Precision System Science) to be used as a nucleic acid extraction reagent (Precision System). -Nucleic acid components containing viral RNA were extracted using MagDEA Dx SV manufactured by Science.

Type A shown by Nakauchi et al. (Journal of Virological Methods, 2011, One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A / H1N1 2009, seasonal influenza A / H1N1, and seasonal influenza A / H3N2 viruses) Primers and probes for detecting the M gene of influenza virus (MP-39-67For, MP-183-153Rev and MP-96-75ProbeAs) and a commercially available quantitative PCR kit (THUNDERBIRD Probe One-step qRT-PCR Kit manufactured by Toyo Spinning Co., Ltd.) It was used and subjected to a quantitative PCR device (7500 Fast Real-Time PCR System manufactured by Applied Biosystems), and the influenza A virus gene contained in the above-extracted nucleic acid component could be quantified.

The embodiments described above are for the purpose of explaining the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is shown not by the embodiment but by the scope of claims. And, various modifications made within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.

The present invention is useful for recovering or isolating nucleic acids from nucleic acid-containing substances such as extracellular vesicles and viruses.

Claims (9)

A recovery kit for recovering the nucleic acid from a nucleic acid-containing substance contained in a nucleic acid-encapsulating substance.
A particulate carrier packed in a solution-permeable bag-like substance that has non-adsorbability to the nucleic acid and has adsorptivity to the nucleic acid-encapsulating substance.
A tubular container for storing the bag-shaped object and
Equipped with
The volume of the bag-shaped object is
100 μl to 5 ml,
Collection kit. The particulate carrier is
Has adsorptivity for one or more selected from the group consisting of proteins, sugar chains and lipids.
The collection kit according to claim 1. The particulate carrier
Clay carrier, agarose carrier, dextran carrier, hydroxyapatite carrier, silica gel carrier, polystyrol resin carrier, polyphenol resin carrier, polyacrylic resin carrier, polyalkyl resin carrier, polyvinyl resin carrier, derivatives of these carriers, and anti-nucleic acid coating on the surface. One or more selected from the group consisting of a carrier in which at least one of a packaging substance antibody and a surface sugar chain recognition molecule is immobilized.
The collection kit according to claim 1 or 2. The average particle size of the particulate carrier is
0.5 μm to 2 mm,
The collection kit according to any one of claims 1 to 3. In the tubular container, the solvent is stored together with the particulate carrier.
The collection kit according to any one of claims 1 to 4. Further equipped with a solution for nucleic acid extraction,
The collection kit according to any one of claims 1 to 5. A liquid sample containing a nucleic acid-containing substance is added to the tubular container of the recovery kit according to any one of claims 1 to 6.
The liquid sample is pipeted to adsorb the nucleic acid-encapsulating substance constituting the nucleic acid-containing substance on the particulate carrier.
Discard the supernatant and
A nucleic acid extraction solution is added to the tubular container to destroy the nucleic acid-encapsulating substance and release the encapsulated nucleic acid.
Collecting the supernatant containing the nucleic acid,
Method for preparing nucleic acid-containing solution. The liquid sample is
A solution containing a virus as the nucleic acid-containing substance.
The method for preparing a nucleic acid-containing solution according to claim 7. The liquid sample is
A solution containing extracellular vesicles as the nucleic acid-containing substance.
The method for preparing a nucleic acid-containing solution according to claim 7.

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