JP2019162037A - Nucleic acid collection member - Google Patents

Abstract

To provide a nucleic acid collection member whereby, a target nucleic acid in a nucleated cell is stored, and when extracted, the cell is concentrated to allow the target nucleic acid to be collected stably, in a short time and with a high purity.SOLUTION: A nucleic acid collection member has a nucleic acid stabilization reagent that deactivates a nucleic acid catabolic enzyme in a sample, and separation means that separates a nucleated cell in the sample.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a nucleic acid collecting member.

  Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are known as biomolecules. DNA and RNA are degraded by a DNA degrading enzyme (deoxyribonuclease, DNase) and an RNA degrading enzyme (ribonuclease, RNase), respectively. Among these, since RNase is a protein that is widely present in all living organisms and is very stable, care must be taken when storing and extracting RNA.

Therefore, for example, a method of storing using a chemical substance that inhibits a degrading enzyme containing RNase, and a method of extracting and purifying the stored sample by multiple times of centrifugation and solid phase adsorption are known.
For the purpose of purifying nucleic acid (DNA or RNA) from a stabilizing reagent, several compositions for purification and a nucleic acid purification method by adsorption to solid phase adsorption have been proposed (for example, see Patent Document 1). ).

  The present invention provides a nucleic acid collecting member capable of storing a target nucleated intracellular nucleic acid and concentrating the cells during extraction to stably recover the target nucleic acid with high purity in a short time. For the purpose.

  The nucleic acid collecting member of the present invention as a means for solving the above-described problems includes a nucleic acid stabilizing reagent that inactivates a nucleolytic enzyme in a sample, and a separating means that separates nucleated cells in the sample. .

  According to the present invention, when a target nucleated intracellular nucleic acid is stored and extracted, the target nucleic acid can be recovered with high purity in a short time by concentrating the cells.

FIG. 1 is a schematic view showing an example of the nucleic acid collecting member of the present invention. FIG. 2 is a diagram for explaining an example of a method for concentrating nucleated cells as a target in a sample using the nucleic acid collecting member of the present invention. FIG. 3A is a photograph showing blood to which a nucleic acid stabilizing reagent has been added. FIG. 3B is an enlarged photograph of FIG. 3A. FIG. 4 is a photograph showing the flow-through after the first centrifugation after adding blood to which a nucleic acid stabilizing reagent has been added on the filter. FIG. 5 is a photograph showing a flow-through after the second centrifugation after adding sterilized water. FIG. 6A is a photograph showing cells remaining on the filter after the second centrifugation. FIG. 6B is an enlarged photograph of FIG. 6A.

(Nucleic acid collection member)
The nucleic acid collecting member of the present invention has a nucleic acid stabilizing reagent that inactivates a nucleolytic enzyme in a sample and a separating means for separating nucleated cells in the sample, and preferably has a lid member, Furthermore, it has another member as needed.
The nucleic acid collecting member is suitably used as a kit or device having functions for storing, concentrating, separating, and collecting the target nucleic acid.

The nucleic acid collecting member of the present invention is based on the knowledge that, in the prior art, the target nucleic acid cannot be recovered with high purity in a short time because the nucleic acid is released into the solution by cell destruction.
In addition, in the conventional method of storage using a chemical substance that inhibits degrading enzyme and extraction / purification by multiple times of centrifugation and solid phase adsorption, unnecessary nucleic acid (DNA not derived from a target cell) is used for centrifugation. And RNA, DNA and RNA floating outside the cell), proteins, lipids, and cell debris are precipitated at the same time. For example, even for the purpose of extracting RNA of nucleated cells (such as leukocytes) in blood, RNA derived from erythrocytes and nucleic acids floating in other liquid components are also simultaneously collected. Further, since the centrifugation operation is repeated a plurality of times in order to wash the precipitate containing the target nucleic acid, there is a problem that the number of steps is increased, which is complicated and takes time.

  Therefore, the nucleic acid collecting member of the present invention includes a nucleic acid stabilizing reagent that inactivates a nucleolytic enzyme in a sample, and a separation unit that separates nucleated cells in the sample, thereby providing a target nucleated substance. When cell nucleic acids are stored and extracted, the cells can be concentrated to recover nucleic acids stably and with high purity in a short time.

<Nucleic acid stabilization reagent>
The nucleic acid stabilizing reagent inactivates the nucleolytic enzyme in the sample. That is, it contains a nucleolytic enzyme inhibitor, a surfactant, and, if necessary, other components.
Examples of the nucleolytic enzyme include a DNA degrading enzyme (deoxyribonuclease, DNase), an RNA degrading enzyme (ribonuclease, RNase), and the like.

  The nucleic acid stabilizing reagent preferably contains at least one of ammonium sulfate, sodium sulfate, and cesium sulfate from the viewpoint of the effect that nuclease (RNase or DNase) does not function due to salting out.

  The nucleic acid stabilizing reagent preferably contains at least one of a ribonuclease inhibitor and a deoxyribonuclease inhibitor in view of the effect that the nuclease (RNase or DNase) does not function.

  The nucleic acid stabilizing reagent preferably contains at least one of tetradecyltrimethylammonium oxalate and tartaric acid from the viewpoint of stabilizing the nucleic acid.

  When the sample is blood containing red blood cells, it is preferable that the nucleic acid stabilizing reagent contains a surfactant from the viewpoint of the effect of destroying the red blood cell membrane. As the surfactant, for example, at least one selected from a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant is preferable.

Examples of the cationic surfactant include dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, cetyltrimethylammonium bromide and the like.
Examples of the anionic surfactant include sodium dodecyl sulfate (SDS), sodium cholate, sodium dodecyl cholate, sodium N-lauroyl sarcosine, and the like.
As the amphoteric surfactant, for example, 3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonate, phosphatidylethanolamine, 3- (3-cholamidepropylyl) dimethylamino-1-propanesulfonate (CHAPS), [ 3-[(3-Cholamidopropyl) dimethylamino] -2-hydroxy-1-propansulfonate (CHAPSO) and the like.
Nonionic surfactants include, for example, polyoxyethylene octyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene-lauryl ester, polyethylene-glycol hexadecyl-ester polyoxyethylene Examples include cetyl ether, saponin, digitonin, n-octyl-β-D-glucoside, n-octyl-β-D-glucopyranoside, n-octylthio-β-D-thioglucopyranoside.
Surfactant may be used individually by 1 type and may use 2 or more types together.

  The nucleic acid stabilizing reagent is preferably in a solid form because it is less likely to leak out of the container as compared with a liquid nucleic acid stabilizing reagent. There is no restriction | limiting in particular as a method of making a nucleic acid stabilization reagent into a solid state, According to the objective, it can select suitably, For example, a freeze-drying method etc. are mentioned.

<< Sample >>
As the sample, blood containing a biomaterial is preferably used.
Here, blood contains blood cell components (cellular components, blood cells), platelets, plasma components (liquid components), and the like.
The blood cell component includes red blood cells, white blood cells, platelets and the like. Leukocytes include neutrophils, eosinophils, basophils, lymphocytes, monocytes and the like. The lymphocytes include NK cells (natural killer cells), B cells (B lymphocytes), T cells (T lymphocytes) and the like.
Plasma components include water, proteins, fats, sugars, inorganic salts, small molecule metabolites, foreign molecules, and the like.

As the sample, blood collected from the subject may be used as it is, or a separated nucleated cell layer obtained by subjecting the collected blood to separation treatment may be used.
The nucleated cell layer separation treatment method is not particularly limited and may be appropriately selected depending on the purpose. For example, a lymphocyte separation medium such as Ficoll (Ficoll-Paque PLUS, manufactured by GE Healthcare Japan Co., Ltd.) is used. Examples include general separation methods used.

  There is no restriction | limiting in particular as a biomaterial, According to the objective, it can select suitably, For example, (1) The substance which contains a nucleotide as a structural component, (2) A cell etc. are mentioned.

(1) Examples of substances containing nucleotides as constituents include nucleic acids such as RNA (ribonucleic acid) containing ribonucleotides and DNA (deoxyribonucleic acid) containing deoxyribonucleotides, fragments of these nucleic acids, or these nucleic acids Or the analog of the fragment | piece etc. are mentioned.
These include, for example, relatively short oligos or polynucleotides used as primers, probes, siRNAs (small interfering RNAs), genes (including mRNA), regardless of length, single strand or double strand And long-chain polynucleotides such as plasmids.
Examples of nucleic acid or nucleic acid fragment analogs include non-nucleic acid components bound to nucleic acids or nucleic acid fragments, nucleic acids or nucleic acid fragments labeled with a labeling agent such as a fluorescent dye or an isotope (for example, fluorescent dyes or radioisotopes) And a primer or probe labeled with a nucleic acid or a nucleic acid or a fragment of a nucleotide constituting a nucleic acid fragment (for example, a peptide nucleic acid). These may be natural products obtained from living organisms or processed products thereof, or may be manufactured using genetic recombination techniques, or may be chemically synthesized. .

(2) The cells include natural cells obtained from living organisms (animals or plants), cell lines, and transformed cells containing recombinant genes.
Examples of animal cells include various cells commonly used in gene recombination techniques (for example, mouse fibroblasts, Chinese hamster ovary cells CHO, monkey COS cells, etc.), or transformed cells thereof.
Examples of plant cells include various cells widely used in gene recombination techniques, and transformed cells thereof.

<Separation means>
The separation means separates nucleated cells in the sample. The separation means can concentrate nucleated cells effectively because nucleated cells (white blood cells or the like) to be analyzed do not pass through.
The separation means is not particularly limited as long as nucleated cells in a sample can be separated, and can be appropriately selected according to the purpose. Examples thereof include a filter and solid phase adsorption. Among these, a filter is preferable from the viewpoint of easy collection of concentrated nucleated cells.
As a filter, what is necessary is just a filter which the cell smaller than a liquid component or a nucleated cell passes without the nucleated cell used as object, for example, a membrane filter etc. are mentioned.

<Cover member>
The nucleic acid collecting member of the present invention preferably has a lid member that covers the opening. By having a lid member, contamination can be prevented and storage stability and safety are improved. The lid member is preferably removable from the viewpoint of operability. The lid member is preferably connected to one end of the container when removed from the viewpoint of preventing the lid member from being lost.

There is no restriction | limiting in particular about the material, a shape, a magnitude | size, a structure, etc. as a cover member, According to the objective, it can select suitably.
There is no restriction | limiting in particular as a material, Although it can select suitably according to the objective, An elastomer is preferable.
The elastomer is excellent in chemical resistance, has a good sealing property as a lid member, and can be used without being attached to a multiwell plate by using an adhesive like a cover sheet.
The elastomer is not particularly limited and may be appropriately selected depending on the intended purpose. For example, various thermoplastic elastomers, butadiene rubber, styrene-butadiene rubber, high styrene rubber, isoprene rubber, acrylic rubber, epichlorohydrin rubber, butyl rubber ethylene -Synthetic rubber such as propylene rubber or natural rubber. These may be used individually by 1 type and may use 2 or more types together.
There is no restriction | limiting in particular as a shape and structure of a cover member, According to the objective, it can select suitably.

The nucleic acid collecting member of the present invention can store the target nucleated intracellular nucleic acid and extract the target nucleic acid in a short time with high purity by concentrating the cells when extracted. The shape, size, material, structure, etc. are not particularly limited and can be appropriately selected according to the purpose.
Examples of the shape of the nucleic acid collecting member include a tube shape, a plate shape, a tray shape, and a cell shape. A connection type member container in which a plurality of members are connected may be used.
Examples of the material of the nucleic acid collecting member include resin, semiconductor, ceramics, metal, glass, and quartz glass. Among these, resin is preferable. Examples of the resin include acrylic resin, polystyrene resin, polyethylene terephthalate resin, polypropylene resin, and polycarbonate resin.
There is no restriction | limiting in particular as a magnitude | size and structure of a nucleic acid collection member, According to the objective, it can select suitably.

  The nucleic acid collecting member has a container shape as a function of preserving the target nucleated intracellular nucleic acid, and the container portion is preferably a blood collection tube, so that the nucleic acid collection member can be used as it is as a blood collection tube.

<Other members>
There is no restriction | limiting in particular as another member, According to the objective, it can select suitably, For example, an identification member, a memory member, etc. are mentioned.
The recognition member is a member for identifying a nucleic acid collecting member, and examples thereof include a barcode, QR code (registered trademark), RFID (Radio Frequency Identifier), characters, symbols, figures, colors, and the like.
The storage member is a member that stores information on the nucleic acid collecting member, and examples thereof include a memory and an IC chip.

  Here, an embodiment of the nucleic acid collecting member of the present invention will be described in detail with reference to the drawings. In addition, in each drawing, the same code | symbol is attached | subjected to the same component and the overlapping description may be abbreviate | omitted. In addition, the number, position, shape, and the like of the following constituent members are not limited to the present embodiment, and can be set to a preferable number, position, shape, and the like in carrying out the present invention.

<First Embodiment>
Figure 1 shows an example of the overall structure of a nucleic acid collection member that concentrates nucleated cells in a liquid sample for the purpose of nucleic acid storage and extraction.
The nucleic acid collecting member (container with nucleic acid stabilizing reagent) 10 of FIG. 1 is a polypropylene tube shape, and a filter (oxyfil filter, manufactured by Toho Co., Ltd.) as a separating means 1 that does not allow nucleated cells to pass through, 1.4 mL of nucleic acid stabilizing reagent 2 (tetradecyltrimethylammonium oxalate and tartaric acid) is contained on the filter.
The nucleic acid stabilizing reagent 2 can penetrate into nucleated cells and preserve the intracellular nucleic acid. In addition, the nucleic acid stabilizing reagent 2 contains a surfactant, and erythrocytes are destroyed by the difference in resistance to the surfactant, but the nucleated cells maintain their shape.

Next, as shown in FIG. 2, a method of concentrating nucleated cells that are targets in blood using the nucleic acid collecting member 10 of FIG. 1 will be described.
First, when a predetermined amount (0.5 mL) of blood as the sample 4 is added to the nucleic acid collecting member 10, the stabilizing reagents (tetradecyltrimethylammonium oxalate and tartaric acid) are contained in the cells by the surfactant in the nucleic acid stabilizing reagent 2. The nucleic acid that permeates into the cell and is present in the cell is stabilized.
Also, erythrocytes are destroyed due to the difference in strength of the membrane with respect to the surfactant, but nucleated cells such as leukocytes remain in shape. Nucleolytic enzymes present outside the cells are also inactivated by the nucleic acid stabilizing reagent 2. In this state, it can be stored for a long time as a blood sample for nucleic acid measurement.
Next, when the nucleic acid is extracted, the blood containing the nucleic acid stabilizing reagent is passed through a filter as the separating means 1 by utilizing a centrifugal operation or a pressure difference. Since the nucleated cells maintain the shape, they cannot pass through the filter, and the target nucleated cells are concentrated on the upper part of the filter.

  The blood (FIGS. 3A and 3B) to which a nucleic acid stabilization reagent (reagent in PAXgene RNA tube (manufactured by Becton Dickinson Co., Ltd.)) was added was treated using a membrane filter and centrifugation. No cells were observed in the flow-through (FIGS. 4 and 5), and the cells were collected on the filter (FIGS. 6A and 6B). Impurities (free RNA, DNA, proteins, lipids, cell debris, etc.) are collected on the flow-through side through the membrane filter, and the collected liquid on the filter is thought to collect nucleated cells with high purity. .

<Second Embodiment>
In the first embodiment, nucleated cells were stored and concentrated in the same manner as in the first embodiment except that the nucleic acid stabilization reagent 2 was in a solid state. Solidification of the nucleic acid stabilizing reagent is performed by a method such as drying, and the amount is adjusted so that an appropriate concentration is obtained when the sample is added.
In the second embodiment, since the nucleic acid stabilizing reagent is in a solid state, there is an advantage that there is a low possibility of leakage outside the nucleic acid collecting member.

As an aspect of this invention, it is as follows, for example.
<1> a nucleic acid stabilizing reagent that inactivates a nucleolytic enzyme in a sample;
Separation means for separating nucleated cells in the sample;
It is a nucleic acid collection member characterized by having.
<2> The nucleic acid collecting member according to <1>, wherein the nucleic acid stabilizing reagent contains at least one of ammonium sulfate, sodium sulfate, and cesium sulfate.
<3> The nucleic acid collecting member according to <1>, wherein the nucleic acid stabilizing reagent contains at least one of a ribonuclease inhibitor and a deoxyribonuclease inhibitor.
<4> The nucleic acid collecting member according to <1>, wherein the nucleic acid stabilizing reagent contains at least one of tetradecyltrimethylammonium oxalate and tartaric acid.
<5> The nucleic acid collecting member according to any one of <1> to <4>, wherein the sample is blood and the nucleic acid stabilizing reagent contains a surfactant.
<6> The <5>, wherein the surfactant is at least one selected from a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant. The nucleic acid collecting member.
<7> The cationic surfactant is dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, or cetyltrimethylammonium bromide,
The anionic surfactant is sodium dodecyl sulfate (SDS), sodium cholate, sodium dodecyl cholate, or sodium N-lauroyl sarcosine, and the amphoteric surfactant is 3-[(3-colamidopropyl). Dimethylammonio] -1-propanesulfonate, phosphatidylethanolamine, 3- (3-cholamidepropyl) dimethylamino-1-propanesulfonate (CHAPS), or [3-[(3-Colamidopropropyloxy) -2-hydroxypropyl-1-dimethylsulfonate-1 ] (CHAPSO)
The nonionic surfactant is polyoxyethylene octyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene-lauryl ester, polyethylene-glycol hexadecyl-ester polyoxyethylene cetyl ether Or a saponin, digitonin, n-octyl-β-D-glucoside, n-octyl-β-D-glucopyranoside, or n-octylthio-β-D-thioglucopyranoside, the nucleic acid collecting member according to the above <7> .
<8> The nucleic acid collecting member according to any one of <1> to <7>, wherein the nucleic acid stabilizing reagent is solid.
<9> The nucleic acid collecting member according to any one of <1> to <8>, wherein the separation unit is a filter.
<10> The nucleic acid collecting member according to any one of <1> to <9>, wherein the container portion is a blood collection tube.
<11> The nucleic acid collecting member according to any one of <1> to <10>, further including a lid member that covers the opening.

  According to the nucleic acid collecting member according to any one of <1> to <11>, the conventional problems can be solved and the object of the present invention can be achieved.

1 Separation means 2 Nucleic acid stabilization reagent 4 Sample 10 Nucleic acid collection member (container with nucleic acid stabilization reagent)

Japanese Patent No. 5390772

Claims (11)

A nucleic acid stabilizing reagent that inactivates a nucleolytic enzyme in the sample;
Separation means for separating nucleated cells in the sample;
A nucleic acid collecting member comprising:   The nucleic acid collecting member according to claim 1, wherein the nucleic acid stabilizing reagent contains at least one of ammonium sulfate, sodium sulfate, and cesium sulfate.   The nucleic acid collection member according to claim 1, wherein the nucleic acid stabilization reagent contains at least one of a ribonuclease inhibitor and a deoxyribonuclease inhibitor.   The nucleic acid collecting member according to claim 1, wherein the nucleic acid stabilizing reagent contains at least one of tetradecyltrimethylammonium oxalate and tartaric acid.   The nucleic acid collection member according to any one of claims 1 to 4, wherein the sample is blood, and the nucleic acid stabilizing reagent contains a surfactant.   The nucleic acid collecting member according to claim 5, wherein the surfactant is at least one selected from a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant. . The cationic surfactant is dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, or cetyltrimethylammonium bromide;
The anionic surfactant is sodium dodecyl sulfate (SDS), sodium cholate, sodium dodecyl cholate, or sodium N-lauroyl sarcosine, and the amphoteric surfactant is 3-[(3-colamidopropyl). Dimethylammonio] -1-propanesulfonate, phosphatidylethanolamine, 3- (3-cholamidepropyl) dimethylamino-1-propanesulfonate (CHAPS), or [3-[(3-Colamidopropropyloxy) -2-hydroxypropyl-1-dimethylsulfonate-1 ] (CHAPSO)
The nonionic surfactant is polyoxyethylene octyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene-lauryl ester, polyethylene-glycol hexadecyl-ester polyoxyethylene cetyl ether The nucleic acid collecting member according to claim 7, which is saponin, digitonin, n-octyl-β-D-glucoside, n-octyl-β-D-glucopyranoside, or n-octylthio-β-D-thioglucopyranoside.   The nucleic acid collecting member according to claim 1, wherein the nucleic acid stabilizing reagent is in a solid form.   The nucleic acid collecting member according to any one of claims 1 to 8, wherein the separating means is a filter.   The nucleic acid collection member according to claim 1, wherein the container portion is a blood collection tube.   The nucleic acid collection member according to claim 1, further comprising a lid member that covers the opening.